Upload
others
View
2
Download
0
Embed Size (px)
Citation preview
How Our Bodies Adapt to Chemicals
Curtis D Klaassen PhD DABT ATS
bull How Our Bodies Adapt to Chemicals or Developing Resistance to Toxicity or Learning to Reprogram the Liver or Progress Depends on Technology
1964
bull Animal ----------------------------------------------------
bull Analytical methods ------------------------------------ bull Colorimeterspectrophotometer
bull Chromatography bull Paper bull TLC
bull Cell fractions (Centrifuge) --------------------------
bull Null mice and rats bull Transgenic mice
bull Computer ----------------------------------------------- bull Oneuniversity
bull Computer bull More than oneperson
bull Radioactivity bull GC bull HPLC-UV bull HPLC-MSMS
bull Tissue culture
present
bull Antibodies bull Radioimmunoassay bull Western blot
bull mRNA
bull Northern blot bull RT-PCR bull Bead assays bull Microarray bull RNA-Seq
bull Transcription factor ndash DNA binding
bull ChIP-qPCR bull ChIP- Seq
1964 2012
-------------------------------------
-------------------------------------
-------------------------------------
1964
bull Drugs and other xenobiotics cross membranes due to their lipid solubility
bull Drugs are biotransformed to water-soluble compounds so they do not pass cell membranes and thus are excreted more readily
bull Microsomal enzyme inducers increase cytochrome P-450s and enhance the elimination of some drugs
bull There might be two cytochrome p450s
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Therefore if the purpose of p450 inducers was to enhance
the elimination of chemicals might the inducers also
increase the elimination of chemicals that are not
biotransformed by enhancing their passage across
membranes
Hypothesis
Plasma clearance of DBSP
Klaassen and Plaa JPET 161361 1968 Time (min)
Pla
sma
DB
SP C
on
cen
trat
ion
(m
g1
00
ml)
1
10
100
0 10 20 30
Control
Phenobarbital
LIVER CELL AS THE CENTRAL PROCESSING UNIT FOR DRUGS
AND NUTRIENTS
Bile Blood Programming
Re-programming
A
BRICK
WALL
PROGRESS
DEPENDS ON
TECHNOLOGY
Effect of PCN on Ouabain uptake in isolated rat hepatocytes
Eaton and Klaassen JPET 208381-385 1979
0
05
1
15
2
25
3
35
0 100 200 300 400 500 600 700 800 900 1000
Ouabain (microM)
V 0
Cont PCN
A
BRICK
WALL
PROGRESS
DEPENDS ON
TECHNOLOGY
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Cd tolerance from Cd pretreatment
Goering and Klaassen JTEH 14803 1984
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and at indicated time
challenged with a lethal dose of CdCl2 (40 mgkg iv) and mortality was
recorded within 48 hrs after Cd challenge
Time after Cd pretreatment n Mortality () No pretreatment 10 100 2 hr 10 100 4 hr 10 100 6 hr 10 60 8 hr 10 50 24 hr 10 0 2 day 10 0 4 day 10 0 8 day 10 0
16 day 10 0
Cd pretreatment protected against acute Cd
hepatotoxicity
Goering and Klaassen TAAP 74308 1984
100
1000
10000
100000
1000000
Control 20 30 40 50
SDH
(S
un
its
ml)
mg Cdkg
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (20-40 mgkg iv) and serum
SDH was determined 10 hrs later Data are Mean plusmn SE of 4-6 rats
Cd pretreatment alters
subcellular Cd distribution
Goering and Klaassen TAAP 70195 1983
0
10
20
30
40
50
60
70
80
Nuclei Mitochondria Microsome Cytosol
C
d d
istr
ibu
tio
n in
th
e li
ver
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and subcellular
Cd distribution was determined 2 hrs later Data are Mean plusmn SE of 6 rats
Cd pretreatment alters cytosolic Cd
distribution
Goering and Klaassen TAAP 70195 1983
0
05
1
15
2
25
3
35
06 10 14 18 22 26
ug
Cd
g li
ver
VeVo
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and
cytosolic Cd distribution was determined 2 hrs later
MT-null mice are highly susceptible to
chronic Cd-induced nephrotoxicity
LiuhelliphellipKlaassen ToxSci 46 197 1998
Wild-type control and MT-null mice were
given CdCl2 (005-24 mgkg sc) daily for 6
weeks and Blood urea nitrogen (BUN) and
renal MT were determined N =6-8 mice
The relationship between human exposure to
cadmium and renal injury
Klaassen et al Ann Rev Pharmacol Toxicol 39 267 1999
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
bull In 1987 a post-doctoral fellow Jie Liu came to my Lab with a ldquosuitcaserdquo of chemicals in Traditional Chinese Medicine that he thought would protect against liver injury
Jie Liu 87-98
OA Oleanolic acid
UA Ursolic acid
UV Uvaol
α-H α-Hederin
HD Hederagenin
GL Glycyrrhizin
18β-GA 18 β-Glycyrrhetinic acid
18 α-GA 18 α-Glycyrrhetinic acid
HAG 19 α-Hydroxyl asiatic acid 29-0- β-glucoside
HA 19 α-Hydroxyl asiatic acid
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
CCl4
AL
T (
UL
)
0
500
1000
1500
2000
2500
3000
SD
H (
U
ml)
0
5000
10000
15000
20000
25000
NE
CR
OS
IS (
grad
e)
00
05
10
15
20
CON Jgs Ngs Ful OA Swe Om DDB
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
AL
T (
UL
)
0
2000
4000
6000
8000
10000
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
SD
H (
kIU
L)
0
5000
10000
15000
20000
25000
Saline
Bromo benz ene
Acetaminophen
Carbon tet
Thioacetamide
Furosemide
Phall oidi n
Colchi cine
Cadmium
D-GalNLPS
Ne
cro
sis
(g
rad
e)
0
1
2
3
VehicleOA
Oleanolic acid
ARE
Maf Nrf2
Oxidative stress and activators
Nrf2
Anti-oxidant and inflammation Cell survival and proliferation
Drug metabolism Lipid metabolism
Ke
ap1
Ke
ap1
Nrf2
CUL3
Modified from DAutreacuteaux and Toledano Nat Rev Mol Cell Biol 8813 2007
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
bull How Our Bodies Adapt to Chemicals or Developing Resistance to Toxicity or Learning to Reprogram the Liver or Progress Depends on Technology
1964
bull Animal ----------------------------------------------------
bull Analytical methods ------------------------------------ bull Colorimeterspectrophotometer
bull Chromatography bull Paper bull TLC
bull Cell fractions (Centrifuge) --------------------------
bull Null mice and rats bull Transgenic mice
bull Computer ----------------------------------------------- bull Oneuniversity
bull Computer bull More than oneperson
bull Radioactivity bull GC bull HPLC-UV bull HPLC-MSMS
bull Tissue culture
present
bull Antibodies bull Radioimmunoassay bull Western blot
bull mRNA
bull Northern blot bull RT-PCR bull Bead assays bull Microarray bull RNA-Seq
bull Transcription factor ndash DNA binding
bull ChIP-qPCR bull ChIP- Seq
1964 2012
-------------------------------------
-------------------------------------
-------------------------------------
1964
bull Drugs and other xenobiotics cross membranes due to their lipid solubility
bull Drugs are biotransformed to water-soluble compounds so they do not pass cell membranes and thus are excreted more readily
bull Microsomal enzyme inducers increase cytochrome P-450s and enhance the elimination of some drugs
bull There might be two cytochrome p450s
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Therefore if the purpose of p450 inducers was to enhance
the elimination of chemicals might the inducers also
increase the elimination of chemicals that are not
biotransformed by enhancing their passage across
membranes
Hypothesis
Plasma clearance of DBSP
Klaassen and Plaa JPET 161361 1968 Time (min)
Pla
sma
DB
SP C
on
cen
trat
ion
(m
g1
00
ml)
1
10
100
0 10 20 30
Control
Phenobarbital
LIVER CELL AS THE CENTRAL PROCESSING UNIT FOR DRUGS
AND NUTRIENTS
Bile Blood Programming
Re-programming
A
BRICK
WALL
PROGRESS
DEPENDS ON
TECHNOLOGY
Effect of PCN on Ouabain uptake in isolated rat hepatocytes
Eaton and Klaassen JPET 208381-385 1979
0
05
1
15
2
25
3
35
0 100 200 300 400 500 600 700 800 900 1000
Ouabain (microM)
V 0
Cont PCN
A
BRICK
WALL
PROGRESS
DEPENDS ON
TECHNOLOGY
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Cd tolerance from Cd pretreatment
Goering and Klaassen JTEH 14803 1984
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and at indicated time
challenged with a lethal dose of CdCl2 (40 mgkg iv) and mortality was
recorded within 48 hrs after Cd challenge
Time after Cd pretreatment n Mortality () No pretreatment 10 100 2 hr 10 100 4 hr 10 100 6 hr 10 60 8 hr 10 50 24 hr 10 0 2 day 10 0 4 day 10 0 8 day 10 0
16 day 10 0
Cd pretreatment protected against acute Cd
hepatotoxicity
Goering and Klaassen TAAP 74308 1984
100
1000
10000
100000
1000000
Control 20 30 40 50
SDH
(S
un
its
ml)
mg Cdkg
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (20-40 mgkg iv) and serum
SDH was determined 10 hrs later Data are Mean plusmn SE of 4-6 rats
Cd pretreatment alters
subcellular Cd distribution
Goering and Klaassen TAAP 70195 1983
0
10
20
30
40
50
60
70
80
Nuclei Mitochondria Microsome Cytosol
C
d d
istr
ibu
tio
n in
th
e li
ver
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and subcellular
Cd distribution was determined 2 hrs later Data are Mean plusmn SE of 6 rats
Cd pretreatment alters cytosolic Cd
distribution
Goering and Klaassen TAAP 70195 1983
0
05
1
15
2
25
3
35
06 10 14 18 22 26
ug
Cd
g li
ver
VeVo
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and
cytosolic Cd distribution was determined 2 hrs later
MT-null mice are highly susceptible to
chronic Cd-induced nephrotoxicity
LiuhelliphellipKlaassen ToxSci 46 197 1998
Wild-type control and MT-null mice were
given CdCl2 (005-24 mgkg sc) daily for 6
weeks and Blood urea nitrogen (BUN) and
renal MT were determined N =6-8 mice
The relationship between human exposure to
cadmium and renal injury
Klaassen et al Ann Rev Pharmacol Toxicol 39 267 1999
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
bull In 1987 a post-doctoral fellow Jie Liu came to my Lab with a ldquosuitcaserdquo of chemicals in Traditional Chinese Medicine that he thought would protect against liver injury
Jie Liu 87-98
OA Oleanolic acid
UA Ursolic acid
UV Uvaol
α-H α-Hederin
HD Hederagenin
GL Glycyrrhizin
18β-GA 18 β-Glycyrrhetinic acid
18 α-GA 18 α-Glycyrrhetinic acid
HAG 19 α-Hydroxyl asiatic acid 29-0- β-glucoside
HA 19 α-Hydroxyl asiatic acid
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
CCl4
AL
T (
UL
)
0
500
1000
1500
2000
2500
3000
SD
H (
U
ml)
0
5000
10000
15000
20000
25000
NE
CR
OS
IS (
grad
e)
00
05
10
15
20
CON Jgs Ngs Ful OA Swe Om DDB
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
AL
T (
UL
)
0
2000
4000
6000
8000
10000
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
SD
H (
kIU
L)
0
5000
10000
15000
20000
25000
Saline
