2005 Micro Array Study

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2378-Tetrachlorodibenzo-p-dioxin (TCDD) alters the mRNA expression

of critical genes associated with cholesterol metabolism bile acid

biosynthesis and bile transport in rat liver A microarray study

Nick Fletcher a David Wahlstr fma Rebecca Lundberga Charlotte B Nilsson bKerstin C Nilsson b Kenneth Stockling b Heike Hellmold b Helen H3 kanssona

a Institute of Environmental Medicine Karolinska Institutet Nobels vag 13 PO Box 210 SE-171 77 Stockholm Sweden bSafety Assessment Astra Zeneca RampD So derta lje SE-151 85 So derta lje Sweden

Received 15 October 2004 accepted 3 December 2004

Available online 19 February 2005

Abstract

2378-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent hepatotoxin that exerts its toxicity through binding to the aryl hydrocarbon

receptor (AhR) and the subsequent induction or repression of gene transcription In order to further identify novel genes and pathways

that may be associated with TCDD-induced hepatotoxicity we investigated gene changes in rat liver following exposure to single oral

doses of TCDD Male SpraguendashDawley rats were administered single doses of 04 Agkg bw or 40 Agkg bw TCDD and killed at 6 h

24 h or 7 days for global analyses of gene expression In general low-dose TCDD exposure resulted in greater than 2-fold induction

of genes coding for a battery of phase I and phase II metabolizing enzymes including cytochrome P450 1a1 (CYP1A1) cytochrome

P450 1a2 (CYP1A2) NAD(P)H dehydrogenase quinone 1 UDP glycosyltransferase 1 family (UGT1A67) and metallothionein 1

However 04 Agkg bw TCDD also altered the expression of growth arrest and DNA-damage-inducible 45 alpha and Cyclin D1

suggesting that even low-dose TCDD exposure can alter the expression of genes indicative of cellular stress or DNA damage andassociated with cell cycle control At the high-dose widespread changes were observed for genes encoding cellular signaling proteins

cellular adhesion cytoskeletal and membrane transport proteins as well as transcripts coding for lipid carbohydrate and nitrogen

metabolism In addition decreased expression of cytochrome P450 7A1 short heterodimer partner (SHP gene designation nr0b2)

farnesoid X receptor (FXR) Ntcp and Slc21a5 (oatp2) were observed and confirmed by RT-PCR analyses in independent rat liver

samples Altered expression of these genes implies major deregulation of cholesterol metabolism and bile acid synthesis and transport

We suggest that these early and novel changes have the potential to contribute significantly to TCDD induced hepatotoxicity and

hypercholesterolemia

D 2004 Elsevier Inc All rights reserved

Keywords Cholesterol metabolism Bile acid Rat liver

Introduction

TCDD is the most potent of the polychlorinated

dibenzo-p-dioxins and the prototypical compound for the

study of aryl hydrocarbon receptor (AhR)-mediated tox-

icity Exposure of laboratory rodents to TCDD elicits a

broad range of biological and toxicological effects includ-

ing delayed mortality associated with a characteristic

wasting syndrome multiple site carcinogenicity teratoge-

nicity immune suppression adverse effects on reproduc-

tion as well as endocrine and neurobehavioral disturban-

ces (Pohjanvirta and Tuomisto 1994 Poland and Knutson

1982)

The initial step in the mechanism of TCDD-toxicity

involves binding to the AhR followed by a subsequent

increase or decrease in the transcription of AhR-regulated

genes (Schmidt and Bradfield 1996) The AhR is a basic

0041-008X$ - see front matter D 2004 Elsevier Inc All rights reserved

doi101016jtaap200412003

Corresponding author Fax +46 8 34 38 49

E-mail address helenhakanssonimmkise (H H3 kansson)

Toxicology and Applied Pharmacology 207 (2005) 1 ndash 24

wwwelseviercomlocateytaap



helixndashloop-helix protein that binds TCDD in the cyto-

plasm and following release of its chaperone proteins

translocates to the nucleus where it associates with

enhancer elements in the 5 V-flanking region of the

CYP1A1 gene known as dioxin-responsive elements

(DRE reviewed in Whitlock 1999 Whitlock et al

1996) The CYP1A1 gene contains multiple copies of the DRE sequence which have been shown to be required

for inducer-dependent transcription in DNA transfection

experiments (Denison et al 1988 1989 Fujisawa-Sehara

et al 1987 Hines et al 1988) Furthermore DRE

elements were well conserved with respect to location

within the CYP1A1 gene for mice rats and humans

(Denison et al 1988 1989 Fujisawa-Sehara et al 1987

Hines et al 1988) DREs have also been found for the

well known battery of AhR responsive genes including

CYP1A2 (Quattr ochi et al 1994) NAD(P)Hquinone

oxidoreductase (Favreau and Pickett 1991) CYP1B1

(Zhang et al 1998) UGT1A16 (Emi et al 1996 Munzelet al 1998) aldehyde dehydrogenase class 3 (Takimoto et

al 1994) and glutathione S-transferase Ya (Paulson et al

1990 Rushmore et al 1990)

More recently global expression studies have been

carried out to investigate other novel genes affected by

TCDD exposure Puga et al (2000) investigated the

transcriptome of human HepG2 cells using commercial

cDNA arrays Exposure to 10 nM TCDD for 8 h altered the

expression of 310 known genes and a similar number of

expressed sequence tags more than 21-fold Of these 310

genes 30 were upregulated and 78 downregulated regard-

less of cycloheximide treatment In another study in HepG2

cells Frueh et al (2001) found that TCDD up or down-

regulated 112 genes two-fold or more It is however

important to consider that these studies were conducted in

vitro in immortalized cell lines and may not necessarily

reflect transcriptional changes occurring in the liver follow-

ing in vivo exposure To that end using serial analyses of

gene expression (SAGE) Kurachi et al (2002) investigated

gene expression changes in mouse liver 7 days after

treatment with a dose of 20 Agkg bw TCDD Together

these studies confirmed the complicated nature of the action

of TCDD on liver cells

If one accepts that TCDD evokes a change in the

transcription of early response genes which subsequently propagate changes in cellular signaling pathways it

should be of importance to identify those genes that

are involved in the initial response Of similar interest is

to identify changes that occur after low-dose exposures

and equally those that are observed after relatively high-

dose exposure In this way it may be possible to

distinguish between adaptive changes to TCDD exposure

or the transcriptional response in a low stress state and

that associated with overt toxicity Therefore in this

study rats were exposed to a low single dose of TCDD

(04 Agkg bw) or a dose intended to elicit moderate

toxicity in SpraguendashDawley rats (40 Agkg bw) Changes

in gene expression were investigated 6 h 24 h and 7

days after TCDD exposure using the Affymetrix U34A

chip Selected novel gene changes were confirmed by

RT-PCR analyses Clinical chemistry and pathological

analyses were also conducted in support of the global

gene expression analyses

Materials and methods

Chemicals

TCDD was obtained from Cambridge Isotope Labs

(ED-901-C)

Animals

Animal experiments were conducted according to GLP at

Gene Logic Inc laboratories Male SpraguendashDawley out- bred CD rats (CRLCD[SD] IGS BR) weighing 250ndash300 g

were obtained from Charles River Laboratories The animals

were singly housed in polycarbonate cages temperatures

were maintained between 180 and 260 8C with a relative

humidity between 30 and 70 Rats were supplied with

feed (2018 Teklad Certified Global diet) and tap water

(routinely analyzed for contaminants and microbes) ad

libitum during the study During a 7-day acclimatization

period rats were observed for general health and suitability

for inclusion in the study

Experimental design

Rats (5dose) received singles doses of TCDD in a

corn oil vehicle (5 mLkg) by oral gavage at 0 04 or 40

Agkg bw on Day 1 Doses were determined in a

preliminary dose-ranging study the high-dose was

designed to elicit moderate toxicity Animals were killed

by decapitation 6 h 24 h and 7 days following

treatment livers were removed snap frozen within

approximately 2 min of death and stored at Agrave80 8C

Blood (approximately 4 mL) samples were taken prior to

termination by puncture of the orbital sinus while under

70 CO230 O2 anesthesia Approximately 1 mL of

blood was collected in serum separator tubes for clinicalchemistry analysis whereas 05 mL of blood was

collected into EDTA tubes for hematological analyses

Microarray experiments

Sample preparation processing and hybridization to

the Rat Genome U34A chip was performed by Gene

Logic Inc as described in the GeneChip Expression

Analysis Technical Manual (Affymetrix Santa Clara CA)

Information on the Rat Genome U34A chip which

analyzes approximately 7000 full-length sequences and

approximately 1000 EST clusters is available on the internet

N Fletcher et al Toxicology and Applied Pharmacology 207 (2005) 1ndash242



(httpwwwaffymetrixcomproductsarraysspecificrgu34

affx) In the experiment one chip was used per animal and

sample

Clinical chemistry

Clinical chemistry and pathological examination wascarried out at Gene Logic Inc laboratories Serum samples

