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ENVR 132/TOXC 142/BIOC142ENVR 132/TOXC 142/BIOC142Biochemical & Molecular ToxicologyBiochemical & Molecular Toxicology
Induction of Metabolism by ToxicantsInduction of Metabolism by Toxicants
Instructor:Stephen S. Ferguson, Ph.D.e-mail: [email protected]
Induction: Definitions and PrinciplesInduction: Definitions and Principles
• The process of increasing the amount or the activity of a protein.
• A homeostatic mechanism for regulating enzyme production in a barrier organ, such as the liver, intestine, kidney.
• In enzymology, an inducer usually combines with and deactivates/activates a regulatory protein which leads to increased gene expression.
P450 Enzyme InductionP450 Enzyme Induction
• Induction can cause marked increases in P450 composition (>20-fold) and chemical clearance or bioactivation.
• As a result, induction can increase tolerance to some toxicants while enhancing the toxicity of others.
• Induction can decrease the therapeutic effect of drugs by increasing the rate and pattern of metabolism.
• Xenobiotics are known to induce enzymes that play a major or no role in their biotransformation (e.g., omeprazole vs. ethanol).
Invitrogen Proprietary & Confid6
Inhibition-Induction
TimeCo
ncen
trat
ion
Ineffective level
Therapeutic Window(drug efficacy)
Toxic / side-effect level
Why Is It Important to Assess Enzyme Why Is It Important to Assess Enzyme Induction?Induction?
• Failure of therapy (e.g. OC’s, epilepsy, HIV)
• Drug tolerance with auto-induction
• Xenobiotic toxicity potentiated
• Complicated dosing regimen
• Chemical carcinogenesis potentiated
• Perturbation of endogenous substrate metabolism/homeostasis
• Hepatomegaly & proliferation of cellular ER & peroxisomes
Internal Exposure to Natural andInternal Exposure to Natural andManMan--made Chemicalsmade Chemicals
• drugs• industrial chemicals • pesticides • pollutants • alkaloids• cigarette smoke
• cruciferous vegetables (indole-3-carbinol)
• secondary plant metabolites
• toxins produced by molds, plants, and animals
• pyrolysis products in cooked food
Types of P450 InducersTypes of P450 Inducers• Many “prototypical” inducers of specific families or
subfamilies of P450 enzymes– CYP1A inducers: 3-MC, BNF, omeprazole, TCDD– CYP3A inducers: rifampin, dexamethasone, troglitazone– CYP2B inducers: phenobarbital, PCBs, phenytoin– CYP4A inducers: fibrates– CYP2E1 inducers: ethanol, isoniazid
• Some overlap in “specificities” of inducers• An inducer for one family of enzymes may also suppress
another family (e.g., BNF)
Induction of Rat Liver P450 Enzymes by Induction of Rat Liver P450 Enzymes by Prototypical Inducers Prototypical Inducers In VivoIn Vivo
CLO
PCN
PB
BNF
Inducer
10,693 ± 620489 ± 52CYP4A
12,693 ± 2,2552,460 ± 780CYP3A
1,460 ± 18024 ± 4CYP2B
3,320 ± 183152 ± 27CYP1A
Induced ActivityControl Activity
In Vivo Induction in Male Rats
P450 Enzyme
CYP1A, EROD; CYP2B, PROD; CYP3A, testosterone 6β-hydroxylation;CYP4A, lauric acid 12-hydroxylation.
Induction and Inhibition of P450 in Mice Treated Induction and Inhibition of P450 in Mice Treated with PB or SKF525A: [with PB or SKF525A: [1414CC--methyl]aminopyrinemethyl]aminopyrine
Serr
umtri
azol
am (n
g/m
l)
Rifampin Effects on Triazolam DispositionRifampin Effects on Triazolam Disposition
Villikka et al., Clin Pharmacol Ther 1997;61:8-14.