Bromo benz ene
Acetaminophen
Carbon tet
Thioacetamide
Furosemide
Phall oidi n
Colchi cine
Cadmium
D-GalNLPS
Ne
cro
sis
(g
rad
e)
0
1
2
3
VehicleOA
Oleanolic acid
ARE
Maf Nrf2
Oxidative stress and activators
Nrf2
Anti-oxidant and inflammation Cell survival and proliferation
Drug metabolism Lipid metabolism
Ke
ap1
Ke
ap1
Nrf2
CUL3
Modified from DAutreacuteaux and Toledano Nat Rev Mol Cell Biol 8813 2007
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
1964
bull Animal ----------------------------------------------------
bull Analytical methods ------------------------------------ bull Colorimeterspectrophotometer
bull Chromatography bull Paper bull TLC
bull Cell fractions (Centrifuge) --------------------------
bull Null mice and rats bull Transgenic mice
bull Computer ----------------------------------------------- bull Oneuniversity
bull Computer bull More than oneperson
bull Radioactivity bull GC bull HPLC-UV bull HPLC-MSMS
bull Tissue culture
present
bull Antibodies bull Radioimmunoassay bull Western blot
bull mRNA
bull Northern blot bull RT-PCR bull Bead assays bull Microarray bull RNA-Seq
bull Transcription factor ndash DNA binding
bull ChIP-qPCR bull ChIP- Seq
1964 2012
-------------------------------------
-------------------------------------
-------------------------------------
1964
bull Drugs and other xenobiotics cross membranes due to their lipid solubility
bull Drugs are biotransformed to water-soluble compounds so they do not pass cell membranes and thus are excreted more readily
bull Microsomal enzyme inducers increase cytochrome P-450s and enhance the elimination of some drugs
bull There might be two cytochrome p450s
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Therefore if the purpose of p450 inducers was to enhance
the elimination of chemicals might the inducers also
increase the elimination of chemicals that are not
biotransformed by enhancing their passage across
membranes
Hypothesis
Plasma clearance of DBSP
Klaassen and Plaa JPET 161361 1968 Time (min)
Pla
sma
DB
SP C
on
cen
trat
ion
(m
g1
00
ml)
1
10
100
0 10 20 30
Control
Phenobarbital
LIVER CELL AS THE CENTRAL PROCESSING UNIT FOR DRUGS
AND NUTRIENTS
Bile Blood Programming
Re-programming
A
BRICK
WALL
PROGRESS
DEPENDS ON
TECHNOLOGY
Effect of PCN on Ouabain uptake in isolated rat hepatocytes
Eaton and Klaassen JPET 208381-385 1979
0
05
1
15
2
25
3
35
0 100 200 300 400 500 600 700 800 900 1000
Ouabain (microM)
V 0
Cont PCN
A
BRICK
WALL
PROGRESS
DEPENDS ON
TECHNOLOGY
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Cd tolerance from Cd pretreatment
Goering and Klaassen JTEH 14803 1984
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and at indicated time
challenged with a lethal dose of CdCl2 (40 mgkg iv) and mortality was
recorded within 48 hrs after Cd challenge
Time after Cd pretreatment n Mortality () No pretreatment 10 100 2 hr 10 100 4 hr 10 100 6 hr 10 60 8 hr 10 50 24 hr 10 0 2 day 10 0 4 day 10 0 8 day 10 0
16 day 10 0
Cd pretreatment protected against acute Cd
hepatotoxicity
Goering and Klaassen TAAP 74308 1984
100
1000
10000
100000
1000000
Control 20 30 40 50
SDH
(S
un
its
ml)
mg Cdkg
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (20-40 mgkg iv) and serum
SDH was determined 10 hrs later Data are Mean plusmn SE of 4-6 rats
Cd pretreatment alters
subcellular Cd distribution
Goering and Klaassen TAAP 70195 1983
0
10
20
30
40
50
60
70
80
Nuclei Mitochondria Microsome Cytosol
C
d d
istr
ibu
tio
n in
th
e li
ver
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and subcellular
Cd distribution was determined 2 hrs later Data are Mean plusmn SE of 6 rats
Cd pretreatment alters cytosolic Cd
distribution
Goering and Klaassen TAAP 70195 1983
0
05
1
15
2
25
3
35
06 10 14 18 22 26
ug
Cd
g li
ver
VeVo
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and
cytosolic Cd distribution was determined 2 hrs later
MT-null mice are highly susceptible to
chronic Cd-induced nephrotoxicity
LiuhelliphellipKlaassen ToxSci 46 197 1998
Wild-type control and MT-null mice were
given CdCl2 (005-24 mgkg sc) daily for 6
weeks and Blood urea nitrogen (BUN) and
renal MT were determined N =6-8 mice
The relationship between human exposure to
cadmium and renal injury
Klaassen et al Ann Rev Pharmacol Toxicol 39 267 1999
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
bull In 1987 a post-doctoral fellow Jie Liu came to my Lab with a ldquosuitcaserdquo of chemicals in Traditional Chinese Medicine that he thought would protect against liver injury
Jie Liu 87-98
OA Oleanolic acid
UA Ursolic acid
UV Uvaol
α-H α-Hederin
HD Hederagenin
GL Glycyrrhizin
18β-GA 18 β-Glycyrrhetinic acid
18 α-GA 18 α-Glycyrrhetinic acid
HAG 19 α-Hydroxyl asiatic acid 29-0- β-glucoside
HA 19 α-Hydroxyl asiatic acid
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
CCl4
AL
T (
UL
)
0
500
1000
1500
2000
2500
3000
SD
H (
U
ml)
0
5000
10000
15000
20000
25000
NE
CR
OS
IS (
grad
e)
00
05
10
15
20
CON Jgs Ngs Ful OA Swe Om DDB
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
AL
T (
UL
)
0
2000
4000
6000
8000
10000
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
SD
H (
kIU
L)
0
5000
10000
15000
20000
25000
Saline
Bromo benz ene
Acetaminophen
Carbon tet
Thioacetamide
Furosemide
Phall oidi n
Colchi cine
Cadmium
D-GalNLPS
Ne
cro
sis
(g
rad
e)
0
1
2
3
VehicleOA
Oleanolic acid
ARE
Maf Nrf2
Oxidative stress and activators
Nrf2
Anti-oxidant and inflammation Cell survival and proliferation
Drug metabolism Lipid metabolism
Ke
ap1
Ke
ap1
Nrf2
CUL3
Modified from DAutreacuteaux and Toledano Nat Rev Mol Cell Biol 8813 2007
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
bull Antibodies bull Radioimmunoassay bull Western blot
bull mRNA
bull Northern blot bull RT-PCR bull Bead assays bull Microarray bull RNA-Seq
bull Transcription factor ndash DNA binding
bull ChIP-qPCR bull ChIP- Seq
1964 2012
-------------------------------------
-------------------------------------
-------------------------------------
1964
bull Drugs and other xenobiotics cross membranes due to their lipid solubility
bull Drugs are biotransformed to water-soluble compounds so they do not pass cell membranes and thus are excreted more readily
bull Microsomal enzyme inducers increase cytochrome P-450s and enhance the elimination of some drugs
bull There might be two cytochrome p450s
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Therefore if the purpose of p450 inducers was to enhance
the elimination of chemicals might the inducers also
increase the elimination of chemicals that are not
biotransformed by enhancing their passage across
membranes
Hypothesis
Plasma clearance of DBSP
Klaassen and Plaa JPET 161361 1968 Time (min)
Pla
sma
DB
SP C
on
cen
trat
ion
(m
g1
00
ml)
1
10
100
0 10 20 30
Control
Phenobarbital
LIVER CELL AS THE CENTRAL PROCESSING UNIT FOR DRUGS
AND NUTRIENTS
Bile Blood Programming
Re-programming
A
BRICK
WALL
PROGRESS
DEPENDS ON
TECHNOLOGY
Effect of PCN on Ouabain uptake in isolated rat hepatocytes
Eaton and Klaassen JPET 208381-385 1979
0
05
1
15
2
25
3
35
0 100 200 300 400 500 600 700 800 900 1000
Ouabain (microM)
V 0
Cont PCN
A
BRICK
WALL
PROGRESS
DEPENDS ON
TECHNOLOGY
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Cd tolerance from Cd pretreatment
Goering and Klaassen JTEH 14803 1984
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and at indicated time
challenged with a lethal dose of CdCl2 (40 mgkg iv) and mortality was
recorded within 48 hrs after Cd challenge
Time after Cd pretreatment n Mortality () No pretreatment 10 100 2 hr 10 100 4 hr 10 100 6 hr 10 60 8 hr 10 50 24 hr 10 0 2 day 10 0 4 day 10 0 8 day 10 0
16 day 10 0
Cd pretreatment protected against acute Cd
hepatotoxicity
Goering and Klaassen TAAP 74308 1984
100
1000
10000
100000
1000000
Control 20 30 40 50
SDH
(S
un
its
ml)
mg Cdkg
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (20-40 mgkg iv) and serum
SDH was determined 10 hrs later Data are Mean plusmn SE of 4-6 rats
Cd pretreatment alters
subcellular Cd distribution
Goering and Klaassen TAAP 70195 1983
0
10
20
30
40
50
60
70
80
Nuclei Mitochondria Microsome Cytosol
C
d d
istr
ibu
tio
n in
th
e li
ver
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and subcellular
Cd distribution was determined 2 hrs later Data are Mean plusmn SE of 6 rats
Cd pretreatment alters cytosolic Cd
distribution
Goering and Klaassen TAAP 70195 1983
0
05
1
15
2
25
3
35
06 10 14 18 22 26
ug
Cd
g li
ver
VeVo
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and
cytosolic Cd distribution was determined 2 hrs later
MT-null mice are highly susceptible to
chronic Cd-induced nephrotoxicity
LiuhelliphellipKlaassen ToxSci 46 197 1998
Wild-type control and MT-null mice were
given CdCl2 (005-24 mgkg sc) daily for 6
weeks and Blood urea nitrogen (BUN) and
renal MT were determined N =6-8 mice
The relationship between human exposure to
cadmium and renal injury
Klaassen et al Ann Rev Pharmacol Toxicol 39 267 1999
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
bull In 1987 a post-doctoral fellow Jie Liu came to my Lab with a ldquosuitcaserdquo of chemicals in Traditional Chinese Medicine that he thought would protect against liver injury
Jie Liu 87-98
OA Oleanolic acid
UA Ursolic acid
UV Uvaol
α-H α-Hederin
HD Hederagenin
GL Glycyrrhizin
18β-GA 18 β-Glycyrrhetinic acid
18 α-GA 18 α-Glycyrrhetinic acid
HAG 19 α-Hydroxyl asiatic acid 29-0- β-glucoside
HA 19 α-Hydroxyl asiatic acid
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
CCl4
AL
T (
UL
)
0
500
1000
1500
2000
2500
3000
SD
H (
U
ml)
0
5000
10000
15000
20000
25000
NE
CR
OS
IS (
grad
e)
00
05
10
15
20
CON Jgs Ngs Ful OA Swe Om DDB
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
AL
T (
UL
)
0
2000
4000
6000
8000
10000
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
SD
H (
kIU
L)
0
5000
10000
15000
20000
25000
Saline
Bromo benz ene
Acetaminophen
Carbon tet
Thioacetamide
Furosemide
Phall oidi n
Colchi cine
Cadmium
D-GalNLPS
Ne
cro
sis
(g
rad
e)
0
1
2
3
VehicleOA
Oleanolic acid
ARE
Maf Nrf2
Oxidative stress and activators
Nrf2
Anti-oxidant and inflammation Cell survival and proliferation
Drug metabolism Lipid metabolism
Ke
ap1
Ke
ap1
Nrf2
CUL3
Modified from DAutreacuteaux and Toledano Nat Rev Mol Cell Biol 8813 2007
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
1964