(5dosetime point) were analyzed on a Roche Hitachi 717

Chemistry Analyzer using commercially available reagents

from Roche Diagnostics Determined endpoints consisted of

calcium phosphorous glucose urea nitrogen creatinine

total protein albumin total bilirubin alanine aminotransfer-

ase alkaline phosphatase aspartate aminotransferase

sodium potassium chloride carbon dioxide triglycerides

cholesterol magnesium sorbitol dehydrogenase and glob-

ulin was calculated as the difference between total protein

and albumin Hematological parameters were measured or

calculated using the ABX 9010TM Haematology AnalyzerInvestigated parameters were white blood cells red blood

cells hemoglobin hematocrit mean corpuscular volume

mean corpuscular hemoglobin and platelets

Pathological examination

Liver samples were preserved in 10 neutral-buffered

formalin Samples were subsequently embedded in paraf-

fin sectioned at approximately 5 Am and stained by

hematoxylin and eosin Samples were then examined

microscopically

Verification of gene changes

Confirmation of gene changes was carried out in rats

treated with single doses of TCDD as previously described

( Nilsson et al 2000) Dose selection was designed to

encompass the dose at which gene changes were observed

using microarray analyses Briefly male SpraguendashDawley

rats (BampK Universal Ab Solentuna Sweden) were housed

3 per cage and received R34 diet (6000 IU vitamin Akg

diet Lactamin Stockholm Sweden) during a four week

acclimatization period Rats (6group 273 F 18 g)

received TCDD in corn oil (1 mLkg bw) at doses of 0

10 and 100 Agkg bw and were killed 3 days following

treatment Anesthesia was carried out using 90 mgkg bw sodium pentobarbital (Mebumal) and death was in-

duced by blood withdrawal from the portal vein Livers

were excised snap frozen in liquid nitrogen and stored at

Agrave70 8C

Real-time PCR (Taqman) experiments

RNA was isolated using the QIAGEN RNeasy Midi Prep

Kit according to the manufacturerrsquos instructions The frozen

tissue samples were homogenized in lysis buffer using a

Fastprep FP120 instrument (Qbiogene Cedex France) The

total RNA was quantified using the NanoDropND-1000

Spectrophotometer (NanoDrop Montchanin USA) The

RNA quality was analyzed on Agilent 2100 Bioanalyzer

using the bRNA 6000 Nano Q Kit (Agilent Technologies

Palo N Alto USA) The procedure was performed according

to the manufacturerrsquos manual Reagent Kit Guide RNA

6000 Nano Assay and Edition 0701 After quantification

the total RNA was stored at Agrave

70 8CTotal RNA was transcribed to cDNA using the High

Capacity cDNA Archive Kit (Applied Biosystems Stock-

holm Sweden)

Real time PCR was performed using an ABI Prism

7700 sequence Detection System (Applied Biosystems

Stockholm Sweden) according to the manufacturerrsquos

protocol and using 5 ngl of template RNA Primers and

probes were supplied by Applied Biosystems Samples

were amplified in triplicate and each run included a

standard curve with known amounts of template RNA 18S

rRNA was used as internal control to which the samples

were normalized

Data analysis

Microarray data analysis Data were analyzed using the

Affymetrix software version MAS 50 (Affymetrix Santa

Clara CA) The RG-U34A Genechip array consists of 8799

probe sets (including 59 control probesets) A total of 37

observations divided into 9 treatment groups were

recorded from individual animals (n = 3ndash5 per treatment

group) Data are contained within GeneLogicrsquos Toxexpress

database

To look for outliers and trends in the data principal

components analysis (PCA Simca-P 81) pairwise correla-

tion analysis and hierarchical clustering (Spotfire version

62) were conducted PCA revealed one outlying sample in

the 6 h 40 Agkg bw dose group This sample was removed

from further analysis Data were also normalized using the

Contrast Normalization routine (Astrand 2003)

To investigate differentially expressed genes ANOVA

models were fitted to each probe set individually with time

and dose as main effects and an interaction term Data were

subjected to a log transform prior to the calculations

Additionally pairwise tests were also carried out within the

model between each dose group against its time-matched

vehicle control The estimated differences in mean levels for the respective group comparisons were then expressed as

fold changes by taking the exponent of the difference

Statistical analysis Statistical analyses of clinical chem-

istry hematological data and RT-PCR experiments was

conducted by one-way analysis of variance (ANOVA) using

Sigmastat Statistical software (Jandel Scientific Erkath

Germany) Where significant differences were indicated

between groups and the data were homogenous (Levene

median test) Least Squares Difference test was used for

pairwise comparisons When tests for homogenous variance

failed the KruskalndashWallis one-way ANOVA on ranks was

N Fletcher et al Toxicology and Applied Pharmacology 207 (2005) 1ndash24 3



used and significant differences were evaluated using

Dunnettrsquos test for multiple comparisons

Results and discussion

Clinical observations

There were no unscheduled deaths during the study

period and no reported clinical signs Body weight was

significantly decreased compared to control at 40 Agkg bw

at 7 days only (18 P b 005 data not shown)

Clinical chemistry

Significant changes in clinical chemistry and hematolog-

ical parameters are shown in Table 1 At 40 Agkg bw TCDD

increased serum cholesterol concentrations at the 24 h and 7-

day time points At 6 h there was a significant decrease inserum cholesterol concentration but the difference between

control values was only minor Serum triglycerides on the

other hand were markedly increased at the high-dose at 24 h

but decreased after 7 days Serum glucose was decreased

significantly only at the high-dose at 7 days Total protein and

globulin concentrations were likewise increased at 7 days

Hemoglobin was increased at the high-dose at all time points

Alanine aminotransferase activity was decreased at the low-

and high-dose at 7 days The absence of significant increases

here is consistent with liver histopathological examination

which revealed no marked signs of hepatotoxicity (below)

Pathology

There were no gross lesions in the livers of control or

treated rats Upon histopathological examination no alter-

ations were evident 6 h after dosing At 24 h minimal

evidence of centrilobular hypertrophy characterized by a loss

of glycogen vacuolization and slight increases in the

eosinophilic matrix were observed in 25 rats given 40 Ag

kg bw TCDD On day 7 centrilobular hypertrophy was

observed in 45 rats given 40 Agkg bw TCDD

Gene expression analyses

Expression of a probeset was considered altered by TCDD

if the change exceeded a 2-fold cut off value and was

statistically significant to P b 001 Applying this criteria a

total of 288 probesets were altered in the liver of male

SpraguendashDawley rats by single oral TCDD exposure at 6 h

24 h andor 7 days (Table 2) Low-dose TCDD exposure

altered the expression of 49 probesets 25 at 6 h (13 up 12

down) 12 (up) at 24 h and 12 (up) at 7 days At 6 h

upregulated genes included CYP1A1 CYP1A2 NAD(P)H

dehydrogenase quinone 1 (Nqo1) UDP glycosyltransferase

1 family (UGT1A6) NF-E2-related factor 2 (Nfe2I2 nrf2)

and growth arrest and DNA damage inducible 45 alpha

(Gadd45a) Nrf2 has been suggested to function as a mediator

of Nqo1 induction following TCDD exposure (Ma et al

2004) and the results here further demonstrate that nrf2 is an

early and sensitive target for TCDD Gadd45a has been

shown to be induced by ionizing radiation as well as in

response to DNA damage as a result of alkylation and

oxidative stress (Hollander and Fornace 2002) In addition

non-genotoxic stresses such as nutrient depletion have also

been shown to induce Gadd45a (Fornace et al 1989 Zhan et

al 1996) While the precise functions of Gadd45a remain to

be determined two studies suggest involvement in the G2Mcell cycle checkpoint (Wang et al 1999b Zhan et al 1999)

Furthermore Gadd45a has been implicated in mechanisms of

DNA damage repair and control of genetic instability

[reviewed in (Hollander and Fornace 2002 Sheikh et al

2000)] Low-dose TCDD exposure also caused down-

regulation of 12 genes at 6 h Interestingly several of these

were transcription factors for instance Onecut1 (codes for

HNF-6) nuclear factor IX (Nfix) and Kruppel-like factor 9

(Klf9) The relevance of these results may be questionable

however since these changes were only seen at the low-dose

and at one time point On the other hand Cyclin D1 (Ccnd1)

which is essential for cell cycle control at G1 was inhibited

Table 1

Clinical chemistry and hematology parameters in the serum of rats treated

with single oral doses of TCDD at 0 04 and 40 Agkg bw and killed at 6 h

24 h and 7 days following treatment

Parameter Dose

Control 04 40

6 hCholesterol (mgdL) 846 F 61 762 F 59 754 F 50

Hemoglobin (gdL) 149 F 05 155 F 03 154 F 03

24 h

Triglycerides (mgdL) 1494 F 279 1186 F 214 260 F 1008

Cholesterol (mgdL) 774 F 81 74 F 109 924 F 73


Absolute neutrophils

(Th AL)

41 F 05 30 F 08 43 F 06

7 days




Red blood cells(mil AL)

67 F 03 68 F 04 76 F 05

Absolute reticulocytes

(mil AL)

02 F 001 018 F 002 012 F 003

Alanine

aminotransferase

(IUL)

58 F 74 452 F 58 462 F 82

Glucose (mgdL) 141 F 129 1286 F 182 1088 F 41

Total protein (gdL) 66 F 02 65 F 01 716 F 03

Globulin (gdL) 23 F 02 24 F 02 27 F 03

P b 005 compared to controls Statistical analysis was by one-way

analysis ANOVA followed by the Least Squares Difference test In cases

where tests for homogenous variance failed analysis was by the Kruskalndash

Wallis one-way ANOVA on ranks and significant differences were

evaluated using Dunnett Ts test for multiple comparisons




Table 2

Probesets altered z 2-fold ( P b 001) compared to control in the liver of rats given TCDD by oral gavage at 0 04 and 40 Agkg bw and killed at 6 h 24 h

or 7 days following exposure

Accession no Gene name Gene Veh 6 h 6 h 24 h 7 days

symbol04 40 04 40 04 40

Detoxificationstress

E00778cds _ s _ at Cytochrome P450 1a1 CYP1A1 28 3133 9973 3458 6903 1709 7017K03241cds _ s _ at Cytochrome P450 1a2 CYP1A2 3883 71 110 87 112 91 185