RifampinPlacebo
Consequences of Cytochrome P450 Enzyme Induction
Consequences of Cytochrome Consequences of Cytochrome P450 Enzyme InductionP450 Enzyme Induction
• Increased toxic effect– Acetaminophen Alcohol, 3-MC– Bromobenzene, CCl4 Phenobarbital
• Increased bioactivation– Cyclophosphamide Macrolides, pesticides
• Increased tumor formation– Altered disposition of endogenous substrates
• Altered cellular and physiological function– proliferation of peroxisomes and SER– increased liver weight– endocrine disruption
• Porphyria, chloracne– PCDDs, azobenzenes, biphenyls (PCBs), naphthalene
Effects of Inducers on Rodent Liver Effects of Inducers on Rodent Liver Physiology and FunctionPhysiology and Function
Acetaminophen Metabolism and ToxicityAcetaminophen Metabolism and Toxicity
~60% ~35%
CYP2E*CYP1A CYP3A
NAPQIN-acetyl-p-benzoquinone imine
*induced by ethanol, isoniazid, phenobarbital
Protein adducts,Oxidative stress,Toxicity
HNCOCH 3
OH
HNCOCH 3
OSO 3H
HNCOCH 3
OO CO 2H
OHOH
HON
O
COCH 3
Endocrine Disruption Endocrine Disruption • Many xenobiotics can mimic certain hormones and
bind to target cellular sites receptive to natural hormones
• Modes of endocrine disruption can result from agonistic or antagonistic receptor binding affecting biosynthesis, transport, storage, release, and clearance of hormones
• Some pesticides have been identified as endocrine disruptors, in particular the thyroid hormone can be affected by: acetochlor, alachlor, fipronil (Frontline), heptachlor, maneb, methomyl, and zineb
• PCBs, mercury, pentachlorophenol are some of the thyroid hormone disruptors that are no longer used as pesticides
• DDT, dieldrin, lindane, methoxychlor, triadimefon are thought to be estrogenic-type environmental endocrine disruptors (EEDs) while atrazine, vinclozolin, and procymidone are thought to be andogenic EEDs
UGT1A
Molecular Mechanisms of P450 Molecular Mechanisms of P450 Enzyme InductionEnzyme Induction
General Mechanisms of P450 InductionGeneral Mechanisms of P450 Induction
• Receptor-mediated transcriptional activation
– Receptor • A macromolecule with which a
hormone, drug, or other chemical interacts to produce a characteristic effect.
– Two key features:• chemical recognition• signal transduction
– Ligand: A chemical that exhibits specific binding to a receptor.
• mRNA stabilization• Protein stabilization
Coordinates: Kumar R, Thompson EB (1999). "The structure of the nuclear hormone receptors". Steroids 64 (5): 310–9
Enzyme InductionEnzyme InductionGeneral mechanism of hepatic enzyme inductionGeneral mechanism of hepatic enzyme induction
proteinprotein
activityactivity
mRNAmRNA
Gene transcriptionGene transcription
XX
Nuclear ReceptorNuclear Receptor
XXRR cytosolcytosol XXRR nucleusnucleus
Phase1Phase1Phase 2 Phase 2
transporterstransporters
cytoplasm
nucleus
Hepatocyte
NRNR’’s and P450 Inductions and P450 Induction
CYP450 genePromoter XREM PBREM
RNA poly IITranscription P450
mRNA
Translation
P450
Increased Drug Metabolism
Drug-OH
Drug
TFs
PXRCAR
RXR NR
SRC-1I
Complex Transcriptional MachineryComplex Transcriptional Machinery
precursor mRNA
mature mRNA
mRNA degradation
micro RNA
protein translation
protein folding
protein degradation
CoCo--regulation of Target Genes by NRregulation of Target Genes by NR’’ss
• Complementary roles of NR’s in protection against xenobiotic exposure.
• Increased expression of the hepatic genes involved in drug metabolism and excretion (e.g., CYP’s, UGT’s, GST’s, transporter proteins).
• These target genes represent redundant but distinct layers of defense.
• There are overlapping similarities and distinct differences in species’ response to activators of NR’s.
Transcription factor
Dimerization partner
Examples of ligands
Genes Regulated
AHR ARNT Dioxins, non-ortho PCBs, some PAHs, bilirubin, etc.