bull Drugs and other xenobiotics cross membranes due to their lipid solubility
bull Drugs are biotransformed to water-soluble compounds so they do not pass cell membranes and thus are excreted more readily
bull Microsomal enzyme inducers increase cytochrome P-450s and enhance the elimination of some drugs
bull There might be two cytochrome p450s
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Therefore if the purpose of p450 inducers was to enhance
the elimination of chemicals might the inducers also
increase the elimination of chemicals that are not
biotransformed by enhancing their passage across
membranes
Hypothesis
Plasma clearance of DBSP
Klaassen and Plaa JPET 161361 1968 Time (min)
Pla
sma
DB
SP C
on
cen
trat
ion
(m
g1
00
ml)
1
10
100
0 10 20 30
Control
Phenobarbital
LIVER CELL AS THE CENTRAL PROCESSING UNIT FOR DRUGS
AND NUTRIENTS
Bile Blood Programming
Re-programming
A
BRICK
WALL
PROGRESS
DEPENDS ON
TECHNOLOGY
Effect of PCN on Ouabain uptake in isolated rat hepatocytes
Eaton and Klaassen JPET 208381-385 1979
0
05
1
15
2
25
3
35
0 100 200 300 400 500 600 700 800 900 1000
Ouabain (microM)
V 0
Cont PCN
A
BRICK
WALL
PROGRESS
DEPENDS ON
TECHNOLOGY
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Cd tolerance from Cd pretreatment
Goering and Klaassen JTEH 14803 1984
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and at indicated time
challenged with a lethal dose of CdCl2 (40 mgkg iv) and mortality was
recorded within 48 hrs after Cd challenge
Time after Cd pretreatment n Mortality () No pretreatment 10 100 2 hr 10 100 4 hr 10 100 6 hr 10 60 8 hr 10 50 24 hr 10 0 2 day 10 0 4 day 10 0 8 day 10 0
16 day 10 0
Cd pretreatment protected against acute Cd
hepatotoxicity
Goering and Klaassen TAAP 74308 1984
100
1000
10000
100000
1000000
Control 20 30 40 50
SDH
(S
un
its
ml)
mg Cdkg
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (20-40 mgkg iv) and serum
SDH was determined 10 hrs later Data are Mean plusmn SE of 4-6 rats
Cd pretreatment alters
subcellular Cd distribution
Goering and Klaassen TAAP 70195 1983
0
10
20
30
40
50
60
70
80
Nuclei Mitochondria Microsome Cytosol
C
d d
istr
ibu
tio
n in
th
e li
ver
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and subcellular
Cd distribution was determined 2 hrs later Data are Mean plusmn SE of 6 rats
Cd pretreatment alters cytosolic Cd
distribution
Goering and Klaassen TAAP 70195 1983
0
05
1
15
2
25
3
35
06 10 14 18 22 26
ug
Cd
g li
ver
VeVo
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and
cytosolic Cd distribution was determined 2 hrs later
MT-null mice are highly susceptible to
chronic Cd-induced nephrotoxicity
LiuhelliphellipKlaassen ToxSci 46 197 1998
Wild-type control and MT-null mice were
given CdCl2 (005-24 mgkg sc) daily for 6
weeks and Blood urea nitrogen (BUN) and
renal MT were determined N =6-8 mice
The relationship between human exposure to
cadmium and renal injury
Klaassen et al Ann Rev Pharmacol Toxicol 39 267 1999
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
bull In 1987 a post-doctoral fellow Jie Liu came to my Lab with a ldquosuitcaserdquo of chemicals in Traditional Chinese Medicine that he thought would protect against liver injury
Jie Liu 87-98
OA Oleanolic acid
UA Ursolic acid
UV Uvaol
α-H α-Hederin
HD Hederagenin
GL Glycyrrhizin
18β-GA 18 β-Glycyrrhetinic acid
18 α-GA 18 α-Glycyrrhetinic acid
HAG 19 α-Hydroxyl asiatic acid 29-0- β-glucoside
HA 19 α-Hydroxyl asiatic acid
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
CCl4
AL
T (
UL
)
0
500
1000
1500
2000
2500
3000
SD
H (
U
ml)
0
5000
10000
15000
20000
25000
NE
CR
OS
IS (
grad
e)
00
05
10
15
20
CON Jgs Ngs Ful OA Swe Om DDB
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
AL
T (
UL
)
0
2000
4000
6000
8000
10000
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
SD
H (
kIU
L)
0
5000
10000
15000
20000
25000
Saline
Bromo benz ene
Acetaminophen
Carbon tet
Thioacetamide
Furosemide
Phall oidi n
Colchi cine
Cadmium
D-GalNLPS
Ne
cro
sis
(g
rad
e)
0
1
2
3
VehicleOA
Oleanolic acid
ARE
Maf Nrf2
Oxidative stress and activators
Nrf2
Anti-oxidant and inflammation Cell survival and proliferation
Drug metabolism Lipid metabolism
Ke
ap1
Ke
ap1
Nrf2
CUL3
Modified from DAutreacuteaux and Toledano Nat Rev Mol Cell Biol 8813 2007
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Therefore if the purpose of p450 inducers was to enhance
the elimination of chemicals might the inducers also
increase the elimination of chemicals that are not
biotransformed by enhancing their passage across
membranes
Hypothesis
Plasma clearance of DBSP
Klaassen and Plaa JPET 161361 1968 Time (min)
Pla
sma
DB
SP C
on
cen
trat
ion
(m
g1
00
ml)
1
10
100
0 10 20 30
Control
Phenobarbital
LIVER CELL AS THE CENTRAL PROCESSING UNIT FOR DRUGS
AND NUTRIENTS
Bile Blood Programming
Re-programming
A
BRICK
WALL
PROGRESS
DEPENDS ON
TECHNOLOGY
Effect of PCN on Ouabain uptake in isolated rat hepatocytes
Eaton and Klaassen JPET 208381-385 1979
0
05
1
15
2
25
3
35
0 100 200 300 400 500 600 700 800 900 1000
Ouabain (microM)
V 0
Cont PCN
A
BRICK
WALL
PROGRESS
DEPENDS ON
TECHNOLOGY
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Cd tolerance from Cd pretreatment
Goering and Klaassen JTEH 14803 1984
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and at indicated time
challenged with a lethal dose of CdCl2 (40 mgkg iv) and mortality was
recorded within 48 hrs after Cd challenge
Time after Cd pretreatment n Mortality () No pretreatment 10 100 2 hr 10 100 4 hr 10 100 6 hr 10 60 8 hr 10 50 24 hr 10 0 2 day 10 0 4 day 10 0 8 day 10 0
16 day 10 0
Cd pretreatment protected against acute Cd
hepatotoxicity
Goering and Klaassen TAAP 74308 1984
100
1000
10000
100000
1000000
Control 20 30 40 50
SDH
(S
un
its
ml)
mg Cdkg
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (20-40 mgkg iv) and serum
SDH was determined 10 hrs later Data are Mean plusmn SE of 4-6 rats
Cd pretreatment alters
subcellular Cd distribution
Goering and Klaassen TAAP 70195 1983
0
10
20
30
40
50
60
70
80
Nuclei Mitochondria Microsome Cytosol
C
d d
istr
ibu
tio
n in
th
e li
ver
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and subcellular
Cd distribution was determined 2 hrs later Data are Mean plusmn SE of 6 rats
Cd pretreatment alters cytosolic Cd
distribution
Goering and Klaassen TAAP 70195 1983
0
05
1
15
2
25
3
35
06 10 14 18 22 26
ug
Cd
g li
ver
VeVo
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and
cytosolic Cd distribution was determined 2 hrs later
MT-null mice are highly susceptible to
chronic Cd-induced nephrotoxicity
LiuhelliphellipKlaassen ToxSci 46 197 1998
Wild-type control and MT-null mice were
given CdCl2 (005-24 mgkg sc) daily for 6
weeks and Blood urea nitrogen (BUN) and
renal MT were determined N =6-8 mice
The relationship between human exposure to
cadmium and renal injury
Klaassen et al Ann Rev Pharmacol Toxicol 39 267 1999
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
bull In 1987 a post-doctoral fellow Jie Liu came to my Lab with a ldquosuitcaserdquo of chemicals in Traditional Chinese Medicine that he thought would protect against liver injury
Jie Liu 87-98
OA Oleanolic acid
UA Ursolic acid
UV Uvaol
α-H α-Hederin
HD Hederagenin
GL Glycyrrhizin
18β-GA 18 β-Glycyrrhetinic acid
18 α-GA 18 α-Glycyrrhetinic acid
HAG 19 α-Hydroxyl asiatic acid 29-0- β-glucoside
HA 19 α-Hydroxyl asiatic acid
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
CCl4
AL
T (
UL
)
0
500
1000
1500
2000
2500
3000
SD
H (
U
ml)
0
5000
10000
15000
20000
25000
NE
CR
OS
IS (
grad
e)
00
05
10
15
20
CON Jgs Ngs Ful OA Swe Om DDB
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
AL
T (
UL
)
0
2000
4000
6000
8000
10000
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
SD
H (
kIU
L)
0
5000
10000
15000
20000
25000
Saline
Bromo benz ene
Acetaminophen
Carbon tet
Thioacetamide
Furosemide
Phall oidi n
Colchi cine
Cadmium
D-GalNLPS
Ne
cro
sis
(g
rad
e)
0
1
2
3
VehicleOA
Oleanolic acid
ARE
Maf Nrf2
Oxidative stress and activators
Nrf2
Anti-oxidant and inflammation Cell survival and proliferation
Drug metabolism Lipid metabolism
Ke
ap1
Ke
ap1
Nrf2
CUL3
Modified from DAutreacuteaux and Toledano Nat Rev Mol Cell Biol 8813 2007
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Therefore if the purpose of p450 inducers was to enhance
the elimination of chemicals might the inducers also
increase the elimination of chemicals that are not
biotransformed by enhancing their passage across
membranes
Hypothesis
Plasma clearance of DBSP
Klaassen and Plaa JPET 161361 1968 Time (min)
Pla
sma
DB
SP C
on
cen
trat
ion
(m
g1
00
ml)
1
10
100
0 10 20 30
Control
Phenobarbital
LIVER CELL AS THE CENTRAL PROCESSING UNIT FOR DRUGS
AND NUTRIENTS
Bile Blood Programming
Re-programming
A
BRICK
WALL
PROGRESS
DEPENDS ON
TECHNOLOGY
Effect of PCN on Ouabain uptake in isolated rat hepatocytes
Eaton and Klaassen JPET 208381-385 1979
0
05
1
15
2
25
3
35
0 100 200 300 400 500 600 700 800 900 1000
Ouabain (microM)
V 0
Cont PCN
A
BRICK
WALL
PROGRESS
DEPENDS ON
TECHNOLOGY
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Cd tolerance from Cd pretreatment
Goering and Klaassen JTEH 14803 1984
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and at indicated time
challenged with a lethal dose of CdCl2 (40 mgkg iv) and mortality was
recorded within 48 hrs after Cd challenge
Time after Cd pretreatment n Mortality () No pretreatment 10 100 2 hr 10 100 4 hr 10 100 6 hr 10 60 8 hr 10 50 24 hr 10 0 2 day 10 0 4 day 10 0 8 day 10 0
16 day 10 0
Cd pretreatment protected against acute Cd
hepatotoxicity
Goering and Klaassen TAAP 74308 1984
100
1000
10000
100000
1000000
Control 20 30 40 50
SDH
(S
un
its
ml)
mg Cdkg
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (20-40 mgkg iv) and serum
SDH was determined 10 hrs later Data are Mean plusmn SE of 4-6 rats
Cd pretreatment alters
subcellular Cd distribution
Goering and Klaassen TAAP 70195 1983
0
10
20
30
40
50
60
70
80
Nuclei Mitochondria Microsome Cytosol
C
d d
istr
ibu
tio
n in
th
e li
ver
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and subcellular
Cd distribution was determined 2 hrs later Data are Mean plusmn SE of 6 rats
Cd pretreatment alters cytosolic Cd
distribution
Goering and Klaassen TAAP 70195 1983
0
05
1
15
2
25
3
35
06 10 14 18 22 26
ug
Cd
g li
ver
VeVo
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and