E01184cds _ s _ at Cytochrome P450 1a2 CYP1A2 7448 58 84 80 100 86 141

M26127 _ s _ at Cytochrome P450 1a2 CYP1A2 7673 36 53 55 74 57 95

rc _ AI176856 _ at Cytochrome P450 subfamily 1B

polypeptide 1

CYP1B1 13 2992 250 13559

U09540 _ at Cytochrome P450 subfamily 1B

polypeptide 1

CYP1B1 80 657 3856

U09540 _ g _ at Cytochrome P450 subfamily 1B

polypeptide 1

CYP1B1 76 750 80 4838

X83867cds _ s _ at Cytochrome P450 subfamily 1B

polypeptide 1

CYP1B1 94 112

E00717UTR1 _ s _ at cDNA encoding cytochrome P-450

from rat liver

No symbol 269 1807 2945 2153 3048 944 2620

J02679 _ s _ at NAD(P)H dehydrogenase quinone 1 Nqo1 1093 23 147 31 105 20 126

M58495mRNA _ at NAD(P)H dehydrogenase quinone 1 Nqo1 41 205 174 111D38061exon _ s _ at UDP glycosyltransferase 1 family

polypeptide A6 arylsulfatase B

Arsb

UGT1A6

303 26 105 86 173 52 193

S56936 _ s _ at UDP glycosyltransferase 1 family


Arsb

UGT1A6

293 23 65 66 174 42 183


polypeptide A6 UDP

glycosyltransferase 1 family

polypeptide A7

UGT1A6

UGT1A7

4926 27 29 55

D83796 _ s _ at UDP glycosyltransferase 1 family

polypeptide A6 UDP


polypeptide A7

UGT1A6

UGT1A7

10508 25 27 48

D38062exon _ s _ at UDP glycosyltransferase 1

family polypeptide A7

UGT1A7 186 78 60 305 20 348

AF039212mRNA _ s _ at UDP glycosyltransferase 1family polypeptide A7

UGT1A7 348 52 31 106 227

J02612mRNA _ s _ at UDP glycosyltransferase 1


UGT1A7 10410 26 26 37

J05132 _ s _ at UDP glycosyltransferase 1


UGT1A7 17473 21 27 37

J03637 _ at Aldehyde dehydrogenase family 3

member A1

Aldh3a1 208 103 569 1058

D38065exon _ s _ at UDP glycosyltransferase 1 family

polypeptide A1

UGT1A1 1777 Agrave25

K00136mRNA _ at Glutathione S-transferase alpha

type 2

GSTA2 18140 21 26 37

S72506 _ s _ at Glutathione S-transferase alpha

type 2

GSTA2 113 87 34

S82820mRNA _ s _ at GSTA5 = glutathione S-transferase

Yc2 subunit [rats Morris hepatomacell line mRNA 1274 nt]

Yc2 subunit

GSTA5

890 40 54 32

X62660mRNA _ at RRGTS8 Rrattus mRNA for

glutathione transferase subunit

GSTA4 1952 23 42

X62660mRNA _ g _ at RRGTS8 Rrattus mRNA for


GSTA4 2770 29 48

rc _ AI102562 _ at Metallothionein Mt1a 69514 96 92

M11794cds2 _ f _ at Metallothionein Mt1a 48512 91 25 91

rc _ AI234950 _ at Acid phosphatase 2 Acp2 1715 20 29

AF045464 _ s _ at Aflatoxin B1 aldehyde reductase Afar 2602 29

J03786 _ s _ at Cytochrome P450 15-beta gene CYP2c12 1528 63

J00728cds _ f _ at Rat cytochrome P-450e

(phenobarbital-inducible)

gene exon 9

No symbol 3892 Agrave20 Agrave25

(continued on next page)




Table 2 (continued )

Accession no Gene name Gene Veh 6 h 6 h 24 h 7 dayssymbol

04 40 04 40 04 40


L00320cds _ f _ at RATCYPB9 Rat

cytochrome P-450b

(phenobarbital-inducible)gene exon 9

Rat CYP2B9 802 Agrave27

M13234cds _ f _ at RATCYPEZ78 Rat cytochrome

P-450e gene exons 7 and 8

No symbol 3027 Agrave21

U40004 _ s _ at cytochrome P450 pseudogene

(CYP2J3P2)

CYP2J3P2 2438 Agrave20

U46118 _ at cytochrome P450 3A9 CYP3A9 1953 Agrave108

M18363cds _ s _ at Cytochrome P450 subfamily IIC

(mephenytoin 4-hydroxylase)

CYP2C 23384 Agrave29

X79081mRNA _ f _ at Cytochrome P450 subfamily IIC


CYP2C 6286 Agrave49

U70825 _ at 5-oxoprolinase Oplah 821 Agrave28

S48325 _ s _ at Cytochrome P450 subfamily 2E

polypeptide 1

CYP2e1 49580 Agrave27

M20131cds _ s _ at Cytochrome P450 subfamily 2E

polypeptide 1


AF056333 _ s _ at Cytochrome P450 subfamily 2E

polypeptide 1


M58041 _ s _ at Cytochrome P450 2c22 CYP2c22 15104 Agrave23

M84719 _ at Flavin-containing

monooxygenase 1

FMO1 2390 Agrave34

U63923 _ at Thioredoxin reductase 1 Txnrd1 1277 25

rc _ AA891286 _ at Thioredoxin reductase 1 Txnrd1 2682 23

rc _ AI172247 _ at Xanthine dehydrogenase Xdh 1956 20

AF037072 _ at Carbonic anhydrase 3 Ca3 5409 Agrave49 Agrave207

L32591mRNA _ at Growth arrest and

DNA-damage-inducible

45 alpha

Gadd45a 382 20 33 40 65

L32591mRNA _ g _ at Growth arrest and


45 alpha

Gadd45a 798 26 25 35

rc _ AI070295 _ g _ at Growth arrest and


45 alpha

Gadd45a 391 49

AF025670 _ g _ at Caspase 6 Casp6 810 21

Lipid metabolism

J05210 _ at ATP citrate-lyase Acly 3890 Agrave30 Agrave29

J05210 _ g _ at ATP citrate-lyase Acly 10875 Agrave24

L07736 _ at Carnitine palmitoyltransferase 1 CPT1 8463 36

J02749 _ at Acetyl-CoA acyltransferase 1

3-oxo acyl-CoA thiolase A

Acaa1 1079 34 25 53

M76767 _ s _ at Fatty acid synthase Fasn 1814 Agrave24

S69874 _ s _ at Fatty acid binding protein 5

epidermal

Fabp 1065 42

rc _ AA799779 _ g _ at Acyl-CoAdihydroxyacetonephosphate

acyltransferase

Gnpat 488 21

U10357 _ at Pyruvate dehydrogenase kinase 2 Pdk2 3263 Agrave33

U10357 _ g _ at Pyruvate dehydrogenase kinase 2 Pdk2 4434 Agrave20

S81497 _ s _ at Lipase A lysosomal acid Lipa 1311 Agrave26

M33648 _ at 3-Hydroxy-3-methylglutaryl-CoA

synthase 2 mitochondrial precursor

Hmgcs2 29290 Agrave20

rc _ AA817846 _ at 3-hydroxybutyrate

dehydrogenase

(heart mitochondrial)

Bdh 4825 Agrave21

AF003835 _ at Isopentenyl-diphosphate

delta isomerase

Idi1 1727 Agrave25







04 40 04 40 04 40

Lipid metabolism

M89945mRNA _ at Farensyl diphosphate synthase Fdps 10239 Agrave23

M00002 _ at Apolipoprotein A-IV Apoa4 7038 Agrave35

J05460 _ s _ at Cytochrome P450 7a1 CYP7A1 4375Agrave

97Agrave

80U18374 _ at Farnesoid X receptor Nr1h4 (FXR) 1563 Agrave23 Agrave20

D86580 _ at Short heterodimer partner SHP (nr0b2) 1390 Agrave36 Agrave36

D86745cds _ s _ at Short heterodimer partner SHP (nr0b2) 1712 Agrave43 Agrave40

M77479 _ at Solute carrier family 10 (sodium

bile acid cotransporter family)

member 1

Slc10a1

(Ntcp)