CYP1A, CYP1B GST, UGT, NQO
CAR
RXR
Phenobarbital (PB), TCPOBOP, chlorinated pesticides, ortho-PCBs, androstanol/ androstenol (inhibits)
CYP2B, CYP3A GST, ABC transporters
PXR (SXR)
RXR
PB, ortho-PCBs, organochlorine pesticides, dexamethasone, pregnenalone, corticosterone, bile acids (lithocholic acid)
CYP3A, CYP2B, CYP7A (repression) GST, ABC transporters
PPAR
RXR
Fibrate drugs, phthalate esters, linoleic acid, arachidonic acid,
CYP4A, CYP7A (repression), CYP8B, LXR, HMGCS2
LXR RXR Cholesterol; (24 S)- hydroxycholesterol CYP7A, ABC transporters, LXR
FXR RXR Bile acids, chenodeoxycholic acid Represses CYP7A, BSEP (ABCB11), CYP8B, CYP27A
ER ER Structurally diverse xenoestrogens CYP19
Receptors Involved in the Regulation of Receptors Involved in the Regulation of CYP Gene ExpressionCYP Gene Expression
Modified from Kast, H. R. et al. J. Biol. Chem. 277:2908-2915, 2002
Coordinate Regulation of P450Coordinate Regulation of P450’’s, s, UGTUGT’’ss and and Transporters by NRTransporters by NR’’ss
UGT’s
MRP3
Role of CAR/PXR in lipid metabolism, Role of CAR/PXR in lipid metabolism, synthesis, and uptake & glucose homeostasissynthesis, and uptake & glucose homeostasis
Moreau et al. 2007 Mol. Pharmaceutics
Induction in cultures of primary Induction in cultures of primary human hepatocyteshuman hepatocytes
CYP2B6 Activity and mRNA with PB & RIF
• Saturable, sigmoidal responses
What is Relevant Induction?What is Relevant Induction?Potency and EfficacyPotency and Efficacy
Dose-Response ‘Window’
(Position → potency)
Magnitude of Response (Efficacy)
EC50
1. Efficacy (e.g. % of PC)
2. Potency (e.g. EC50)
Emax
Relationship between In Vitro Potency and Induction In VivoEC50 Cmax [Cmax]/EC50 Clinical
RelevanceNifedione 8 0.008 0.001 No knownLovastatin 1-6 0.008 0.008-0.002 No knownRosiglitazone 5-10 0.3-1.2 0.05-0.12 No knownSimvastatin 0.14 0.024 0.17 No knownTroglitazone 3-6 7 2.3 YesPhenytoin 25 80 3.2 YesAvasimibe 0.2 1-6 5-30 YesRifampicin 0.8 14 17.5 YesCarbamazepine 0.9 25 28 YesClotrimazole 1-5 Topical (Inhibition)
[Cmax]/EC50 < 0.1, induction not likely
1< [Cmax]/ EC50 < 0.1, induction possible
[Cmax]/ EC50 > 1, induction likely
Aryl Hydrocarbon Receptor Aryl Hydrocarbon Receptor (AhR)(AhR)
• Aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix (bHLH) protein belonging to the Per-Arnt-Sim (PAS) family of transcription factors
• It transcriptionally induces expression of hepatic CYP1A1, CYP1A2, and CYP1B1 , as well as several other genes, including some phase II metabolizing enzymes
• AHR ligands include PAHs and TCDD
AhR Signaling PathwayAhR Signaling Pathway
Cytoplasm Nucleus
9090
X
AhR
L
L
9090
X
L
9090
X
L
L
9090
X
L
or Arnt
From: Anne Mullen, Advanced Pharmacology, McMaster University, Ontario, CA
AhR Signaling PathwayAhR Signaling Pathway
XRE promoter gene (CYP1A1)
Translation
Increased expression CYP1A1 protein
Increased expression of other gene products
+
AhR/Arntheterodimer
mRNA
IC
+
TNGCGTG
Amino Carboxy
AF-1 DBD LBD AF-2
Modulators interactwith some cofactors
Binding to responseelements of target genes
Ligand and coactivatorbinding pockets
Translocaseactivity
5’ 3’ 5’ 3’ 5’ 3’
Monomers RXR Heterodimers Homodimers
LBD
DBD
NR-LBD RXR-LBD
DBD DBD DBD DBD
NR-LBD NR-LBD
RORTLXERRNGFI-B
PXRCARPPARLXRFXRRAR
GRERRXRCOUP-TFHNF4Rev-ErbGCNF
Nuclear Hormone ReceptorsNuclear Hormone Receptors
Nuclear Receptor PXRNuclear Receptor PXRPB
CA
R
PXR
cytoplasm
nucleus
HSP
90
PXR
RIF
RXR
PXR
RXR
XREM CYP3A
?translocation?-mouse-yes-human-?