cytosolic Cd distribution was determined 2 hrs later
MT-null mice are highly susceptible to
chronic Cd-induced nephrotoxicity
LiuhelliphellipKlaassen ToxSci 46 197 1998
Wild-type control and MT-null mice were
given CdCl2 (005-24 mgkg sc) daily for 6
weeks and Blood urea nitrogen (BUN) and
renal MT were determined N =6-8 mice
The relationship between human exposure to
cadmium and renal injury
Klaassen et al Ann Rev Pharmacol Toxicol 39 267 1999
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
bull In 1987 a post-doctoral fellow Jie Liu came to my Lab with a ldquosuitcaserdquo of chemicals in Traditional Chinese Medicine that he thought would protect against liver injury
Jie Liu 87-98
OA Oleanolic acid
UA Ursolic acid
UV Uvaol
α-H α-Hederin
HD Hederagenin
GL Glycyrrhizin
18β-GA 18 β-Glycyrrhetinic acid
18 α-GA 18 α-Glycyrrhetinic acid
HAG 19 α-Hydroxyl asiatic acid 29-0- β-glucoside
HA 19 α-Hydroxyl asiatic acid
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
CCl4
AL
T (
UL
)
0
500
1000
1500
2000
2500
3000
SD
H (
U
ml)
0
5000
10000
15000
20000
25000
NE
CR
OS
IS (
grad
e)
00
05
10
15
20
CON Jgs Ngs Ful OA Swe Om DDB
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
AL
T (
UL
)
0
2000
4000
6000
8000
10000
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
SD
H (
kIU
L)
0
5000
10000
15000
20000
25000
Saline
Bromo benz ene
Acetaminophen
Carbon tet
Thioacetamide
Furosemide
Phall oidi n
Colchi cine
Cadmium
D-GalNLPS
Ne
cro
sis
(g
rad
e)
0
1
2
3
VehicleOA
Oleanolic acid
ARE
Maf Nrf2
Oxidative stress and activators
Nrf2
Anti-oxidant and inflammation Cell survival and proliferation
Drug metabolism Lipid metabolism
Ke
ap1
Ke
ap1
Nrf2
CUL3
Modified from DAutreacuteaux and Toledano Nat Rev Mol Cell Biol 8813 2007
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Plasma clearance of DBSP
Klaassen and Plaa JPET 161361 1968 Time (min)
Pla
sma
DB
SP C
on
cen
trat
ion
(m
g1
00
ml)
1
10
100
0 10 20 30
Control
Phenobarbital
LIVER CELL AS THE CENTRAL PROCESSING UNIT FOR DRUGS
AND NUTRIENTS
Bile Blood Programming
Re-programming
A
BRICK
WALL
PROGRESS
DEPENDS ON
TECHNOLOGY
Effect of PCN on Ouabain uptake in isolated rat hepatocytes
Eaton and Klaassen JPET 208381-385 1979
0
05
1
15
2
25
3
35
0 100 200 300 400 500 600 700 800 900 1000
Ouabain (microM)
V 0
Cont PCN
A
BRICK
WALL
PROGRESS
DEPENDS ON
TECHNOLOGY
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Cd tolerance from Cd pretreatment
Goering and Klaassen JTEH 14803 1984
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and at indicated time
challenged with a lethal dose of CdCl2 (40 mgkg iv) and mortality was
recorded within 48 hrs after Cd challenge
Time after Cd pretreatment n Mortality () No pretreatment 10 100 2 hr 10 100 4 hr 10 100 6 hr 10 60 8 hr 10 50 24 hr 10 0 2 day 10 0 4 day 10 0 8 day 10 0
16 day 10 0
Cd pretreatment protected against acute Cd
hepatotoxicity
Goering and Klaassen TAAP 74308 1984
100
1000
10000
100000
1000000
Control 20 30 40 50
SDH
(S
un
its
ml)
mg Cdkg
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (20-40 mgkg iv) and serum
SDH was determined 10 hrs later Data are Mean plusmn SE of 4-6 rats
Cd pretreatment alters
subcellular Cd distribution
Goering and Klaassen TAAP 70195 1983
0
10
20
30
40
50
60
70
80
Nuclei Mitochondria Microsome Cytosol
C
d d
istr
ibu
tio
n in
th
e li
ver
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and subcellular
Cd distribution was determined 2 hrs later Data are Mean plusmn SE of 6 rats
Cd pretreatment alters cytosolic Cd
distribution
Goering and Klaassen TAAP 70195 1983
0
05
1
15
2
25
3
35
06 10 14 18 22 26
ug
Cd
g li
ver
VeVo
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and
cytosolic Cd distribution was determined 2 hrs later
MT-null mice are highly susceptible to
chronic Cd-induced nephrotoxicity
LiuhelliphellipKlaassen ToxSci 46 197 1998
Wild-type control and MT-null mice were
given CdCl2 (005-24 mgkg sc) daily for 6
weeks and Blood urea nitrogen (BUN) and
renal MT were determined N =6-8 mice
The relationship between human exposure to
cadmium and renal injury
Klaassen et al Ann Rev Pharmacol Toxicol 39 267 1999
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
bull In 1987 a post-doctoral fellow Jie Liu came to my Lab with a ldquosuitcaserdquo of chemicals in Traditional Chinese Medicine that he thought would protect against liver injury
Jie Liu 87-98
OA Oleanolic acid
UA Ursolic acid
UV Uvaol
α-H α-Hederin
HD Hederagenin
GL Glycyrrhizin
18β-GA 18 β-Glycyrrhetinic acid
18 α-GA 18 α-Glycyrrhetinic acid
HAG 19 α-Hydroxyl asiatic acid 29-0- β-glucoside
HA 19 α-Hydroxyl asiatic acid
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
CCl4
AL
T (
UL
)
0
500
1000
1500
2000
2500
3000
SD
H (
U
ml)
0
5000
10000
15000
20000
25000
NE
CR
OS
IS (
grad
e)
00
05
10
15
20
CON Jgs Ngs Ful OA Swe Om DDB
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
AL
T (
UL
)
0
2000
4000
6000
8000
10000
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
SD
H (
kIU
L)
0
5000
10000
15000
20000
25000
Saline
Bromo benz ene
Acetaminophen
Carbon tet
Thioacetamide
Furosemide
Phall oidi n
Colchi cine
Cadmium
D-GalNLPS
Ne
cro
sis
(g
rad
e)
0
1
2
3
VehicleOA
Oleanolic acid
ARE
Maf Nrf2
Oxidative stress and activators
Nrf2
Anti-oxidant and inflammation Cell survival and proliferation
Drug metabolism Lipid metabolism
Ke
ap1
Ke
ap1
Nrf2
CUL3
Modified from DAutreacuteaux and Toledano Nat Rev Mol Cell Biol 8813 2007
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
LIVER CELL AS THE CENTRAL PROCESSING UNIT FOR DRUGS
AND NUTRIENTS
Bile Blood Programming
Re-programming
A
BRICK
WALL
PROGRESS
DEPENDS ON
TECHNOLOGY
Effect of PCN on Ouabain uptake in isolated rat hepatocytes
Eaton and Klaassen JPET 208381-385 1979
0
05
1
15
2
25
3
35
0 100 200 300 400 500 600 700 800 900 1000
Ouabain (microM)
V 0
Cont PCN
A
BRICK
WALL
PROGRESS
DEPENDS ON
TECHNOLOGY
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Cd tolerance from Cd pretreatment
Goering and Klaassen JTEH 14803 1984
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and at indicated time
challenged with a lethal dose of CdCl2 (40 mgkg iv) and mortality was
recorded within 48 hrs after Cd challenge
Time after Cd pretreatment n Mortality () No pretreatment 10 100 2 hr 10 100 4 hr 10 100 6 hr 10 60 8 hr 10 50 24 hr 10 0 2 day 10 0 4 day 10 0 8 day 10 0
16 day 10 0
Cd pretreatment protected against acute Cd
hepatotoxicity
Goering and Klaassen TAAP 74308 1984
100
1000
10000
100000
1000000
Control 20 30 40 50
SDH
(S
un
its
ml)
mg Cdkg
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (20-40 mgkg iv) and serum
SDH was determined 10 hrs later Data are Mean plusmn SE of 4-6 rats
Cd pretreatment alters
subcellular Cd distribution
Goering and Klaassen TAAP 70195 1983
0
10
20
30
40
50
60
70
80
Nuclei Mitochondria Microsome Cytosol
C
d d
istr
ibu
tio
n in
th
e li
ver
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and subcellular
Cd distribution was determined 2 hrs later Data are Mean plusmn SE of 6 rats
Cd pretreatment alters cytosolic Cd
distribution
Goering and Klaassen TAAP 70195 1983
0
05
1
15
2
25
3
35
06 10 14 18 22 26
ug
Cd
g li
ver
VeVo
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and
cytosolic Cd distribution was determined 2 hrs later
MT-null mice are highly susceptible to
chronic Cd-induced nephrotoxicity
LiuhelliphellipKlaassen ToxSci 46 197 1998
Wild-type control and MT-null mice were
given CdCl2 (005-24 mgkg sc) daily for 6
weeks and Blood urea nitrogen (BUN) and
renal MT were determined N =6-8 mice
The relationship between human exposure to
cadmium and renal injury
Klaassen et al Ann Rev Pharmacol Toxicol 39 267 1999
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
bull In 1987 a post-doctoral fellow Jie Liu came to my Lab with a ldquosuitcaserdquo of chemicals in Traditional Chinese Medicine that he thought would protect against liver injury
Jie Liu 87-98
OA Oleanolic acid
UA Ursolic acid
UV Uvaol
α-H α-Hederin
HD Hederagenin
GL Glycyrrhizin
18β-GA 18 β-Glycyrrhetinic acid
18 α-GA 18 α-Glycyrrhetinic acid
HAG 19 α-Hydroxyl asiatic acid 29-0- β-glucoside
HA 19 α-Hydroxyl asiatic acid
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
CCl4
AL
T (
UL
)
0
500
1000
1500
2000
2500
3000
SD
H (
U
ml)
0
5000
10000
15000
20000
25000
NE
CR
OS
IS (
grad
e)
00
05
10
15
20
CON Jgs Ngs Ful OA Swe Om DDB
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
AL
T (
UL
)
0
2000
4000
6000
8000
10000
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
SD
H (
kIU
L)
0
5000
10000
15000
20000
25000
Saline
Bromo benz ene
Acetaminophen
Carbon tet
Thioacetamide
Furosemide
Phall oidi n
Colchi cine
Cadmium
D-GalNLPS
Ne
cro
sis
(g
rad
e)
0
1
2
3
VehicleOA
Oleanolic acid
ARE
Maf Nrf2
Oxidative stress and activators
Nrf2
Anti-oxidant and inflammation Cell survival and proliferation
Drug metabolism Lipid metabolism
Ke
ap1
Ke
ap1
Nrf2
CUL3
Modified from DAutreacuteaux and Toledano Nat Rev Mol Cell Biol 8813 2007
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
A
BRICK
WALL
PROGRESS
DEPENDS ON
TECHNOLOGY
Effect of PCN on Ouabain uptake in isolated rat hepatocytes
Eaton and Klaassen JPET 208381-385 1979
0
05
1
15
2
25
3
35
0 100 200 300 400 500 600 700 800 900 1000
Ouabain (microM)
V 0
Cont PCN
A
BRICK
WALL
PROGRESS
DEPENDS ON
TECHNOLOGY
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Cd tolerance from Cd pretreatment
Goering and Klaassen JTEH 14803 1984
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and at indicated time
challenged with a lethal dose of CdCl2 (40 mgkg iv) and mortality was
recorded within 48 hrs after Cd challenge
Time after Cd pretreatment n Mortality () No pretreatment 10 100 2 hr 10 100 4 hr 10 100 6 hr 10 60 8 hr 10 50 24 hr 10 0 2 day 10 0 4 day 10 0 8 day 10 0
16 day 10 0
Cd pretreatment protected against acute Cd
hepatotoxicity
Goering and Klaassen TAAP 74308 1984
100
1000
10000
100000
1000000
Control 20 30 40 50
SDH
(S
un
its
ml)
mg Cdkg
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (20-40 mgkg iv) and serum
SDH was determined 10 hrs later Data are Mean plusmn SE of 4-6 rats
Cd pretreatment alters
subcellular Cd distribution
Goering and Klaassen TAAP 70195 1983
0
10
20
30
40
50
60
70
80
Nuclei Mitochondria Microsome Cytosol
C
d d
istr
ibu
tio
n in
th
e li
ver