11044 Agrave21

U88036 _ at Solute carrier family 21

(organic anion

transporter) member 5

Slc21a5

oatp2

4630 Agrave32 Agrave29

D10262 _ at Choline kinase Chk 726 24 29 23

E04239cds _ s _ at Choline kinase Chk 129 31

L14441 _ at Phosphatidylethanolamine

N-methyltransferase

PEMT 6315 Agrave27

D28560 _ at Ectonucleotide

pyrophosphatase phosphodiesterase 2

Enpp2 2917 27 41

D28560 _ g _ at Ectonucleotide

pyrophosphatase

phosphodiesterase 2

Enpp2 1615 37 34

D78588 _ at Diacylglycerol kinase zeta Dgkz 534 Agrave23

AB009372 _ at Lysophospholipase LOC246266 943 Agrave48 Agrave156

Carbohydrate metabolism

X53588 _ at Glucokinase Gck 748 Agrave32 Agrave30

AF080468 _ at Glucose-6-phosphatase

transport protein

G6pt1 6645 Agrave26 Agrave26

AF080468 _ g _ at Glucose-6-phosphatase

transport protein


X07467 _ at Glucose-6-phosphate

dehydrogenase

G6pd 728 33 35

rc _ AI008020 _ at Malic enzyme 1 Me1 275 26 43 20

rc _ AI171506 _ g _ at Malic enzyme 1 Me1 752 43 45

M26594 _ at Malic enzyme 1 Me1 430 41 36

rc _ AI171506 _ at Malic enzyme 1 Me1 388 51 44

rc _ AI059508 _ s _ at Transketolase Tkt 1164 Agrave25

K03243mRNA _ s _ at Phosphoenolpyruvate

carboxykinase

PEPCK 24179 Agrave32 Agrave42

U32314 _ at Pyruvate carboxylase Pc 3515 Agrave22 Agrave22

U32314 _ g _ at Pyruvate carboxylase Pc 3111 Agrave20

Nitrogen metabolism

AB003400 _ at d-Amino acid oxidase Dao1 1239 Agrave76

X12459 _ at Arginosuccinate synthetase Ass 26118 Agrave21 Agrave31

rc _ AI179613 _ at Glutamate dehydrogenase 1 Glud1 17405 Agrave24

rc _ AI233216 _ at Glutamate dehydrogenase 1 Glud1 6905Agrave

24Agrave

21rc _ AA852004 _ s _ at Glutamine synthetase Glul 902 Agrave31 Agrave31

M91652complete _ seq _ at Glutamine synthetase Glul 2570 Agrave24 Agrave21

rc _ AI232783 _ s _ at Glutamine synthetase Glul 6552 Agrave23

J05499 _ at Liver mitochondrial glutaminase Ga 2030 Agrave39

M58308 _ at Histidine ammonia lyase Hal 3436 Agrave44

D10354 _ s _ at Alanine aminotransferase Alat 2570 Agrave32

D13667cds _ s _ at Serine pyruvate aminotransferase Spat 967 Agrave27

X06357cds _ s _ at Serine pyruvate aminotransferase Spat 4465 Agrave22

X13119cds _ s _ at Serine dehydratase Sds 271 105

X06150cds _ at Glycine methyltransferase Gnmt 2359 Agrave20

E03229cds _ s _ at Cytolosic cysteine dioxygenase Cdo1 24819 Agrave33 Agrave29

AF056031 _ at Kynurenine 3-hydroxylase Kmo 2683 Agrave22

Z50144 _ at Kynurenine aminotransferase 2 Kat2 1164 Agrave25






04 40 04 40 04 40

Nitrogen metabolism

Z50144 _ g _ at Kynurenine aminotransferase 2 Kat2 2458 Agrave22

J04171 _ at Aspartate aminotransferase Asat 1687 25 22

AF038870 _ at Betaine-homocysteinemethyltransferase

Bhmt 22602 20

J03959 _ g _ at Urate oxidase Uox 622 22

rc _ AA900413 _ at Dihydrofolate reductase 1

(active)

Dhfr1 2389 22

AJ000347 _ g _ at 3(2)5-bisphosphate

nucleotidase

Bpnt1 577 31

D90404 _ at cathepsin C Ctsc 11757 Agrave24

Mitochondrial electron transport chain

X15030 _ at Cytochrome c oxidase

subunit Va

Cox5a 9753 31

Retinoid metabolism

X65296cds _ s _ at Carboxylesterase 3

(carboxylesterase ES10)

CES3 4719 Agrave21 Agrave81

L46791 _ at Carboxylesterase 3


CES3 2496 Agrave64

D00362 _ s _ at Esterase 2 ES2 17296 Agrave52

M20629 _ s _ at Esterase 2 ES2 20350 Agrave28

AF016387 _ at Retinoid X receptor gamma Rxrg 410 21

Steroid metabolism

S81448 _ s _ at Steroid 5 alpha-reductase 1 Srd5a1 3288 Agrave332

J05035 _ g _ at Steroid 5 alpha-reductase 1 Srd5a1 8835 Agrave177

J05035 _ at Steroid 5 alpha-reductase 1 Srd5a1 4560 Agrave131

M31363mRNA _ f _ at (Ad) M31363mRNA

RATHSST Rat hydroxysteroid

sulfotransferase mRNA


rc _ AA818122 _ f _ at Sulfotransferase hydroxysteroid

gene 2

Sth2 18559 Agrave37

D14988 _ f _ at Sulfotransferase hydroxysteroid

gene 2

Sth2 29775 Agrave36


gene 2

Sth2 11993 Agrave31

D14989 _ f _ at Rat mRNA for hydroxysteroid

sulfotransferase subunit

complete cds


M67465 _ at Hydroxy-delta-5-steroid

dehydrogenase

3 beta- and steroid

delta-isomerase

Hsd3b 7019 Agrave24

X57999cds _ at Deiodinase iodothyronine

type 1

Dio 1 826 Agrave43

X91234 _ at 17-beta hydroxysteroid

dehydrogenase type 2

Hsd17b2 15271 20

M33312cds _ s _ at Cytochrome P450 IIA1

(hepatic steroid

hydroxylase IIA1) gene

CYP2A1 13452 36

L24207 _ i _ at (Ad) L24207 Rattus

norvegicus testosterone

6-beta-hydroxylase

(CYP3A1) mRNA

CYP3A1 1659 24

L24207 _ r _ at Rattus norvegicus testosterone

6-beta-hydroxylase

(CYP3A1) mRNA

Cyp3A1 1068 27

D13912 _ s _ at Cytochrome P-450PCN

(PNCN inducible)

cytochrome P450 subfamily

3A poypeptide 3

Cyp3A1

Cyp3a3

6993 25







04 40 04 40 04 40

Kinases

rc _ AI145931 _ at UDP-N-acetylglucosamine-

2-epimerase

N-acetylmannosamine kinase

Uae1 2653 Agrave23

Circadian rhythm

AB016532 _ at Period homolog 2 Per2 65 44

Membrane bound proteins

AF004017 _ at Solute carrier family 4

member 4

Slc4a4 493 70


(vesicular glutamate transporter)

member 1

Slc17a1 915 24 30

U28504 _ g _ at Solute carrier family 17


member 1

Slc17a1 428 36 56

AB015433 _ s _ at Solute carrier family 3 member 2 Slc3a2 1577 21 40

X89225cds _ s _ at Solute carrier family 3 member 2 Slc3a2 1046 30D84450 _ at ATPase Na+K+

transporting beta

polypeptide 3

Atp1b3 968 29

M74494 _ g _ at ATPase Na+K+

transporting alpha 1

Atp1a1 2444 Agrave31




rc _ AA799645 _ g _ at FXYD domain-containing

ion transport regulator 1

Fxyd1 1307 Agrave20 Agrave29

L27651 _ at Solute carrier family 22

(organic anion transporter)

member 7

Slc22a7 3174 Agrave21


(iron-regulated transporter)

member 1


rc _ AI145680 _ s _ at Solute carrier 16

(monocarboxylic acid




A2 (glucose transporter

type 2)


U76379 _ s _ at Solute carrier family 22

member 1


AJ011656cds _ s _ at Claudin 3 Cldn3 3533 Agrave25

S61865 _ s _ at Syndecan Synd1 2061 Agrave20

X60651mRNA _ s _ at Syndecan Synd1 937 Agrave29

M31322 _ g _ at Sperm membrane protein

(YWK-II)

LOC64312 3213 21

AF097593 _ at Cadherin 2 Cdh2 950 Agrave24

U23056 _ at C-CAM4 protein LOC287009 240 25 544U23055cds _ s _ at Partial cds C-CAM4 protein

carcinoembryonic

antigen-related cell

adhesion molecule 1

Ceacam1 322 676

J04963 _ at Carcinoembryonic


adhesion molecule 1

Ceacam1 787 23

U32575 _ at Sec6 Sec6 181 44 62

U32575 _ g _ at Sec6 Sec6 342 20 45 94

rc _ AA926292 _ s _ at Trans-Golgi network protein 1 Ttgn1 919 20 26

rc _ AA859954 _ at Vacuole membrane protein 1 Vmp1 1472 26

rc _ AA892759 _ at Synaptosomal-associated protein

23 kDa

Snap23 205 34






04 40 04 40 04 40

Cell cycle

X75207 _ s _ at Cyclin D1 Ccnd1 696 Agrave20 Agrave24

D14014 _ g _ at Cyclin D1 Ccnd1 1307 Agrave36

D14014 _ at Cyclin D1 Ccnd1 1238Agrave

33Agrave

24

RNA processing

AF041066 _ at Ribonuclease RNase A family 4 Rnase4 15183 Agrave23

Cell signaling

X52140 _ at Integrin alpha 1 Itga1 1069 Agrave21

M83680 _ at GTPase Rab14 Rab14 735 Agrave23

L19180 _ g _ at Protein tyrosine phosphatase

receptor type D

Ptprd 935 Agrave59 Agrave81

L19933 _ s _ at Protein tyrosine phosphatase

receptor type D

Ptprd 835 Agrave21

K03249 _ at G protein-coupled receptor

37-like 1 enoyl-Coenzyme A

hydratase3-hydroxyacyl

Coenzyme A dehydrogenase

Ehhadh 2219 Agrave36

M63122 _ at Tumor necrosis factor receptor

super family member 1a

Tnfrsf1a 1906 20

rc _ AA892251 _ at Arginine vasopressin receptor 1A Avpr1a 2064 25 27

D85435 _ g _ at PKC-delta binding protein Prkcdbp 4280 28 24

rc _ AA900505 _ at RhoB gene Arhb 310 40

rc _ AA874794 _ g _ at Nerve growth factor receptor

(TNFRSF16) associated protein 1

Ngfrap1 308

L19699 _ g _ at V-ral simian leukemia viral

oncogene homolog B (ras related)

Ralb 282 20

AJ010828 _ at Chemokine orphan receptor 1 Rdc1 49 133

AF017437 _ g _ at Integrin-associated protein Cd47 187 25

Transcription factors

Y14933mRNA _ s _ at One cut domain family member 1

alternative name hepatocytenuclear factor 6 beta

Onecut1 1084 Agrave73

AB012234 _ g _ at Nuclear factor IX Nfix 732 Agrave45

D12769 _ at Kruppel-like factor 9 Klf 9 1886 Agrave20

AB017044exon _ at AB017044exon Rattus

norvegicus gene for hepatocyte

nuclear factor 3 gamma

partial cds

HNF3-G 631 Agrave27

X84210complete _ seq _ s _ at Nuclear factor IA Nfia 752 Agrave24

rc _ AI234146 _ at Cysteine rich protein 1 Csrp1 1397 Agrave27 Agrave63

rc _ AI014091 _ at Cbpp300-interacting

transactivator with GluAsp-rich

carboxy-terminal domain 2

Cited2 or

MRG1

864 Agrave36

L25785 _ at Transforming growth factor beta

1 induced transcript 4

(stimulated clone 22 homologue)