Activator/Agonist CYP TargetHuman RIF CYP3A4Rat PCN CYP3A1/2Mouse PCN Cyp3a11
Nuclear Receptor CAR: PB InductionNuclear Receptor CAR: PB Induction--Constitutively ActiveConstitutively Active
CAR
cytoplasmnucleus
HSP
90
HSP
90
CAR
PP2A
PB
CCRP
RXR
CA
R
RXR
CCRP
OA
PBREM CYP2B
?
Activator/Agonist Inhibitor/Antagonist CYP TargetHuman CITCO, PB, DPH Clotrimazole?, Miclizine? CYP2B6Rat PB, TCPOBOP Androstenol CYP2B1Mouse PB, TCPOBOP Androstenol Cyp2b10
In cell lines spontaneously translocates to the nucleus
5’ 3’
nnn
DRn
IRn
ERn
CYP2B Response elements
CYP2B6 TGTACT n=4 TGACCCCYP2b10 TGTACT n=4 TGACCTCYP2B1 TCTACT n=4 TGACCT CYP2B2 TGTACT n=5 TGACCT
NR1s
CYP2B6 TGGACT n=4 TGAACCCYP2b10 TCAACT n=4 TGACACCYP2B1 TCAACT n=4 TGACAC CYP2B2 TCAACT n=4 TGACAC
NR2s
NR3CYP2B6 TGGACT n=4 TGACCC
CYP3A Response elements
CYP3A4 TGAACT n=3 TGACCCCYP3A2 TGACCT n=3 TGAGCTCYP3A23 TGACCT n=4 TGAGTT CYP3A2 TGAACT n=3 TGAACT
DRs
CYP3A4 TGAAAT n=6 GGTTCA CYP3A4 TGAACT n=6 AGGTCACYP3A23 TTAACT n=6 AGGTCA CYP3A5 TGAACT n=6 AGGTAACYP3A7 TTAACT n=6 AGGTCA CYP3A7 TGAAAT n=6 AGTTCA
ERs
Other GenesUGT1A1 TGAGTT n=4 TAACCT MDR1 TGAGAT n=6 AGTTCArMRP2 TGAACT n=8 AGTTCA CYP2C9 CAAACT n=4 TGACCT
2C9-1839 2C8-8806
21 3 4 5 6 7 21 3 4 5 6 7
1. RXR2. CAR3. PXR4. CAR/RXR5. CAR/RXR-20Xcc6. PXR/RXR7. PXR/RXR-20Xcc
PXR/RXRCAR/RXR
PXR Binding Sites
agTCAACTttgaTGACCCca
aaTGAACTtgc.TGACCCtc
2C8-8806
3A4-7733
NR Binding (PXR and CAR) to NR Binding (PXR and CAR) to Promoter Response ElementsPromoter Response Elements
NR Binding (PXR and CAR) to Promoter NR Binding (PXR and CAR) to Promoter Response Elements (CYP3A4)Response Elements (CYP3A4)
Goodwin et al., Mol. Pharmacol., 2001
Differential Binding of PXR and Differential Binding of PXR and CAR to Other Promoter RegionsCAR to Other Promoter Regions
NR3-2B6 ER6-3A4PXR + + + + + + CAR + + + + + +RXR + + + + + + + + + + + +
PXR/RXRCAR/RXR
GR/Dexamethasone Role in Basal & Induced Expression GR/Dexamethasone Role in Basal & Induced Expression via CAR/PXR (Master Regulator)via CAR/PXR (Master Regulator)
Pascussi et al. 2001, Eur J. Biochem, v. 268, p.6346
Wang & LeCluyse 2003, Clin Pharmacokin, v. 32, p.1331
Molecular Basis for the Species Molecular Basis for the Species Differences in Enzyme InductionDifferences in Enzyme Induction
Rabbit
Human
Rat
0.1%
DM
SO
5μM
PC
N
10μM
Rifa
mpi
cin
10μM
SR
1281
3
10μM
DTB
A
CYP3A6
CYP3A4
CYP3A23
Species Differences in the Regulation of CYP3A Enzymes
Species Differences in the Regulation Species Differences in the Regulation of CYP3A Enzymesof CYP3A Enzymes
Species Differences in CYP2B Species Differences in CYP2B Induction by PhenobarbitalInduction by Phenobarbital
Species Differences in CYP1A Species Differences in CYP1A Induction by XenobioticsInduction by Xenobiotics
CYP1A1/2 Activity in Rat Hepatocytes as a Function of Treatment with Drug 'X'
0
100
200
300
400
500
600
700
800
900
1000
Contro
l (0.1%
DMSO)
3-MC 1µ
M
Drug 'X
' 0.6µ
M
Drug 'X
' 2µM
Drug 'X
' 6µM
Drug 'X
' 20µ
M
Phen
acet
in O
-Dea
lkyl
atio
n (p
mol
/min
/mg
CYP1A Activity in Dog Hepatocytes as a Function of Treatment with Drug 'X'
0
100
200
300
400
500
600
Contro
l (0.1%
DMSO)
3-MC 2µ
M
Drug 'X
' 0.6µ
M
Drug 'X
' 2µM
Drug 'X
' 6µM
Drug 'X
' 20µ
M
Phen
acet