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and subcellular
Cd distribution was determined 2 hrs later Data are Mean plusmn SE of 6 rats
Cd pretreatment alters cytosolic Cd
distribution
Goering and Klaassen TAAP 70195 1983
0
05
1
15
2
25
3
35
06 10 14 18 22 26
ug
Cd
g li
ver
VeVo
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and
cytosolic Cd distribution was determined 2 hrs later
MT-null mice are highly susceptible to
chronic Cd-induced nephrotoxicity
LiuhelliphellipKlaassen ToxSci 46 197 1998
Wild-type control and MT-null mice were
given CdCl2 (005-24 mgkg sc) daily for 6
weeks and Blood urea nitrogen (BUN) and
renal MT were determined N =6-8 mice
The relationship between human exposure to
cadmium and renal injury
Klaassen et al Ann Rev Pharmacol Toxicol 39 267 1999
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
bull In 1987 a post-doctoral fellow Jie Liu came to my Lab with a ldquosuitcaserdquo of chemicals in Traditional Chinese Medicine that he thought would protect against liver injury
Jie Liu 87-98
OA Oleanolic acid
UA Ursolic acid
UV Uvaol
α-H α-Hederin
HD Hederagenin
GL Glycyrrhizin
18β-GA 18 β-Glycyrrhetinic acid
18 α-GA 18 α-Glycyrrhetinic acid
HAG 19 α-Hydroxyl asiatic acid 29-0- β-glucoside
HA 19 α-Hydroxyl asiatic acid
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
CCl4
AL
T (
UL
)
0
500
1000
1500
2000
2500
3000
SD
H (
U
ml)
0
5000
10000
15000
20000
25000
NE
CR
OS
IS (
grad
e)
00
05
10
15
20
CON Jgs Ngs Ful OA Swe Om DDB
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
AL
T (
UL
)
0
2000
4000
6000
8000
10000
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
SD
H (
kIU
L)
0
5000
10000
15000
20000
25000
Saline
Bromo benz ene
Acetaminophen
Carbon tet
Thioacetamide
Furosemide
Phall oidi n
Colchi cine
Cadmium
D-GalNLPS
Ne
cro
sis
(g
rad
e)
0
1
2
3
VehicleOA
Oleanolic acid
ARE
Maf Nrf2
Oxidative stress and activators
Nrf2
Anti-oxidant and inflammation Cell survival and proliferation
Drug metabolism Lipid metabolism
Ke
ap1
Ke
ap1
Nrf2
CUL3
Modified from DAutreacuteaux and Toledano Nat Rev Mol Cell Biol 8813 2007
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Effect of PCN on Ouabain uptake in isolated rat hepatocytes
Eaton and Klaassen JPET 208381-385 1979
0
05
1
15
2
25
3
35
0 100 200 300 400 500 600 700 800 900 1000
Ouabain (microM)
V 0
Cont PCN
A
BRICK
WALL
PROGRESS
DEPENDS ON
TECHNOLOGY
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Cd tolerance from Cd pretreatment
Goering and Klaassen JTEH 14803 1984
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and at indicated time
challenged with a lethal dose of CdCl2 (40 mgkg iv) and mortality was
recorded within 48 hrs after Cd challenge
Time after Cd pretreatment n Mortality () No pretreatment 10 100 2 hr 10 100 4 hr 10 100 6 hr 10 60 8 hr 10 50 24 hr 10 0 2 day 10 0 4 day 10 0 8 day 10 0
16 day 10 0
Cd pretreatment protected against acute Cd
hepatotoxicity
Goering and Klaassen TAAP 74308 1984
100
1000
10000
100000
1000000
Control 20 30 40 50
SDH
(S
un
its
ml)
mg Cdkg
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (20-40 mgkg iv) and serum
SDH was determined 10 hrs later Data are Mean plusmn SE of 4-6 rats
Cd pretreatment alters
subcellular Cd distribution
Goering and Klaassen TAAP 70195 1983
0
10
20
30
40
50
60
70
80
Nuclei Mitochondria Microsome Cytosol
C
d d
istr
ibu
tio
n in
th
e li
ver
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and subcellular
Cd distribution was determined 2 hrs later Data are Mean plusmn SE of 6 rats
Cd pretreatment alters cytosolic Cd
distribution
Goering and Klaassen TAAP 70195 1983
0
05
1
15
2
25
3
35
06 10 14 18 22 26
ug
Cd
g li
ver
VeVo
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and
cytosolic Cd distribution was determined 2 hrs later
MT-null mice are highly susceptible to
chronic Cd-induced nephrotoxicity
LiuhelliphellipKlaassen ToxSci 46 197 1998
Wild-type control and MT-null mice were
given CdCl2 (005-24 mgkg sc) daily for 6
weeks and Blood urea nitrogen (BUN) and
renal MT were determined N =6-8 mice
The relationship between human exposure to
cadmium and renal injury
Klaassen et al Ann Rev Pharmacol Toxicol 39 267 1999
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
bull In 1987 a post-doctoral fellow Jie Liu came to my Lab with a ldquosuitcaserdquo of chemicals in Traditional Chinese Medicine that he thought would protect against liver injury
Jie Liu 87-98
OA Oleanolic acid
UA Ursolic acid
UV Uvaol
α-H α-Hederin
HD Hederagenin
GL Glycyrrhizin
18β-GA 18 β-Glycyrrhetinic acid
18 α-GA 18 α-Glycyrrhetinic acid
HAG 19 α-Hydroxyl asiatic acid 29-0- β-glucoside
HA 19 α-Hydroxyl asiatic acid
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
CCl4
AL
T (
UL
)
0
500
1000
1500
2000
2500
3000
SD
H (
U
ml)
0
5000
10000
15000
20000
25000
NE
CR
OS
IS (
grad
e)
00
05
10
15
20
CON Jgs Ngs Ful OA Swe Om DDB
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
AL
T (
UL
)
0
2000
4000
6000
8000
10000
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
SD
H (
kIU
L)
0
5000
10000
15000
20000
25000
Saline
Bromo benz ene
Acetaminophen
Carbon tet
Thioacetamide
Furosemide
Phall oidi n
Colchi cine
Cadmium
D-GalNLPS
Ne
cro
sis
(g
rad
e)
0
1
2
3
VehicleOA
Oleanolic acid
ARE
Maf Nrf2
Oxidative stress and activators
Nrf2
Anti-oxidant and inflammation Cell survival and proliferation
Drug metabolism Lipid metabolism
Ke
ap1
Ke
ap1
Nrf2
CUL3
Modified from DAutreacuteaux and Toledano Nat Rev Mol Cell Biol 8813 2007
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
A
BRICK
WALL
PROGRESS
DEPENDS ON
TECHNOLOGY
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Cd tolerance from Cd pretreatment
Goering and Klaassen JTEH 14803 1984
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and at indicated time
challenged with a lethal dose of CdCl2 (40 mgkg iv) and mortality was
recorded within 48 hrs after Cd challenge
Time after Cd pretreatment n Mortality () No pretreatment 10 100 2 hr 10 100 4 hr 10 100 6 hr 10 60 8 hr 10 50 24 hr 10 0 2 day 10 0 4 day 10 0 8 day 10 0
16 day 10 0
Cd pretreatment protected against acute Cd
hepatotoxicity
Goering and Klaassen TAAP 74308 1984
100
1000
10000
100000
1000000
Control 20 30 40 50
SDH
(S
un
its
ml)
mg Cdkg
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (20-40 mgkg iv) and serum
SDH was determined 10 hrs later Data are Mean plusmn SE of 4-6 rats
Cd pretreatment alters
subcellular Cd distribution
Goering and Klaassen TAAP 70195 1983
0
10
20
30
40
50
60
70
80
Nuclei Mitochondria Microsome Cytosol
C
d d
istr
ibu
tio
n in
th
e li
ver
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and subcellular
Cd distribution was determined 2 hrs later Data are Mean plusmn SE of 6 rats
Cd pretreatment alters cytosolic Cd
distribution
Goering and Klaassen TAAP 70195 1983
0
05
1
15
2
25
3
35
06 10 14 18 22 26
ug
Cd
g li
ver
VeVo
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and
cytosolic Cd distribution was determined 2 hrs later
MT-null mice are highly susceptible to
chronic Cd-induced nephrotoxicity
LiuhelliphellipKlaassen ToxSci 46 197 1998
Wild-type control and MT-null mice were
given CdCl2 (005-24 mgkg sc) daily for 6
weeks and Blood urea nitrogen (BUN) and
renal MT were determined N =6-8 mice
The relationship between human exposure to
cadmium and renal injury
Klaassen et al Ann Rev Pharmacol Toxicol 39 267 1999
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
bull In 1987 a post-doctoral fellow Jie Liu came to my Lab with a ldquosuitcaserdquo of chemicals in Traditional Chinese Medicine that he thought would protect against liver injury
Jie Liu 87-98
OA Oleanolic acid
UA Ursolic acid
UV Uvaol
α-H α-Hederin
HD Hederagenin
GL Glycyrrhizin
18β-GA 18 β-Glycyrrhetinic acid
18 α-GA 18 α-Glycyrrhetinic acid
HAG 19 α-Hydroxyl asiatic acid 29-0- β-glucoside
HA 19 α-Hydroxyl asiatic acid
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
CCl4
AL
T (
UL
)
0
500
1000
1500
2000
2500
3000
SD
H (
U
ml)
0
5000
10000
15000
20000
25000
NE
CR
OS
IS (
grad
e)
00
05
10
15
20
CON Jgs Ngs Ful OA Swe Om DDB
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
AL
T (
UL
)
0
2000
4000
6000
8000
10000
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
SD
H (
kIU
L)
0
5000
10000
15000
20000
25000
Saline
Bromo benz ene
Acetaminophen
Carbon tet
Thioacetamide
Furosemide
Phall oidi n
Colchi cine
Cadmium
D-GalNLPS
Ne
cro
sis
(g
rad
e)
0
1
2
3
VehicleOA
Oleanolic acid
ARE
Maf Nrf2
Oxidative stress and activators
Nrf2
Anti-oxidant and inflammation Cell survival and proliferation
Drug metabolism Lipid metabolism
Ke
ap1
Ke
ap1
Nrf2
CUL3
Modified from DAutreacuteaux and Toledano Nat Rev Mol Cell Biol 8813 2007
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Cd tolerance from Cd pretreatment
Goering and Klaassen JTEH 14803 1984
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and at indicated time
challenged with a lethal dose of CdCl2 (40 mgkg iv) and mortality was
recorded within 48 hrs after Cd challenge
Time after Cd pretreatment n Mortality () No pretreatment 10 100 2 hr 10 100 4 hr 10 100 6 hr 10 60 8 hr 10 50 24 hr 10 0 2 day 10 0 4 day 10 0 8 day 10 0
16 day 10 0
Cd pretreatment protected against acute Cd
hepatotoxicity
Goering and Klaassen TAAP 74308 1984
100
1000
10000
100000
1000000
Control 20 30 40 50
SDH
(S
un
its
ml)
mg Cdkg
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (20-40 mgkg iv) and serum
SDH was determined 10 hrs later Data are Mean plusmn SE of 4-6 rats
Cd pretreatment alters
subcellular Cd distribution
Goering and Klaassen TAAP 70195 1983
0
10
20
30
40
50
60
70
80
Nuclei Mitochondria Microsome Cytosol
C
d d
istr
ibu
tio
n in
th
e li
ver
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and subcellular
Cd distribution was determined 2 hrs later Data are Mean plusmn SE of 6 rats
Cd pretreatment alters cytosolic Cd
distribution
Goering and Klaassen TAAP 70195 1983
0
05
1
15
2
25
3
35
06 10 14 18 22 26
ug
Cd
g li
ver
VeVo
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and
cytosolic Cd distribution was determined 2 hrs later
MT-null mice are highly susceptible to
chronic Cd-induced nephrotoxicity
LiuhelliphellipKlaassen ToxSci 46 197 1998
Wild-type control and MT-null mice were
given CdCl2 (005-24 mgkg sc) daily for 6
weeks and Blood urea nitrogen (BUN) and
renal MT were determined N =6-8 mice
The relationship between human exposure to