Tgfb1i4 Agrave 4734 Agrave28 Agrave36 Agrave37

rc _ AI177161 _ g _ at NF-E2-related factor 2 Nfe2l2nrf2 406 26 41 47 53

rc _ AI177161 _ at NF-E2-related factor 2 Nfe2l2nrf2 648 25 33 37 59

Heme synthesis

J03190 _ at Aminolevulinic acid synthase 1 Alas1 3005 Agrave43

J03190 _ g _ at Aminolevulinic acid synthase 1 Alas1 1924 Agrave23

D86297 _ at Aminolevulinic acid synthase 2 Alas2 1122 Agrave27

rc _ AI178971 _ at Hemoglobin alpha 1 Hba1 610 Agrave52

X56325mRNA _ s _ at Hemoglobin alpha 1 Hba1 40041 Agrave28

M94918mRNA _ f _ at Hemoglobin beta Hbb 28954 Agrave29

M94919mRNA _ f _ at mRNA RATBETGLOY Rat

beta-globin gene exons 1ndash3








04 40 04 40 04 40

Immune

D10729 _ s _ at Proteasome (prosome

macropain) subunit beta type

8 (low molecular mass polypeptide 7)

Psmb8 3232 Agrave21

M64795 _ f _ at M64795 Rat MHC class I

antigen gene


M33025 _ s _ at Parathymosin Ptms 4727 Agrave30

rc _ AI136977 _ g _ at FK506 binding protein 4 59kDa Fkbp4 907 Agrave23

rc _ AI136977 _ at FK506 binding protein 4 59kDa Fkbp4 473 Agrave131

M86564 _ at Prothymosin alpha Ptma 1858 Agrave21

D88250 _ at Complement component 1

s subcomponent

C1s 6862 29

M31038 _ at RT1 class Ib gene RT1Aw2 438 Agrave27

rc _ AA945608 _ at Serum amyloid P-component Sap 14151 Agrave25

Cell differentiation

rc _ AI231292 _ at Cystatin C Cst3 2235 Agrave20

rc _ AA858673 _ at Pancreatic secretory trypsininhibitor type II (PSTI-II)

LOC266602 14588Agrave

31

M15481 _ g _ at Insulin-like growth factor 1 Igf1 30347 Agrave26

M15481 _ at Insulin-like growth factor 1 Igf1 4548 Agrave26

X06107 _ i _ at Insulin-like growth factor 1 Igf1 1871 Agrave23

M81183Exon _ UTR _ g _ at M81183Exon _ UTR

RATINSLGFA Rat insulin-like

growth factor I gene

3 end of exon 6


rc _ AA924289 _ s _ at Insulin-like growth factor binding

protein acid labile subunit

Igfals 2535 Agrave24

S46785 _ at Insulin-like growth factor binding



M31837 _ at Insulin-like growth factor binding

protein 3

Igfbp3 1325 Agrave26

M58634 _ at Insulin-like growth factor binding protein 1

Igfbp1 516 25 47 29 49

Cytoskeleton

U31463 _ at Myosin heavy polypeptide 9

non-muscle

Myh9 1052 Agrave38

X52815cds _ f _ at X52815cds RRGAMACT Rat

mRNA for cytoplasmic-gamma

isoform of actin


rc _ AI179012 _ s _ at Actin beta Actb 25868 Agrave32

X70706cds _ at Plastin 3 (T-isoform) Pls3 1033 Agrave20

U05784 _ s _ at Microtubule-associated proteins

1A1B light chain 3

MPL3 3218 33 26

rc _ AA944422 _ at Calponin 3 acidic Cnn3 794 22

rc _ AA892814 _ s _ at Calpain small subunit Capns1 3395 Agrave22

L24776 _ at tropomyosin 3 gamma Tpm3 446 20

Poorly characterized andor unknown function in liver

X12355 _ s _ at Glucose regulated protein 58 kDa Grp58 4725 Agrave29

rc _ AI234604_s _ at Heat shock cognate protein 70 Hsc70 10189 Agrave22

D30649mRNA _ s _ at Alkaline phosphodiesterase LOC54410 789 Agrave21 Agrave35

U62897 _ at Carboxypeptidase D Cpd 880 Agrave24

rc _ AA859837 _ g _ at Guanine deaminase Gda 2956 Agrave21

J00738 _ s _ at Alpha-2u globulin PGCL4 LOC259247 9926 Agrave1176

AB000199 _ at CCA2 protein Cca2 3793 Agrave23

U55765 _ at Serine (or cysteine) proteinase

inhibitor clade A (alpha-1

antiproteinase antitrypsin)

member 10

Serpina 10

Rasp-1

5175 22






04 40 04 40 04 40


X96437mRNA _ g _ at X96437mRNA RNPRG1

Rnorvegicus PRG1 gene

No symbol 661 29

X96437mRNA _ at X96437mRNA RNPRG1Rnorvegicus PRG1 gene

No symbol 888 22

S61960 _ s _ at Cysteine conjugate beta-lyase No symbol 869 25 24 42

rc _ AA893239 _ at 2-hydroxyphytanoyl-CoA lyase Hpcl2 3090 Agrave23

S85184 _ at S85184 Cyclic Protein-2 =

cathepsin L proenzyme [rats

Sertoli cells mRNA 1790 nt]

CP-2 804 23 30

S77494 _ s _ at Lysyl oxidase Lox 558 Agrave43

X61381cds _ s _ at RRIIMRNA R rattus interferon

induced mRNA


rc _ AI172293 _ at Sterol-C4-methyl oxidase-like Sc4mol 6850 Agrave21

E12625cds _ at Sterol-C4-methyl oxidase-like Sc4mol 3679 Agrave25

rc _ AA891916 _ at Membrane interacting protein

of RGS16

Mir16 1508 21

rc _ AA891916 _ g _ at Membrane interacting protein

of RGS16

Mir16 2135 20

rc _ AA859981 _ at Inositol (myo)-1(or 4)-

monophosphatase 2

Impa2 385 32

D17809 _ at Beta-4N-

acetylgalactosaminyltransferase

Galgt1 1455 Agrave23 Agrave25

X14848cds12 _ at MIRNXX Rattus norvegicus

mitochondrial genome

No symbol 362 2

rc _ AI639029 _ s _ at Rat mixed-tissue library Rattus

norvegicus cDNA

clone rx05067 3 mRNA

sequence [Rattus norvegicus]

No symbol 323 4

rc _ AI638989 _ at Rat mixed-tissue library

Rattus norvegicus cDNA clone

rx01268 3 mRNA sequence

[Rattus norvegicus]


rc _ AI639162 _ at Rat mixed-tissue libraryRattus norvegicus cDNA



No symbol 113 5

rc _ AA955983 _ at rc _ AA955983 UI-R-E1-fb-e-

12-0-UIs1 Rattus norvegicus

cDNA 3 endclone = UI-R-E1-

fb-e-12-0-UIclone _ end =

3 gb = AA955983 Ag =

Rn7854len = 542

No symbol 5035 2

U47312 _ s _ at U47312 RNU47312 Rat R2

cerebellum DDRT-T-PCR


LIARCD-3 mRNA sequence

[Rattus norvegicus]


rc _ AA875171 _ at rc _ AA875171 UI-R-E0-ce-f-12-0-UIs1 Rattus norvegicus


ce-f-12-0-UIclone _ end =

3 gb = AA875171gi =

2980119 Ag = Rn2814

len = 458

No symbol 863 2

rc _ AA817987 _ f _ at rc _ AA817987 UI-R-A0-ah-a-


cDNA 3 endclone = UI-R-A0-

ah-a-06-0-UIclone _ end = 3 gb =

AA817987gi = 2887867 Ag =

Rn23920len = 373








04 40 04 40 04 40


rc _ AA859899 _ at rc _ AA859899 UI-R-E0-

cg-a-03-0-UIs1 Rattus

norvegicus cDNA 3 end clone = UI-R-E0-cg-a-03-

0-UIclone _ end = 3 gb =

AA859899gi = 2949419 Ag =

Rn810len = 353



Rattus norvegicus cDNA



No symbol 99 54

ESTs

rc _ AI236601 _ at EST233163 Rattus

norvegicus cDNA

643 28

rc _ AA892246 _ at EST196049 Rattus

norvegicus cDNA

722 22 20 20

rc _ AA799700 _ at EST189197 Rattusnorvegicus cDNA

1300 24


norvegicus cDNA

10889 22 26


norvegicus cDNA

268 28


norvegicus cDNA

42918 116 110

rc _ AA892888 _ g _ at EST196691 Rattus

norvegicus cDNA

21687 22


norvegicus cDNA

421 22

rc _ AI014135 _ g _ at EST207690 Rattus

norvegicus cDNA

2863 34


norvegicus cDNA

1472 20


norvegicus cDNA

553 20


norvegicus cDNA

808 22


norvegicus cDNA

2143 20


norvegicus cDNA

2686 20 41


norvegicus cDNA

550 21


norvegicus cDNA

291 20


norvegicus cDNA

1054 25


479 23

rc _ H33001 _ at EST108598 Rattus

norvegicus cDNA

1380 Agrave22


norvegicus cDNA

1628 Agrave24


norvegicus cDNA

788 21


norvegicus cDNA

875 Agrave23


norvegicus cDNA

593 Agrave35


norvegicus cDNA





2-fold in one probeset at 6 h and subsequent changes wereseen at the high-dose and at a latter time point Therefore

similar to Gadd45a this result suggests that even low single-

dose TCDD exposure can influence critical genes associated

with cell cycle control

While low-dose TCDD exposure appeared in the main

to induce genes associated with xenobiotic metabolism

and excretion high-dose TCDD exposure resulted in more

widespread changes in gene expression At 40 Agkg bw

TCDD altered the expression of 57 probesets greater than

2-fold at 6 h (44 up 13 down) 97 probesets at 24 h (61

up 36 down) and 236 probesets (107 up 129 down) at 7

days Therefore these results in particular the large

increase in the number of affected genes at 7 days imply

a time dependence for the effects of TCDD in the liver

Thus it appears that an initial adaptation to TCDD may

provide the signal for a cascade of secondary changes

Together the affected probesets represented approximately

185 genes with known or inferred function in for

instance cellular signaling cellular adhesion cytoskeletal

arrangement and membrane transport In addition tran-

scripts coding for proteins associated with steroid and

retinoid metabolism immune function and intermediary

metabolism were markedly affected These changes are

discussed in more detail in subsequent sections In

particular discussion is focused on TCDD inducedchanges in intermediary metabolism with a view to