in O
-Dea
lkyl
atio
n (p
mol
/min
/mg)
CYP1A2 Activity in Human Hepatocytes as a Function of Treatment with Drug 'X'
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Contro
l (0.1% D
MSO)
3-MC 2µM
Drug 'X
' 0.2µM
Drug 'X
' 2µM
Drug 'X
' 6µM
Drug 'X
' 20µ
M
CY
P1A
2 A
ctiv
ity (n
mol
/min
/mg
Species Differences in CYP4A Species Differences in CYP4A Induction by Clofibric AcidInduction by Clofibric Acid
Rat Human
CTL 1 10 100 500 1000
CYP
4A1
Fold
Indu
ctio
n
0
10
20
30
40
50
60
Clofibric Acid (µM)
CTL 1 10 100 500 1000
CYP
4A11
Fol
d In
duct
ion
0
10
20
30
40
50
60
Clofibric Acid (µM)
CTL 1 10 100 500 1000
CYP
4A11
Fol
d In
duct
ion
0
1
2
3
4
Clofibric Acid (µM)
Rat Hepatocytes Human Hepatocytes
Laur
ic a
cid
12-h
ydro
xyla
tion
PAH Inducers in Rat vs. Human
Rat TCDD 1A1 mRNA
EC50 = 0.00767 +/- 0.00409
EC50 = 0.0107 +/- 0.043
CYP1A1 mRNA Hu497
Species difference in potency and efficacy
EC50 = 0.00767 +/- 0.00409
Observations and QuestionsObservations and Questions• Significant species differences are observed
in response to inducers.• All major subfamilies of inducible CYP’s
(CYP1A, CYP2B, CYP3A, CYP4A) exhibit this behavior.
• What is the molecular basis of the species-specific responses?
• What is the significance of these differences to predicting human toxicity?
PXR ExpressionPlasmid
RXR PXR
PXRE Reporter Gene
Drug
Reporter Plasmid
Transfection Assay for P450 Transfection Assay for P450 Enzyme InductionEnzyme Induction
CV-1HuH7 cell
PXRRXR
Differential Activation of Differential Activation of Human,Human, Rabbit,Rabbit,andand RatRat PXR by CYP3A InducersPXR by CYP3A Inducers
PCN
rifampicin
lovastatin
clotrimazole
Normalized Reporter Activity
0 20 40 60 80 100 300 350 400
OH
OHO
NN
NMe
NH
OO
O
HO
AcO
MeO
OHHO
N N
Cl
O
O
H
O
HO O
H
HO
O
HCN
PXR Sequence HomologyPXR Sequence Homology1 41 107 141 434
Human PXR1
Rat PXR11 38 104 138 431
Xenopus ONR11 37 102 136 386
Human VDR1 24 89 122 427
LigandDNA
96
69
63 37
76
42
Mouse PXR11 38 104 138 431
96 76
Rabbit PXR11 41 107 141 434
8294 Variation in ligand binding domain consistent with in vivo species differ-ences in response to inducers
Amino Acid Differences in the Amino Acid Differences in the Ligand Binding Domain of PXRLigand Binding Domain of PXR
Zhang et al., Arch. Biochem. Biophys., 1999
Ser187 Leu213 Asp266 Glu337 Ile417
Gly181 Leu206 Tyr263 His333
hPXR
Phe184 Leu210 Asp263 Lys334 Ser414
Gly178 Arg203 Tyr260 Arg333
mPXR
Val184 Val210 Glu263 Glu333 Thr414
Asp178 Ser203 His260 Arg333
rPXR
ATTTAAGGAAAgGGGTCAGACC------AACTAGGGTAaAGTTCAGTG
+1 (gene)
Rat CYP4A1
-2kb-10kb
-4466-4850384 bpDR1 (9/12) DR1 (9/12)
Rat CYP4A1 Response ElementsRat CYP4A1 Response Elements
Proximal PPRE Identified by Aldridge et. al. Biochem. J. 306, 473-479, 1995
Element 1 not functional Element 2 is a Functional PPRE
+1
Human CYP411
-2kb-5kb
AAACAAGGGAATAGCCCAAAAG
-4493DR1 (8/12)
-4472
-7kb-10kb
AAAAGTGGGCAAAGGATATGCADR1 (8/12)
-7238 -7217
Analysis of the Human CYP4A11 GeneAnalysis of the Human CYP4A11 Gene
Upstream analysis of the CYP4A11 gene located on chromosome 1 revealed two possible PPRE’s
Kawashima et. al., Archives of Biochemistry and Biophysics (2000) 378(2), 333-339Sequenced -2251 bp upstream of gene, no PPRE identified.