cadmium and renal injury
Klaassen et al Ann Rev Pharmacol Toxicol 39 267 1999
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
bull In 1987 a post-doctoral fellow Jie Liu came to my Lab with a ldquosuitcaserdquo of chemicals in Traditional Chinese Medicine that he thought would protect against liver injury
Jie Liu 87-98
OA Oleanolic acid
UA Ursolic acid
UV Uvaol
α-H α-Hederin
HD Hederagenin
GL Glycyrrhizin
18β-GA 18 β-Glycyrrhetinic acid
18 α-GA 18 α-Glycyrrhetinic acid
HAG 19 α-Hydroxyl asiatic acid 29-0- β-glucoside
HA 19 α-Hydroxyl asiatic acid
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
CCl4
AL
T (
UL
)
0
500
1000
1500
2000
2500
3000
SD
H (
U
ml)
0
5000
10000
15000
20000
25000
NE
CR
OS
IS (
grad
e)
00
05
10
15
20
CON Jgs Ngs Ful OA Swe Om DDB
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
AL
T (
UL
)
0
2000
4000
6000
8000
10000
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
SD
H (
kIU
L)
0
5000
10000
15000
20000
25000
Saline
Bromo benz ene
Acetaminophen
Carbon tet
Thioacetamide
Furosemide
Phall oidi n
Colchi cine
Cadmium
D-GalNLPS
Ne
cro
sis
(g
rad
e)
0
1
2
3
VehicleOA
Oleanolic acid
ARE
Maf Nrf2
Oxidative stress and activators
Nrf2
Anti-oxidant and inflammation Cell survival and proliferation
Drug metabolism Lipid metabolism
Ke
ap1
Ke
ap1
Nrf2
CUL3
Modified from DAutreacuteaux and Toledano Nat Rev Mol Cell Biol 8813 2007
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Cd tolerance from Cd pretreatment
Goering and Klaassen JTEH 14803 1984
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and at indicated time
challenged with a lethal dose of CdCl2 (40 mgkg iv) and mortality was
recorded within 48 hrs after Cd challenge
Time after Cd pretreatment n Mortality () No pretreatment 10 100 2 hr 10 100 4 hr 10 100 6 hr 10 60 8 hr 10 50 24 hr 10 0 2 day 10 0 4 day 10 0 8 day 10 0
16 day 10 0
Cd pretreatment protected against acute Cd
hepatotoxicity
Goering and Klaassen TAAP 74308 1984
100
1000
10000
100000
1000000
Control 20 30 40 50
SDH
(S
un
its
ml)
mg Cdkg
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (20-40 mgkg iv) and serum
SDH was determined 10 hrs later Data are Mean plusmn SE of 4-6 rats
Cd pretreatment alters
subcellular Cd distribution
Goering and Klaassen TAAP 70195 1983
0
10
20
30
40
50
60
70
80
Nuclei Mitochondria Microsome Cytosol
C
d d
istr
ibu
tio
n in
th
e li
ver
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and subcellular
Cd distribution was determined 2 hrs later Data are Mean plusmn SE of 6 rats
Cd pretreatment alters cytosolic Cd
distribution
Goering and Klaassen TAAP 70195 1983
0
05
1
15
2
25
3
35
06 10 14 18 22 26
ug
Cd
g li
ver
VeVo
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and
cytosolic Cd distribution was determined 2 hrs later
MT-null mice are highly susceptible to
chronic Cd-induced nephrotoxicity
LiuhelliphellipKlaassen ToxSci 46 197 1998
Wild-type control and MT-null mice were
given CdCl2 (005-24 mgkg sc) daily for 6
weeks and Blood urea nitrogen (BUN) and
renal MT were determined N =6-8 mice
The relationship between human exposure to
cadmium and renal injury
Klaassen et al Ann Rev Pharmacol Toxicol 39 267 1999
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
bull In 1987 a post-doctoral fellow Jie Liu came to my Lab with a ldquosuitcaserdquo of chemicals in Traditional Chinese Medicine that he thought would protect against liver injury
Jie Liu 87-98
OA Oleanolic acid
UA Ursolic acid
UV Uvaol
α-H α-Hederin
HD Hederagenin
GL Glycyrrhizin
18β-GA 18 β-Glycyrrhetinic acid
18 α-GA 18 α-Glycyrrhetinic acid
HAG 19 α-Hydroxyl asiatic acid 29-0- β-glucoside
HA 19 α-Hydroxyl asiatic acid
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
CCl4
AL
T (
UL
)
0
500
1000
1500
2000
2500
3000
SD
H (
U
ml)
0
5000
10000
15000
20000
25000
NE
CR
OS
IS (
grad
e)
00
05
10
15
20
CON Jgs Ngs Ful OA Swe Om DDB
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
AL
T (
UL
)
0
2000
4000
6000
8000
10000
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
SD
H (
kIU
L)
0
5000
10000
15000
20000
25000
Saline
Bromo benz ene
Acetaminophen
Carbon tet
Thioacetamide
Furosemide
Phall oidi n
Colchi cine
Cadmium
D-GalNLPS
Ne
cro
sis
(g
rad
e)
0
1
2
3
VehicleOA
Oleanolic acid
ARE
Maf Nrf2
Oxidative stress and activators
Nrf2
Anti-oxidant and inflammation Cell survival and proliferation
Drug metabolism Lipid metabolism
Ke
ap1
Ke
ap1
Nrf2
CUL3
Modified from DAutreacuteaux and Toledano Nat Rev Mol Cell Biol 8813 2007
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Cd pretreatment protected against acute Cd
hepatotoxicity
Goering and Klaassen TAAP 74308 1984
100
1000
10000
100000
1000000
Control 20 30 40 50
SDH
(S
un
its
ml)
mg Cdkg
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (20-40 mgkg iv) and serum
SDH was determined 10 hrs later Data are Mean plusmn SE of 4-6 rats
Cd pretreatment alters
subcellular Cd distribution
Goering and Klaassen TAAP 70195 1983
0
10
20
30
40
50
60
70
80
Nuclei Mitochondria Microsome Cytosol
C
d d
istr
ibu
tio
n in
th
e li
ver
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and subcellular
Cd distribution was determined 2 hrs later Data are Mean plusmn SE of 6 rats
Cd pretreatment alters cytosolic Cd
distribution
Goering and Klaassen TAAP 70195 1983
0
05
1
15
2
25
3
35
06 10 14 18 22 26
ug
Cd
g li
ver
VeVo
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and
cytosolic Cd distribution was determined 2 hrs later
MT-null mice are highly susceptible to
chronic Cd-induced nephrotoxicity
LiuhelliphellipKlaassen ToxSci 46 197 1998
Wild-type control and MT-null mice were
given CdCl2 (005-24 mgkg sc) daily for 6
weeks and Blood urea nitrogen (BUN) and
renal MT were determined N =6-8 mice
The relationship between human exposure to
cadmium and renal injury
Klaassen et al Ann Rev Pharmacol Toxicol 39 267 1999
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
bull In 1987 a post-doctoral fellow Jie Liu came to my Lab with a ldquosuitcaserdquo of chemicals in Traditional Chinese Medicine that he thought would protect against liver injury
Jie Liu 87-98
OA Oleanolic acid
UA Ursolic acid
UV Uvaol
α-H α-Hederin
HD Hederagenin
GL Glycyrrhizin
18β-GA 18 β-Glycyrrhetinic acid
18 α-GA 18 α-Glycyrrhetinic acid
HAG 19 α-Hydroxyl asiatic acid 29-0- β-glucoside
HA 19 α-Hydroxyl asiatic acid
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
CCl4
AL
T (
UL
)
0
500
1000
1500
2000
2500
3000
SD
H (
U
ml)
0
5000
10000
15000
20000
25000
NE
CR
OS
IS (
grad
e)
00
05
10
15
20
CON Jgs Ngs Ful OA Swe Om DDB
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
AL
T (
UL
)
0
2000
4000
6000
8000
10000
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
SD
H (
kIU
L)
0
5000
10000
15000
20000
25000
Saline
Bromo benz ene
Acetaminophen
Carbon tet
Thioacetamide
Furosemide
Phall oidi n
Colchi cine
Cadmium
D-GalNLPS
Ne
cro
sis
(g
rad
e)
0
1
2
3
VehicleOA
Oleanolic acid
ARE
Maf Nrf2
Oxidative stress and activators
Nrf2
Anti-oxidant and inflammation Cell survival and proliferation
Drug metabolism Lipid metabolism
Ke
ap1
Ke
ap1
Nrf2
CUL3
Modified from DAutreacuteaux and Toledano Nat Rev Mol Cell Biol 8813 2007
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Cd pretreatment alters
subcellular Cd distribution
Goering and Klaassen TAAP 70195 1983
0
10
20
30
40
50
60
70
80
Nuclei Mitochondria Microsome Cytosol
C
d d
istr
ibu
tio
n in
th
e li
ver
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and subcellular
Cd distribution was determined 2 hrs later Data are Mean plusmn SE of 6 rats
Cd pretreatment alters cytosolic Cd
distribution
Goering and Klaassen TAAP 70195 1983
0
05
1
15
2
25
3
35
06 10 14 18 22 26
ug
Cd
g li
ver
VeVo
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and
cytosolic Cd distribution was determined 2 hrs later
MT-null mice are highly susceptible to
chronic Cd-induced nephrotoxicity
LiuhelliphellipKlaassen ToxSci 46 197 1998
Wild-type control and MT-null mice were
given CdCl2 (005-24 mgkg sc) daily for 6
weeks and Blood urea nitrogen (BUN) and
renal MT were determined N =6-8 mice
The relationship between human exposure to
cadmium and renal injury
Klaassen et al Ann Rev Pharmacol Toxicol 39 267 1999
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
bull In 1987 a post-doctoral fellow Jie Liu came to my Lab with a ldquosuitcaserdquo of chemicals in Traditional Chinese Medicine that he thought would protect against liver injury
Jie Liu 87-98
OA Oleanolic acid
UA Ursolic acid
UV Uvaol
α-H α-Hederin
HD Hederagenin
GL Glycyrrhizin
18β-GA 18 β-Glycyrrhetinic acid
18 α-GA 18 α-Glycyrrhetinic acid
HAG 19 α-Hydroxyl asiatic acid 29-0- β-glucoside
HA 19 α-Hydroxyl asiatic acid
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
CCl4
AL
T (
UL
)
0
500
1000
1500
2000
2500
3000
SD
H (
U
ml)
0
5000
10000
15000
20000
25000
NE
CR
OS
IS (
grad
e)
00
05
10
15
20
CON Jgs Ngs Ful OA Swe Om DDB
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
AL
T (
UL
)
0
2000
4000
6000
8000
10000
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
SD
H (
kIU
L)
0
5000
10000
15000
20000
25000
Saline
Bromo benz ene
Acetaminophen
Carbon tet
Thioacetamide
Furosemide
Phall oidi n
Colchi cine
Cadmium
D-GalNLPS
Ne
cro
sis
(g
rad
e)
0
1
2
3
VehicleOA
Oleanolic acid
ARE
Maf Nrf2
Oxidative stress and activators
Nrf2
Anti-oxidant and inflammation Cell survival and proliferation
Drug metabolism Lipid metabolism
Ke
ap1
Ke
ap1
Nrf2
CUL3
Modified from DAutreacuteaux and Toledano Nat Rev Mol Cell Biol 8813 2007
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Cd pretreatment alters cytosolic Cd
distribution
Goering and Klaassen TAAP 70195 1983
0
05
1
15
2
25
3
35
06 10 14 18 22 26
ug
Cd
g li
ver
VeVo
No pretreatment
Cd pretreatment
Rats were pretreated with CdCl2 (20 mg Cdkg sc) and 24 hrs later were
challenged with hepatotoxic doses of CdCl2 (35 mg Cdkg iv) and
cytosolic Cd distribution was determined 2 hrs later
MT-null mice are highly susceptible to
chronic Cd-induced nephrotoxicity
LiuhelliphellipKlaassen ToxSci 46 197 1998
Wild-type control and MT-null mice were
given CdCl2 (005-24 mgkg sc) daily for 6
weeks and Blood urea nitrogen (BUN) and
renal MT were determined N =6-8 mice
The relationship between human exposure to
cadmium and renal injury
Klaassen et al Ann Rev Pharmacol Toxicol 39 267 1999