further elucidating mechanisms that may be associated

with TCDD-induced wasting and alterations of interme-

diary metabolism Specific attention is drawn to novel

findings in the cholesterol metabolismbile acid biosyn-

thesis pathway

Detoxification

The commonly reported members of the AhR gene battery

(CYP1A1 CYP1A2 cytochrome P450 subfamily 1B

polypeptide 1 CYP1B1 UGT1A6 Nqo1 glutathione S -

transferase GSTA2 and aldehyde dehydrogenase family 3member A1 Aldh3a1) all showed increased expression

following TCDD exposure however marked differences

were observed with respect to the time of induction and doses

at which induction was observed CYP1A1 was increased at

all time points to a maximum of about 1000-fold at 6 h

following a dose of 40 Ag kg bw TCDD using RT-PCR

Vanden Heuvel et al (1994) previously reported that TCDD

increased relative CYP1A1 mRNA expression 4000- to

7000-fold following single doses of 1 and 10 Agkg bw

respectively CYP1A2 induction was approximately 100-fold

less than for CYP1A1 UGT1A67 mRNA expression was

increased 2- to 20-fold at all time points dependent on

probeset Here CYP1B1 induction was largely a high-dose

effect and not observed at the low dose at 6- or 24-h time

points in agreement with the suggestion that induction of

CYP1B1 is less sensitive to TCDD compared with

CYP1A1 at the protein level following acute exposure

to TCDD in rats (Santostefano et al 1997 Walker et al

1998) Walker et al (1999) also showed that the

constitutive expression of CYP1B1 in female rat liver

was much lower than that of CYP1A1 and CYP1A2

however the present results in male rats indicated that the

constitutive expression of CYP1A1 and CYP1B1 were

comparable whereas basal expression of CYP1A2 was

higher than both CYP1A1 and CYP1B1 (Table 2) Effectson Aldh3a1 were likewise high-dose effects as were

altered expression of GSTA4 and GSTA2 mRNA

TCDD-induced wasting-altered intermediary

metabolism

TCDD-treated rats display a peculiar wasting syndrome

characterized by a 2- to 5-week period of decreased body

weight gain and hypophagia that has been suggested to

contribute to the ultimate lethality of TCDD The time-

course and dose-dependence of these events has previously

been characterized (Christian et al 1986 Kelling et al



04 40 04 40 04 40

ESTs

rc _ AA892799 _ s _ at EST196602 Rattus

norvegicus cDNA

2262 Agrave21

rc _ AI169695 _ f _ at EST215591 Rattusnorvegicus cDNA

3426Agrave

22


norvegicus cDNA

1340 Agrave21


norvegicus cDNA

6360 Agrave24


norvegicus cDNA

438 23


norvegicus cDNA

391 Agrave39

Values at 6 h 24 h and 7 days represent fold-changes compared to corresponding vehicle control values following dosing with TCDD at 04 and 40 Agkg bw

Veh 6 h = mean expression in rat liver after Affymetrix scaling following vehicle (corn oil) only Expression of a probeset was considered altered by TCDD if

the change was z 2-fold compared to controls and the result was significant to P b 001 See Microarray data analysis




1985 Peterson et al 1984 Seefeld et al 1984) Briefly

single doses of 5 and 15 Agkg bw TCDD were shown to

decrease body weight gain in SpraguendashDawley rats whereas

doses of 25 and 50 Agkg bw reduced body weight over a 35-

day monitoring period Progressive weight loss was

observed from the first few days following TCDD exposure

such that after 2 weeks rats treated at 50 Agkg bw had lost approximately 25 of their original body weight (Seefeld et

al 1984) Similarly Fischer F-344 rats exposed to 100 Ag

kg bw TCDD lost about 40ndash50 of their initial body weight

by day 14 (Kelling et al 1985) Lethality was observed from

about 2-weeks in both studies and continued to increase up

to around 5 weeks such that mortality was about 25 at 25

Agkg bw 75 at 50 Agkg bw and 95 at 100 Agkg bw

Pair-fed animals matched to TCDD-treated animals also

exhibited high rates of mortality but there appears to be

some species-specific differences in the contribution of

weight loss to acute lethality (Kelling et al 1985)

Associated with the wasting syndrome appears to be changesin parameters related to lipid carbohydrate and perhaps

though less studied nitrogen metabolism [for a comprehen-

sive review of early studies into TCDD-induced wasting the

reader is referred to Pohjanvirta and Tuomisto (1994)] The

overall picture of the effects of TCDD on intermediary

metabolism however has not been elucidated and is

complicated by contrasting results in separate studies and

incomparable study designs investigating vastly different

doses as well as time points While it is likely that many of

the previously identified genes and proteins are involved in

the bodyrsquos adaptation to TCDD insult there are probably

several hitherto unidentified genes involved Therefore gene

array technology offers a unique opportunity to gain insight

into the relationships between these genes at a particular time

point and also to identify other genes that could contribute to

the wasting syndrome

Hepatic lipid synthesis

Fatty acid synthase (Fasn) mRNA expression was

decreased 24-fold at 7 days (Agrave18 P b 001 at 6 h)

consistent with previous results that showed decreased Fasn

activity following TCDD exposure (Lakshman et al 1989)

These data therefore suggest that the effects of TCDD on

Fasn may be mediated at the level of transcription and

furthermore the early time point suggests that the effects of TCDD on Fasn could be a direct effect of the chemical and

not secondary to decreased feed intake On the other hand

acetyl-CoA carboxylase expression was not affected in this

study consistent with previous observations that decreased

acetyl-CoA carboxylase activity was not associated with

decreased protein levels (McKim et al 1991) Importantly

we also observed decreased expression of ATP citrate-lyase

(Acly) (approximately 3-fold) at 6 h and 7 days dependent

upon probeset (Table 2) Acly catalyzes citrate cleavage to

yield acetyl-CoA and oxaloacetate thus supplying the

precursor for cytosolic lipogenesis Acly levels have been

shown to be dependent on diet markedly decreased by

starvation and induced by refeeding a high-carbohydrat e

low-fat diet (Elshourbagy et al 1990 Gibson et al 1972)

However similar to Fasn effects at the early time point

suggest that decreased expression of Acly could be

mediated by TCDD and not secondary to hypophagia

Acly inhibitors markedly decrease the synthesis of fatty

acids and cholesterol indicating a central role for Acly inhepatic de novo lipid synthesis (Pearce et al 1998

Sullivan et al 1974) These results therefore suggest that

decreased Acly expression could contribute to decreased

fatty acid and cholesterol synthesis observed in rats

following high-dose TCDD exposure (Lakshman et al

1988 1989) by limiting the availability of cytosolic

acetyl-CoA

Lipid metabolism and ketone body formation

TCDD exposure altered the expression of several hitherto

unidentified genes associated with lipid metabolism and

ketone body formation (Table 2) Peroxisomal acetyl-CoAacyltransferase 1 (Acaa1) which functions in theh-oxidation

of long chain fatty acids in peroxisomes [reviewed in

Mannaerts et al (2000)] was markedly increased at the high

doseat6h24hand7days(Table 2) Upregulation already at

6 h suggests that Acaa1 could be directly regulated by TCDD

and not secondary to changes elicited by TCDD-induced

hypophagia In addition carnitine palmitoyltransferase 1

(CPT1) which is localized in the outer mitochondrial

membrane and is generally considered to be the rate limiting

enzyme for oxidation of long chain fatty acids in the liver

(McGarry and Brown 1997) was increased 36-fold at the

high-dose at 7 days Upregulation at 7 days only together

with results that have shown that CPT1 expression is

increased in response to starvation (McGarry and Brown

1997 Louet et al 2002) suggests that the effect may be due

to decreased food intake in TCDD-treated rats Regardless

upregulation of CPT1 suggests that 7 days following TCDD-

dosing high-dose rats were in a ketotic state and that free

fatty acids should be undergoing h-oxidation This hypoth-

esis is consistent with previous studies that have shown

TCDD treated rats had lower respiratory quotients than did

pair-fed rats approximately 2ndash3 weeks after dosing (Muzi et

al 1989 Potter et al 1986) suggesting greater utilization of

fat for energy Similarly respiratory quotients were decreased

in chicken embryos exposed to TCDD (Lentnek et al 1991)These changes occurred entirely independent of food intake

thus demonstrating that TCDD alters intermediary metabo-

lism independent of hypophagia On the other hand other

investigators have found that fatty acid oxidation was

unchanged in mitochondrial and peroxisomal fractions in

the livers of male Fischer F344 rats exposed to 160 Agkg bw

TCDD versus controls fed ad libitum (Tomaszewski et al

1988) Similarly h-oxidation was normal in livers of rats

given 20 Agkg bw TCDD (Lakshman et al 1991) In

addition the ketogenic rate was increased from fatty acids but

decreased from glycerol The authors interpreted the decrease

in the hepatic ketogenic rate from glycerol as suggestive of




altered activity of the pyruvate dehydrogenase complex

Although we found no effect of TCDD on the expression of

pyruvate dehydrogenase pyruvate dehydrogenase kinase 2

(Pdk2) expression was decreased up to 33-fold (depend-

ent on probe set) at the high dose at 7 days (19-fold P b

001 at 24 h) Pyruvate dehydrogenase kinases inactivate

pyruvate dehydrogenase decreasing the synthesis of acetyl-CoA thus preserving three carbon substrates for

gluconeogenesis Therefore this result should seemingly

favor activation of the pyruvate dehydrogenase complex

and suggests a potential novel target that could contribute

to TCDD-inhibited gluconeogenesis

Although decreased Pdk2 expression decreased fatty acid

synthesis and increased h-oxidation of fatty acids could be

expected to result in increased substrate for ketone body

production expression of mitochondrial 3-hydroxy-3-meth-

ylglutaryl-CoA synthase 2 (Hmgcs2) was decreased approx-

imately 2-fold compared to controls at 24 h (Agrave18 P b 001)

and 7 days This enzyme is responsible for production of 3-hydroxy-3-methylglutaryl-CoA produced inside the mito-

chondria Altered expression of mitochondrial Hmgcs2 may

thus explain the previously observed decrease in circulating

ketone bodies in the plasma of TCDD-treated rats (Christian

et al 1986 Sweatlock and Gasiewicz 1985) A failure to

increase ketone body formation in cases of decreased food

intakestarvation would constitute an inappropriate r esponse

to reduced caloric intake Indeed Serra et al (1993) showed

that starvation (24 h) increased both mRNA levels (c4-fold)

and the amount of 3-hydroxy-3-methylglutaryl-CoA syn-

thase protein (c2-fold) It may then be suggested that the

failure of the liver to respond appropriately to decreased food

intake through the synthesis of ketone bodies could contrib-

ute to the wasting syndrome and thus TCDD-induced

lethality In particular this result which showed Hmgcs2

expression is not increased but actually decreased compared

to ad libitum fed controls suggests that decreased Hmgcs2

expression may play an important role in this process

Cholesterol metabolism and bile acid transport

CYP7A1 mRNA expression was persistently and mark-

edly decreased at 6 h 24 h (Agrave51 fold ns P = 0012) and 7

days following exposure to 40 Agkg bw TCDD (Table 2)