Gel Shift AssayGel Shift Assay
PPARα + - .5 1 2 + - .5 1 2 + - .5 1 2RXRα - + .5 1 2 - + .5 1 2 - + .5 1 2
Rat Human -4.5 kb Human -7.5 kb
PPRE/PPARα/RXR
SummarySummary• Induction of metabolism is caused by many
structurally unrelated xenobiotics.• Induction occurs mainly by transcriptional
regulation of metabolizing enzymes and transporter proteins.
• Nuclear receptors mediate the induction response by most xenobiotics.
• Amino acid differences in the ligand-binding domain of the receptors are mainly responsible for the species differences in the induction of CYP450 enzymes.
Additional ReadingAdditional Reading• Parkinson, A.: Biotransformation of xenobiotics. In: Casarett and Doull’s
Toxicology. The Basic Science of Poisons. Sixth edition (edited by C.D. Klaassen). McGraw Hill, New York, 2001.
• Wang, H. and Negishi, M. (2003) Transcriptional regulation of cytochrome p450 2B genes by nuclear receptors. Curr Drug Metab. 4(6):515-25.
• Bertilsson, G., Heidrich, J., Svensson, K., Asman, M., Jendeberg, L., Sydowbackman, M., Ohlsson, R., Postlind, H., Blomquist, P. and Berkenstam, A. (1998) Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction. Proc. Natl. Acad. USA.95:12208-12213.
• Blumberg, B., and Evans, R.M. (1998) Orphan nuclear receptors – new ligands and new possibilities. Genes Dev. 12:3149-3155.
• Geick A., Eichelbaum M., and Burk O. (2001) Nuclear receptor response elements mediate induction of intestinal MDR1 by rifampin. J Biol Chem.276(18):14581-14587.
• Moreau, A, Vilarem, MJ, Maurel, P; and Pascussi, JM. (2007) Xenoreceptors CAR and PXR Activation and Consequences on Lipid Metabolism, Clucose Homeostasis, and Inflammatory Response. Mol. Pharmaceutics 5(1):35-41
Additional ReadingAdditional Reading• Goodwin B., Hodgson E., and Liddle C. (1999) The orphan human
pregnane X receptor mediates the transcriptional activation of CYP3A4 by rifampicin through a distal enhancer module. Mol Pharmacol56:1329-1339.
• Honkakoski P. and Negishi M. (1998) Regulatory DNA elements of phenobarbital-responsive cytochrome P450 CYP2B genes. J Biochem Mol Toxicol 12:3-9.
• Jones, S. A., Moore, L. B., Shenk, J. L., Wisely, G.B., Hamilton, G. A., McKee, D. D., Tomkinson, N. C. O., LeCluyse, E. L., Wilson, T. M., Kliewer, S. A. and Moore, J. T. 2000. The pregnane X receptor, a promiscuous xenobiotic receptor that has diverged during evolution. Mol. Endocrinol. 14: 27-39.
• Wang, H., and LeCluyse E. L. 2003. Role of orphan nuclear receptors in the regulation of drug metabolising enzymes. Clin. Pharmacokinet. 42: 1331-1357.