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
bull In 1987 a post-doctoral fellow Jie Liu came to my Lab with a ldquosuitcaserdquo of chemicals in Traditional Chinese Medicine that he thought would protect against liver injury
Jie Liu 87-98
OA Oleanolic acid
UA Ursolic acid
UV Uvaol
α-H α-Hederin
HD Hederagenin
GL Glycyrrhizin
18β-GA 18 β-Glycyrrhetinic acid
18 α-GA 18 α-Glycyrrhetinic acid
HAG 19 α-Hydroxyl asiatic acid 29-0- β-glucoside
HA 19 α-Hydroxyl asiatic acid
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
CCl4
AL
T (
UL
)
0
500
1000
1500
2000
2500
3000
SD
H (
U
ml)
0
5000
10000
15000
20000
25000
NE
CR
OS
IS (
grad
e)
00
05
10
15
20
CON Jgs Ngs Ful OA Swe Om DDB
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
AL
T (
UL
)
0
2000
4000
6000
8000
10000
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
SD
H (
kIU
L)
0
5000
10000
15000
20000
25000
Saline
Bromo benz ene
Acetaminophen
Carbon tet
Thioacetamide
Furosemide
Phall oidi n
Colchi cine
Cadmium
D-GalNLPS
Ne
cro
sis
(g
rad
e)
0
1
2
3
VehicleOA
Oleanolic acid
ARE
Maf Nrf2
Oxidative stress and activators
Nrf2
Anti-oxidant and inflammation Cell survival and proliferation
Drug metabolism Lipid metabolism
Ke
ap1
Ke
ap1
Nrf2
CUL3
Modified from DAutreacuteaux and Toledano Nat Rev Mol Cell Biol 8813 2007
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
MT-null mice are highly susceptible to
chronic Cd-induced nephrotoxicity
LiuhelliphellipKlaassen ToxSci 46 197 1998
Wild-type control and MT-null mice were
given CdCl2 (005-24 mgkg sc) daily for 6
weeks and Blood urea nitrogen (BUN) and
renal MT were determined N =6-8 mice
The relationship between human exposure to
cadmium and renal injury
Klaassen et al Ann Rev Pharmacol Toxicol 39 267 1999
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
bull In 1987 a post-doctoral fellow Jie Liu came to my Lab with a ldquosuitcaserdquo of chemicals in Traditional Chinese Medicine that he thought would protect against liver injury
Jie Liu 87-98
OA Oleanolic acid
UA Ursolic acid
UV Uvaol
α-H α-Hederin
HD Hederagenin
GL Glycyrrhizin
18β-GA 18 β-Glycyrrhetinic acid
18 α-GA 18 α-Glycyrrhetinic acid
HAG 19 α-Hydroxyl asiatic acid 29-0- β-glucoside
HA 19 α-Hydroxyl asiatic acid
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
CCl4
AL
T (
UL
)
0
500
1000
1500
2000
2500
3000
SD
H (
U
ml)
0
5000
10000
15000
20000
25000
NE
CR
OS
IS (
grad
e)
00
05
10
15
20
CON Jgs Ngs Ful OA Swe Om DDB
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
AL
T (
UL
)
0
2000
4000
6000
8000
10000
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
SD
H (
kIU
L)
0
5000
10000
15000
20000
25000
Saline
Bromo benz ene
Acetaminophen
Carbon tet
Thioacetamide
Furosemide
Phall oidi n
Colchi cine
Cadmium
D-GalNLPS
Ne
cro
sis
(g
rad
e)
0
1
2
3
VehicleOA
Oleanolic acid
ARE
Maf Nrf2
Oxidative stress and activators
Nrf2
Anti-oxidant and inflammation Cell survival and proliferation
Drug metabolism Lipid metabolism
Ke
ap1
Ke
ap1
Nrf2
CUL3
Modified from DAutreacuteaux and Toledano Nat Rev Mol Cell Biol 8813 2007
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
The relationship between human exposure to
cadmium and renal injury
Klaassen et al Ann Rev Pharmacol Toxicol 39 267 1999
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
bull In 1987 a post-doctoral fellow Jie Liu came to my Lab with a ldquosuitcaserdquo of chemicals in Traditional Chinese Medicine that he thought would protect against liver injury
Jie Liu 87-98
OA Oleanolic acid
UA Ursolic acid
UV Uvaol
α-H α-Hederin
HD Hederagenin
GL Glycyrrhizin
18β-GA 18 β-Glycyrrhetinic acid
18 α-GA 18 α-Glycyrrhetinic acid
HAG 19 α-Hydroxyl asiatic acid 29-0- β-glucoside
HA 19 α-Hydroxyl asiatic acid
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
CCl4
AL
T (
UL
)
0
500
1000
1500
2000
2500
3000
SD
H (
U
ml)
0
5000
10000
15000
20000
25000
NE
CR
OS
IS (
grad
e)
00
05
10
15
20
CON Jgs Ngs Ful OA Swe Om DDB
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
AL
T (
UL
)
0
2000
4000
6000
8000
10000
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
SD
H (
kIU
L)
0
5000
10000
15000
20000
25000
Saline
Bromo benz ene
Acetaminophen
Carbon tet
Thioacetamide
Furosemide
Phall oidi n
Colchi cine
Cadmium
D-GalNLPS
Ne
cro
sis
(g
rad
e)
0
1
2
3
VehicleOA
Oleanolic acid
ARE
Maf Nrf2
Oxidative stress and activators
Nrf2
Anti-oxidant and inflammation Cell survival and proliferation
Drug metabolism Lipid metabolism
Ke
ap1
Ke
ap1
Nrf2
CUL3
Modified from DAutreacuteaux and Toledano Nat Rev Mol Cell Biol 8813 2007
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
bull In 1987 a post-doctoral fellow Jie Liu came to my Lab with a ldquosuitcaserdquo of chemicals in Traditional Chinese Medicine that he thought would protect against liver injury
Jie Liu 87-98
OA Oleanolic acid
UA Ursolic acid
UV Uvaol
α-H α-Hederin
HD Hederagenin
GL Glycyrrhizin
18β-GA 18 β-Glycyrrhetinic acid
18 α-GA 18 α-Glycyrrhetinic acid
HAG 19 α-Hydroxyl asiatic acid 29-0- β-glucoside
HA 19 α-Hydroxyl asiatic acid
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
CCl4
AL
T (
UL
)
0
500
1000
1500
2000
2500
3000
SD
H (
U
ml)
0
5000
10000
15000
20000
25000
NE
CR
OS
IS (
grad
e)
00
05
10
15
20
CON Jgs Ngs Ful OA Swe Om DDB
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
AL
T (
UL
)
0
2000
4000
6000
8000
10000
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
SD
H (
kIU
L)
0
5000
10000
15000
20000
25000
Saline
Bromo benz ene
Acetaminophen
Carbon tet
Thioacetamide
Furosemide
Phall oidi n
Colchi cine
Cadmium
D-GalNLPS
Ne
cro
sis
(g
rad
e)
0
1
2
3
VehicleOA
Oleanolic acid
ARE
Maf Nrf2
Oxidative stress and activators
Nrf2
Anti-oxidant and inflammation Cell survival and proliferation
Drug metabolism Lipid metabolism
Ke
ap1
Ke
ap1
Nrf2
CUL3
Modified from DAutreacuteaux and Toledano Nat Rev Mol Cell Biol 8813 2007
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
bull In 1987 a post-doctoral fellow Jie Liu came to my Lab with a ldquosuitcaserdquo of chemicals in Traditional Chinese Medicine that he thought would protect against liver injury
Jie Liu 87-98
OA Oleanolic acid
UA Ursolic acid
UV Uvaol
α-H α-Hederin
HD Hederagenin
GL Glycyrrhizin
18β-GA 18 β-Glycyrrhetinic acid
18 α-GA 18 α-Glycyrrhetinic acid
HAG 19 α-Hydroxyl asiatic acid 29-0- β-glucoside
HA 19 α-Hydroxyl asiatic acid
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
CCl4
AL
T (
UL
)
0
500
1000
1500
2000
2500
3000
SD
H (
U
ml)
0
5000
10000
15000
20000
25000
NE
CR
OS
IS (
grad
e)
00
05
10
15
20
CON Jgs Ngs Ful OA Swe Om DDB
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
AL
T (
UL
)
0
2000
4000
6000
8000
10000
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
SD
H (
kIU
L)
0
5000
10000
15000
20000
25000
Saline
Bromo benz ene
Acetaminophen
Carbon tet
Thioacetamide
Furosemide
Phall oidi n
Colchi cine
Cadmium
D-GalNLPS
Ne
cro
sis
(g
rad
e)
0
1
2
3
VehicleOA
Oleanolic acid
ARE
Maf Nrf2
Oxidative stress and activators
Nrf2
Anti-oxidant and inflammation Cell survival and proliferation
Drug metabolism Lipid metabolism
Ke
ap1
Ke
ap1
Nrf2
CUL3
Modified from DAutreacuteaux and Toledano Nat Rev Mol Cell Biol 8813 2007
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
CCl4
AL
T (
UL
)
0
500
1000
1500
2000
2500
3000
SD
H (
U
ml)
0
5000
10000
15000
20000
25000
NE
CR
OS
IS (
grad
e)
00
05
10
15
20
CON Jgs Ngs Ful OA Swe Om DDB
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
AL
T (
UL
)
0
2000
4000
6000
8000
10000
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
SD
H (
kIU
L)
0
5000
10000
15000
20000
25000
Saline
Bromo benz ene
Acetaminophen
Carbon tet
Thioacetamide
Furosemide
Phall oidi n
Colchi cine
Cadmium
D-GalNLPS
Ne
cro
sis
(g
rad
e)
0
1
2
3
VehicleOA
Oleanolic acid
ARE
Maf Nrf2
Oxidative stress and activators
Nrf2
Anti-oxidant and inflammation Cell survival and proliferation
Drug metabolism Lipid metabolism
Ke
ap1
Ke
ap1
Nrf2
CUL3
Modified from DAutreacuteaux and Toledano Nat Rev Mol Cell Biol 8813 2007
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Liu and Klaassen Fundam Appl Toxicol 2234-40 1994
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
AL
T (
UL
)
0
2000
4000
6000
8000
10000
SalineBromobenzeneAcetaminophenCarbon tetThioacetamideFurosemidePhalloidinColchicineCadmiumD-GalNLPS
SD
H (
kIU
L)
0
5000
10000
15000
20000
25000
Saline
Bromo benz ene
Acetaminophen
Carbon tet
Thioacetamide
Furosemide
Phall oidi n
Colchi cine
Cadmium
D-GalNLPS
Ne
cro
sis
(g
rad
e)
0
1
2
3
VehicleOA
Oleanolic acid
ARE
Maf Nrf2
Oxidative stress and activators
Nrf2
Anti-oxidant and inflammation Cell survival and proliferation
Drug metabolism Lipid metabolism
Ke
ap1
Ke
ap1
Nrf2
CUL3
Modified from DAutreacuteaux and Toledano Nat Rev Mol Cell Biol 8813 2007
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Oleanolic acid
ARE
Maf Nrf2
Oxidative stress and activators
Nrf2
Anti-oxidant and inflammation Cell survival and proliferation
Drug metabolism Lipid metabolism
Ke
ap1
Ke
ap1
Nrf2
CUL3
Modified from DAutreacuteaux and Toledano Nat Rev Mol Cell Biol 8813 2007
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
ARE
Maf Nrf2
Oxidative stress and activators
Nrf2
Anti-oxidant and inflammation Cell survival and proliferation
Drug metabolism Lipid metabolism
Ke
ap1
Ke
ap1
Nrf2
CUL3
Modified from DAutreacuteaux and Toledano Nat Rev Mol Cell Biol 8813 2007
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Oleanolic acid is a Nrf2 activator N
rf2 P
rote
in
Rela
tive t
o C
on
tro
l
No
rmalized
to
-a
cti
n
0
1
2
3
4
5
6
7
8
ND ND
Oleanolic Acid - + - +
Wild-type Nrf2-nullL
iver
mR
NA
Exp
ressio
n(N
orm
alized
to
Co
ntr
ol)
0
2
4
6
8
10
12
14
16
18Ho-1Nqo1 GclcNrf2
Oleanolic Acid - + - +
nullWT
- + - +
nullWT
- + - +
nullWT
- + - +
nullWT
A single dose of oleanolic acid (30mgkg ip) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild-type but not Nrf2-null mice
Reisman and Klaassen Biochem Pharmacol 771273-82 2009
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
What Genes Might the Keap1-Nrf2 Pathway Alter
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Nrf2-null Wild-type Keap1-KD Keap1-HKO
Nrf2
Generation of ldquoGene dose-responserdquo Model
Col 2 Col 2 Col 2 Col 2 Nrf2
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Pro
tein
exp
ressio
n
0
1
2
3
4
NA
Wu and Klaassen Toxicol Sci 123590-600 2011