Subsequently RT-PCR on independent samples showed a

dose-dependent and approximate 6-fold decrease inCYP7A1 expression following a dose of 100 Agkg bw

TCDD however large standard deviations in controls

precluded a statistically significant result (Fig 1a) CYP7A1

is the rate limiting enzyme that catalyzes the conversion of

cholesterol into bile acids representing one of the major

pathways for disposal of cholesterol in mammals (Russell

and Setchell 1992) Dietary cholesterol induces both

CYP7A1 mRNA expression and activity (Jelinek et al

1990 Russell and Setchell 1992) In contrast bile acids

have been shown to decrease CYP7A1 expression and

activity (Jelinek et al 1990) The mechanisms of this

inhibitory pathway have only recently been delineated

whereby bile acids bind to the farnesoid X receptor (FXR)

an orphan nuclear receptor that heterodimerizes with the

retinoid X receptor (RXR) (Makishima et al 1999 Parks et

al 1999 Wang et al 1999a) An activated FXR then

induces expression of the short heterodimer partner orphan

nuclear receptor (SHP) which interacts with the liver

receptor homolog 1 (LRH-1) repressing the transcriptionof CYP7A1 (Goodwin et al 2000 Lu et al 2000)

Furthermore induction of SHP eventually represses the

SHP promoter itself (Lu et al 2000) In this study

expression of both FXR and SHP were markedly decreased

FXR was downregulated approximately 2-fold at 6 h and 7

days whereas SHP was downregulated 22-f old (ns) at 6 h

and 36-fold at 24 h and 7 days (Table 2) Confirmatory

analyses by RT-PCR showed that FXR expression was

decreased approximately 2-fold at 100 Agkg bw and

slightly but significantly at 10 Agkg bw (Fig 1 b)

Likewise SHP mRNA expression was decreased approx-

imately 10-fold at 100 Agkg bw (Fig 1c) SHP is an orphannuclear receptor that lacks a DNA- binding domain but

contains a ligand binding domain (Seol et al 1996) In

addition to its role in bile acid synthesis SHP has been

shown to suppress the transcriptional activity of retinoid

estrogen and thyroid hormone receptors (Goodwin et al

2000 Johansson et al 1999 Masuda et al 1997 Seol et

al 1996 1998) thereby functioning as a general repressor

of nuclear receptor function Furthermore in vitro SHP has

been shown to suppress TCDD-induced reporter activity

from CYP1A1 and UGT1A6 gene promoters (Klinge et al

2001) Therefore identification of SHP as a target for

transcriptional regulation by TCDD in vivo has significant

potential to explain aspects of TCDD-toxicity

In addition to the roles outlined above SHP gene

activation has also been shown to correlate with bile acid

induced down-regulation of Ntcp by a mechanism sug-

gested to involve FXR dependent suppression of the Ntcp

RARRXR response element (Denson et al 2001) In this

study expression of Ntcp the principal hepatic basolateral

bile salt transporter was also found to be downregulated

approximately 17- and 2-fold at the high dose at 24 h and 7

days using microarray (Table 2) Using RT-PCR analyses

we confirmed significantly decreased Ntcp expression at 10

and 100 Agkg bw 3 days after TCDD exposure (Fig 1d) It

is interesting that Ntcp which is inducible by retinoidsshould be downregulated in rat liver following acute dose

TCDD-treatment which increased hepatic retinoic acid

levels in these rat livers (Schmidt et al 2003) These

results may therefore suggest that TCDD could influence

retinoid-dependent expression of Ntcp an event that has

been observed for other genes including transglutaminase in

vitro (Krig et al 2002)

Slc21a5 (oatp2) mRNA expression was decreased at 24 h

and 7 days (Agrave32 and Agrave29 fold respectively) RT-PCR

analysis confirmed down-regulation of oatp2 at 10 and 100

Agkg bw the change at the high-dose approximately 8-fold

(Fig 1e) Oatp2 is localized to the basolateral membrane of




hepatocytes and is predominately expressed in perivenous

pericentral hepatocytes (Kakyo et al 1999 Reichel et al

1999) This membrane transport protein carries a widevariety of structurally unrelated compounds including bile

salts (Kullak-Ublick et al 2000 Meier et al 1997) and has

a particularly high affinity for the cardiac glycosides

ouabain and digoxin ( Noe et al 1997 Reichel et al

1999) This result showing markedly decreased oatp2

mRNA expression contrasts to that of Guo et al (2002)

where a single dose of 39 Agkg bw markedly decreased

oatp2 protein levels but mRNA levels were unaffected

Regardless decreased oatp2 protein expression and altered

transcriptional regulation could contribute to the well

known decreased transport of ouabain into bile following

TCDD exposure (Yang et al 1977)

Therefore together these data showing decreased expres-

sion of CYP7A1 FXR SHP Ntcp and oatp2 imply marked

alterations to cholesterol metabolism bile acid synthesis andtransport Decreased CYP7A1 expression andor activity has

previously been associated with increased circulating cho-

lesterol levels in CYP7A1 knockout mice (Erickson et al

2003) a strain of hyperlipidemic rats (Brassil et al 1998)

and humans with a dysfunctional CYP7A1 gene (Pullinger et

al 2002) Therefore these results offer a likely explanation

for increased cholesterol observed in serum following TCDD

exposure (Table 1)

In addition decreased expression of CYP7A1 and Ntcp

may suggest altered concentrations of signaling bile acids in

the liver Indeed previous studies have shown that the

expression of both CYP7A1 and Ntcp are downregulated

Fig 1 CYP7A1 (a) FXR (b) SHP (c) Ntcp (d) and oatp2 (e) mRNA expression relative to 18S RNA in rat liver 3 days following exposure to 0 10 and 100

Agkg bw TCDD (n = 6) Statistical analyses were as described in Materials and methods Indicates significantly different from controls at P b 005 Primers

and probes were supplied by Applied Biosystems CYP7A1 (accession no J05460 ID Rn00564065 _ m1) FXR (accession no U18374 ID Rn00572658 _ ml)

SHP short heterodimer partner (accession no D86580 ID Rn00589173 _ m1) Ntcp (accession no M77479 ID Rn00566894 _ m1) and oatp2 (accession no

U88036 ID Rn00756233 _ m1) and Eukaryotic 18S rRNA endogenous control (accession no X03205 ID Hs99999901 _ s1)




following exposure to bile acids (Jelinek et al 1990 Sinal et

al 2000) Furthermore increased serum bile acid concen-

trations were observed in earlier studies following exposure

to compounds that elicit TCDD-like toxicity (Brewster et al

1988a 1988b Couture et al 1988) whereas other studies

showed that bile flow was decreased in a dose-dependent

manner from the liver of TCDD-treated rats (Yang et al1977 1983) Since bile acids are well-known hepatotoxins

this pathway may thus represent a novel mechanism to

explain TCDD-induced liver toxicity In addition recent

evidence showing that ursodeoxycholic acid an antichole-

static drug conferred a remarkable resistance to TCDD-

induced body weight loss in mice (Kwon et al 2004) and

bile acids are potent suppressors of phosphoenolpyruvat e

carboxykinase (PEPCK) expression (De Fabiani et al 2003)

suggest that altered bile acid synthesis and transport could

contribute to the wasting syndrome In terms of metabolic

significance it may be pointed out that changes related to

cholesterol metabolism occurred relatively early (already at 6 h) in comparison to other well-established metabolic

changes assumed critical in TCDD-induced toxicity (ie

inhibition of PEPCK and pyruvate carboxylase (Pc) expres-

sion) Further analyses of CYP7A1 FXR and SHP protein

levels are ongoing to clarify the role of these proteins in

altered cholesterol metabolism and bile acid synthesis

following TCDD exposure


Expression of glucokinase the enzyme responsible for

conversion of glucose to glucose-6-phosphate was de-

creased approximately 3-fold at 6 h and 7 days Gluco-

kinase mRNA expression has previously been shown to be

downregulated in cases of feed deprivation (Chauhan and

Dakshinamurti 1991) but the marked early effects suggest

that the effect of TCDD on glucokinase expression may be a

direct effect of chemical exposure In addition the hepato-

cyte-specific glucokinase promoter appears to be under

complex hormonal control Insulin increases glucokinase

expression whereas glucagon decreases glucokinase gene

transcription (Iynedjian et al 1989) Thyroid hormone

biotin and retinoic acid have also been shown to influence

glucokinase mRNA expression ( Narkewicz et al 1990

Chauhan and Dakshinamurti 1991 Decaux et al 1997

Cabrera-Valladares et al 2001) In the serum TCDD has been shown to decrease insulin levels (Potter et al 1983)