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Constitutively Induced or Suppressed Genes
Induced genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Suppressed genes
Nrf2-n
ull
Wild
-type
Keap1-KD
Keap1-HKO
Fo
ld-i
nd
ucti
on
0
2
4
6
Wu and Klaassen Toxicol Sci 123590-600 2011
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
What Drug Processing Genes are Altered by Nrf2 activation
bull Uptake transporters
bull Phase-I enzymes
bull Phase-II enzymes
bull Efflux transporters
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Phase-II enzymes Glutathione Conjugation
Glutathione S-transferase
Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3
Fo
ld-i
nd
uc
tio
n
0
2
4
6
8
10
30
32
34 Nrf2-null
Wild-type
Keap1-KD
Keap1-HKO
Not altered Gstk1 Gstm5 Gstm7 Gsto1 Gstp1 Gstt1 Gstt2
Wu hellip Klaassen PLoS one 7 e39006 2012
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Yeager and Klaassen Toxicol Sci 111238 2009
AhR and Nrf2 interactions
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
UPTAKEPhaseICyps
Diffusion
BIOTRANSFORMATION
Nucleophiles EFFLUX
Detoxific
aon
PhaseINqo1Eh-1
Hydrophilicityeacute
PhaseIIUgtsSults
ElectrophilesOxida veStressand
Forma onofAdducts
Mrps
GSHConjuga on
Conjugates
Gsts
Glutamate
Cysteine
γ-glutamylcysteine GSH
Glycine
Gcl Gs Nrf2
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Expression of Which Antioxidant Genes are altered by Nrf2
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Antioxidant Genes were Induced with Graded Nrf2 Activation
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
H2O2
H2O
Prdx (red)
Prdx (oxi)
Txn (red)
Txn (oxi)
NADPH
NADP+
Txnrd1
Prdx (red)
Prdx (oxi)
Sulfiredoxin
GSSG
GSH
NADP+
NADPH
Gr
glutamate cysteine glucine
Gcl Gs
Gpx
OH
Fe2+
Ferritin
Biliverdin
Bvrb
Bilirubin
NADP+
NADPH
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Michael Sporn ldquothe father of chemopreventionrdquo asked for our Oleanolic Acid so he could make
more potent analogues
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
CDDO$ CDDOMe$ CDDOIm$
Oleanolic$acid$
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Transcription factor
DNA
RNA
Nucleus
TRANSCRIPTION FACTORS AND GENE REGULATION
Transcription
factor
DNA
RNA
Nucleus
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
DBD LBD
Dimerization
nuclear receptor structurefunction
ligand coactivator
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Prototypical Target Genes of Xeno-sensors
Chemicals Transcription Factor
Prototypical target genes in liver
TCDD AhR Cyp1a1
PB CAR Cyp2b10
Rifampicin
PCN
PXR Cyp3a11
Clofibrate PPARα Cyp4a14
OA Nrf2 Nqo1
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Transporters in Liver B
loo
d (
Sin
us
oid
)
Bil
e C
an
ali
cu
lus
Blo
od
(S
inu
so
id)
Mrp4
Abca1
Mrp3
Mrp6
Oatp1a4
Ntcp
Oatp1a1
Oatp1b2
Ent1
Oct1
Oat2
Uptake Efflux
Apical SideBasolateral Basolateral
OATP1A2
Mdr2MDR3
Abcg5
Bcrp
Mrp2
Mate1
Mdr1
Bsep
Abcg8Atp8b1
OATP1B1
OATP1B3
Atp7b
Oatp1a4
Klaassen and Lu Toxicol Sci 101 186 2008
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Hepatic induction of rat Oatp1a4 protein expression
000
010
020
030
040
050
060
070
Oat
p1
a4 p
rote
in (
OD
50m
g p
rote
in)
AhR CAR PXR PPARα Nrf2
Rausch-Derra helliphellip Klaassen Hepatology 331469 2001
Guo helliphellip Klaassen JPET 300206 2002
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Mouse Oatp1a4 induction by PCN is PXR-dependent
0
100
200
300
400
CON PCN CON PCN
Oat
p1
a4 m
RN
A e
xpre
ssio
n
(RLU
10
mg
tota
l RN
A)
WT mice PXR-null mice
Cheng and Klaassen DMD 341863 2006
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Summary of TF-dependent gene battery using wild-type and TF-null mice
Transcription Factor TF-dependent gene battery in liver
AhR Phase I Cyp1a2Cyp2b10Nqo1Aldh1b1 Aldh7a1
Phase II Gstm2 mGst3 Sult5a1 Ugt1a1 Ugt1a6 Ugt1a9 Ugt2b35
Transporters Mrp4 Mate1 Oatp1a1 Oatp1a4
CAR Phase I Cyp1a2 Cyp2b10 Cyp3a11 Nqo1 Aldh1a1 Aldh1a7
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt1 Sult1e1
Sult2a2 Sult3a1 Sult5a1 Papss2 Ugt1a1 Ugt1a9 Ugt2a3 Ugt2b1
Ugt2b34 Ugt2b35 Ugt2b36
Transporters Mrp2 Mrp3 Mrp4 Oatp1a1 Oatp1a4
PXR Phase I Cyp2b10 Cyp3a11 Aldh1a1 Aldh1a7
Phase II Gsta1 Gstm1 Gstm2 Gstm3 Gstm4 Sult2a2 Sult3a1 Ugt1a1
Ugt1a5 Ugt1a9 Papss2
Transporters Mrp2 Mrp3 Oatp1a4 Abcg5
PPARα Phase I Cyp3a11 Cyp4a14 Nqo1 Aldh1a1 Aldh1a7 Aldh3a2 Aldh9a1
Phase II Gsta1 Gstm3 Gstm4
Transporters Mrp4
Nrf2 Phase I Cyp1a2 Cyp2b10 Nqo1 Aldh1a1
Phase II Gsta1 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstt2 mGst3
Ugt2b35 Ugt2b36
Transporters Mrp3 Mrp4 Oatp1a1
Aleksunes and Klaassen DMD 40 1191 2012
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
PXR-Response Elements
AGGTCA AGGTCA
TGAACT AGGTCA
AGGTCA TGAACT
Direct repeat
Everted repeat
Inverted repeat
Spacers (n=123hellip)
bull It is generally recognized that DR3 and ER6 are the prototypical DNA-binding motifs
for PXR based on data obtained from a few genes
bull Examples CYP3A4 in humans ER6 (AG(GT)TCAnnnnnnAG(GT)TCA) -1kb of TSS
CYP3A23 in rats DR3 (AGTTCAtgaAGTTCA) 5rsquo-flanking region (-133bp of TSS)
El-Sankary et al DMD 28 493 2000
Schuetz et al Mol Pharm 54 1113 1998
Is this still true on a genome-wide scale
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Hunting for specific nucleotides targeted by a TF within the entire genome
If typed 1500000 pages
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
PXR-binding to Cyp3a11 gene locus in mouse liver
Corn Oil
PCN
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Control
PCN
Cyp3a11
36
31
31
33
35 40
Chr 5
66
34
29
10894
29
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR3 DR4 etc
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
PX
R b
ind
ing
fo
ld-e
nri
ch
men
t
Corn Oil
PCN
Oatp1a4
Examples for Transporters
PXR-binding to Oatp1a4 gene locus in mouse liver
277
549
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
PXR-response element DR-9 only Location 10kb upstream of the transcription site
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
DR4
DR9
DR14
DR19
DR5n+4
(n=0123hellip)
Novel DR5n+4 Periodic DNA-binding Patterns of PXR
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
The DR5n+4 Periodic Pattern for PXR-binding to DNA 1 helical turn 34 Aring 10 intervals
Watson and Crick (1953 CSH Symp Quant Biol)
DR4
DR9
DR14
n=0
n=1
n=2
hellip
(n+1) half turns
DR5n+4
DNA interacts with PXR in the unit of half a helical turn
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
The DR5n+4 Periodic Pattern for PXR-binding to DNA
An ldquoAccordionrdquo Model
Cui hellip Klaassen Nucleic Acids Res 38 7943 2010
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
The binding of TFs to DNA does not explain entirely the re-programming of cells
bull Why not all genes expressed every tissue
bull Not always a good correlation between activation of TFs and function of liver cells
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
The Freedom of Expression is Under Tight Control by Epigenetic Mechanisms
Epigenetic Factors
bull DNA methylation
bull Histone modifications
bull microRNAs
Functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence
Epigentic events control the freedom of gene expression without altering the underlying DNA sequence
Epigenetics
microRNAs
CpG
DNA methylation Histone modification
RNAPol-II
TFs
RISC
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Methylated gene
DNA Methylation as a Suppression Signal for Gene Expression
Un-methylated gene
Gene Activation
Gene Silencing
CH
3
CH
3
CH
3
CH
3
STOP
GO
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Histone Modifications Determine whether the DNA is ldquoLooserdquo Enough to be Accessed by Transcription Factors
Me Me
H3K4
Me Me
H3K27
Me
Me Me
H3K27
Me
PcG
Me Me
H3K4
H3K4Me2
H3K27Me3
STOP
GO
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
microRNA complex
mRNA
3rsquoUTR
microRNAs usually lead to decreased mRNA stability and suppression of protein synthesis
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Comparison of 24hr LD50 of Ouabain in rats of
varying age
( Klaassen JPET 1972)
AGE (days old)
3 9 15 21 27 33 39 45
LE
TH
AL
DO
SE
50 (
mg
kg
)
0
20
40
60
80
100
120
140
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
( Klaassen JPET 1972)
39 DAY OLD RATS
0 5 10 15 20 25 30 35 40 45
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
7 DAY OLD RATS
TIME (minutes)
0 5 10 15 20 25 30 35 40 45
TIS
SU
E C
ON
C (
gg
)
0
2
4
6
8
10
12
14
16
18
20
22
KID
NEY
PLASM
A
LUNG
LIVER
MUSC
LE
HEA
RT
SPLEEN
BRAIN
Differential distribution of Ouabain in Livers of young rats
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Three Patterns of Liver Transporters during Development
-2 0 1 3 5 10 15 20 25 30 45 60
Age (Days)
Peri
nata
lA
do
les
cen
tA
du
lt
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
20
40
60
80
100
120
Perinatal
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ss
ion
(F
PK
M)
0
200
400
600
800
1000 Adolescent
Age (Days)
-2 0 1 3 5 10 15 20 25 30 45 60
Ge
ne
Ex
pre
ssio
n (
FP
KM
)
0
100
200
300
400Adult
CuihellipKlaassenToxicolSci2012
62cri caldrugtransporters
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Examples of Liver-Reprogramming
bull P-450 inducers bull Heavy metals bull Plants bull PXR activation bull Development of liver after birth bull Diet
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
ReprogrammingtheLiverbyDiets
HighCholesterolampCA
HighFat
HighSatFatampSucrose
LabChow
EFADeficient
AIN-93Purified
DHADeficient
HighFructose
DietRestric on
ColorKey
HighLow
5282geneshadalteredmRNAsinatleastonediet
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Significance and Conclusion bull Liver can be programmed and re-programmed bull This is the way the liver adapts to its environment bull All billion liver cells have the hardware and software to be re-
programmed bull It is chemicals that alter the programming bull There are no drugs for liver failure bull If we can learn which chemicals will turn on and off the various
programs we will have drugs for liver diseases Cirrhosis and fatty liver Nutritional functions obesity and diabetes Excretory functions jaundice and hyper-bilirubinemia Clotting factors heart attacks stroke Cholesterol synthesis and degradation atherosclerosis
Thank you
curtisklaassenphdgmailcom
Thank you
curtisklaassenphdgmailcom