whereas in vitro studies have shown that nuclear protein

binding to a T3-responsive element is increased but

decreased to a retinoic acid-responsive element in guinea

pig liver (Ashida and Matsumura 1998) Therefore it is

possible that the effects of TCDD on the glucokinase

promoter following TCDD exposure may be a complex

multifactoral event The expression of glucose-6-phospha-

tase transport protein 1 (G6pt1) mRNA was decreased

approximately 25-fold at the 24-h and 7-day time points

(two probe sets Table 2) The function of this gene in the rat

remains to be determined but G6pt1 is a putative homologue

of human glucose-6-phosphate translocase which has been

associated with glycogen storage disease (Gerin et al 1997

Lin et al 1998) This gene codes for a transmembrane

protein that purportedly transports glucose-6-phosphate to

the inner lumen of the endoplasmic reticulum where the

active site of glucose-6-phosphatase is positioned (Pan et al

1998 Chen et al 2000) It seems plausible then that alteredexpression of G6pt1 could influence glucose-6-phosphatase

activity which has also previously been shown t o b e

decreased following high-dose TCDD exposure (Weber et

al 1991a) Therefore together these results showing

persistent changes to the regulation of glucokinase and

G6pt1 suggest further novel mechanisms to explain altered

glucose and glycogen production in the liver of TCDD-

treated rats

Glucose-6-phosphate dehydrogenase (G6pd) the key

regulatory enzyme of the pentose phosphate pathway was

increased 33-fold and 35-fold at 24 h and 7 days

respectively In addition to hormonal regulation it has been suggested that G6pd could be responsive to oxidative

stress with the ability to rapidly meet the need to maintain

cellular redox state (Kletzien et al 1994) For example

hepatic G6pd has also been shown to be induced by

chemicals that induce oxidative stress including diquat and

thioacetamide (Cramer et al 1995 Diez-Fernandez et al

1996) as well as common substances such as alcohol

(Stumpo and Kletzien 1985) Likewise Hori et al (1997)

showed that G6pd activity was increased in mice and rats

following PCB126 exposure In addition expression of

mRNA for malic enzyme another NADPH generating

enzyme was markedly increased at 24 h and 7 days

Increased mRNA expression of malic enzyme was con-

sistent with increased hepatic malic enzyme activity that

has previously been observed in TCDD-treated rats but

only in the presence of thyroid hormone (Kelling et al

1987 Roth et al 1988 Schuur et al 1997) therefore

these results suggest that TCDD may affect malic enzyme

at the level of transcription Another enzyme crucial for the

flux of carbohydrate through the pentose phosphate path-

way transketolase was downregulated 25 and 18 times

(ns data not shown) at the high doses at 7 days and 24 h

respectively Transketolase catalyzes the transformation of

xylulose 5-phosphate and ribose 5-phosphate into sedo-

heptulose 7-phosphate and glyceraldehyde 3-phosphatewhich are then integrated into the glycolytic pathway

Similar to glucose-6-phosphate dehydrogenase PCB126

has also been shown to decrease transketolase activity at

doses sufficient to induce wasting (Ishii et al 2001)

Further minor changes (b2-fold) were observed in the

glycolytic pathway at the high dose at 7 days Therefore

together the effects described above appear to suggest a

shift away from liver glycogen synthesis and the classical

glycolytic pathway with perhaps more carbon units directed

towards the pentose phosphate pathway in order to obtain

reducing equivalents such that a cellular redox state can be

maintained




Nitrogen metabolism

Exposure to TCDD at 40 Agkg bw altered the expres-

sion of several genes associated with amino acid metabo-

lismnitrogen balance The changes consisted predominately

of down-regulation and occurred mainly at or following

the 24-h time period (Table 2) Significantly altered

expression of genes coding for amino acid metabolizingenzymes was not unexpected given previous studies that

have shown markedly altered levels of circulating levels of

amino acids in TCDD-treated rats (Christian et al 1986

Viluksela et al 1999) However together these results

which show substantial changes to expression of amino acid

metabolizing enzymes several of which are directly

involved in gluconeogenesis suggest that altered degrada-

tion of amino acids to form glutamate could contribute to

inhibited gluconeogenesis in TCDD-treated animals

Accordingly it is the failure to maintain gluconeogenesis

that has received most attention as a possible cause of

TCDD-induced lethality Much of the focus has beendirected towards the inhibition of PEPCK the expression

andor activity of which has repeatedly been shown to be

decreased following TCDD exposure (Stahl et al 1993

Weber et al 1991a 1991b 1995 Viluksela et al 1995)

Likewise herein PEPCK mRNA expression was decreased

approximately 3- and 4-fold at 24 h and 7 days Interest-

ingly PEPCK expression was not affected at 6 h suggesting

that the TCDD-elicited change in PEPCK expression may

be due to secondary or other metabolic changes Likewise

Pc mRNA expression was decreased approximately 2-fold

at 24 h and 7-days following exposure to 40 Agkg bw

TCDD suggesting similarly that changes to Pc expression

may not be directly regulated by TCDD but secondary to

some other metabolic changes Decreased Pc expression in

addition to playing a role in decreased gluconeogenesis

could also contribute to decreased substrate export to the

cytosol for lipogenesis

Together these results showing marked changes to the

mRNA expression of several amino acid metabolizing

enzymes in addition to key enzymes of gluconeogenesis

as well as lipid and carbohydrate metabolism suggest that

the wasting syndrome is a complex multifactoral event In

that regard they are consistent with studies in different

species or strains that thus far have failed to identify a single

enzyme that can adequately explain different sensitivities toTCDD-induced wasting and lethality (Unkila et al 1995

Viluksela et al 1999 Weber et al 1995) Investigation of

changes at the substrate and protein level could help further

elucidate the pertinent changes in gene expression relevant

to TCDD-induced wasting

The retinoid pathway

TCDD has been shown to elicit widespread disruption

to retinoid homeostasis including decreased accumulation

of hepatic retinyl esters enhanced mobilization of hepatic

retinoids and increased metabolism and excretion of

retinoid species [reviewed in Nilsson and Hakansson

(2002)] Using kinetic analyses Kelley et al (1998

2000) predicted that the initial event in TCDD-altered

retinoid homeostasis was the mobilization of hepatic

retinoids and secretion into the plasma This result implies

that metabolism of retinyl esters by retinyl ester hydrolases

or carboxylesterases could be an initiating event in TCDD-

altered retinoid homeostasis Regardless investigation of retinyl ester hydrolase activities in rat liver following

TCDD exposure has revealed no differences from controls

( Nilsson et al 2000) which may imply that retinoid

homeostasis is disrupted downstream in the pathway or

that other carboxylesterase enzymes could be involved

In this study a number of probesets for carboxylesterase

enzymes were markedly down regulated The expression of

D50580 which was recently shown to metabolise retinyl

palmitate (Sanghani et al 2002) was decreased about 6- and

7-fold respectively at 7 days following low- and high-doses

respectively the results significant at P b 005 (data not

shown) Likewise esterase 2 (ES2) expression wasdecreased up to 52-fold at 7 days ES2 is secreted from

the liver (Alexson et al 1994) and is likely to play a role in

the metabolism of retinyl esters (Sun et al 1997) It has

therefore been suggested that ES2 could play a role in the

metabolism of retinyl esters in the space of Disse although

t here is no dir ect evidence to support this hypothesis

(Harrison 2000) There were no significant effects on the

expression of lipoprotein lipase hepatic lipase or carboxyl

ester lipase which have also been suggested to be involved in

retinyl ester metabolism Thus these results which show

decreased expression of carboxylesterases offer little

explanation of the early and low-dose TCDD-dependent

depletion of hepatic retinyl esters

Likewise there were few other significant effects in the

retinoid-metabolizing pathway including Raldh1a2 for

which the mouse promoter has been shown to contain

DREs (Wang et al 2001) Together these results indicating

no significant changes to the transcription of putatively

specific retinoid metabolizing enzymes serve to strengthen

the hypothesis that TCDD-induced hepatic retinoid deple-

tion could be mediated by TCDD-induced metabolizing

enzymes such as CYP1A12 or perhaps UGT1A67

enzymes CYP1A1 has been shown to metabolize various

retinoid species there are no data to date on the activity of

UGT1A67 on retinoid species but increased retinoidglucuronidation has previously been observed following

TCDD exposure (Bank et al 1989) Interestingly how-

ever the Affymetrix U34A chip does not contain a probe

for lecithin retinol acyltransferase (LRAT) an important

mediator of hepatic retinyl ester levels and likely to play a

central role in decreased hepatic retinyl ester concentra-

tions following TCDD exposure ( Nilsson et al 1996)

Another interesting and previously unreported effect

was the 2-fold induction of RXR g at 6 h following high-dose

TCDD exposure A second probeset also showed significant

increases in RXR g expression but the results failed to

achieve the 2-fold cut-off On the basis of these data it is too




early to speculate as to the relevance of increased RXR g

expression but changes in the number and types of receptors

in the cell could lead to changes in pattern of gene expression

following exposure to both RXR ligands and ligands for

r eceptors that form heterodimeric complexes with the RXR

(Ahuja et al 2003)

Heme synthesis

High-dose TCDD exposure resulted in a marked reduction

in the expression of aminolevulinic acid synthase 1 (ALAS1)

and aminolevulinic acid synthase 2 (ALAS2) at 7 days Early

studies showed that TCDD either increased or had no effect

on ALAS activity (Goldstein et al 1973 Woods 1973)

therefore the relevance of decreased mRNA expression

observed herein is unclear Interestingly genes coding for the

alpha and beta subunits of hemoglobin were also markedly

decreased following high-dose TCDD exposure at 7 days

These results would appear to be in contrast with clinicalchemistry results that showed slightly increased serum

hemoglobin at all time points and red blood cell concentration

at 7 days A possible explanation for increased values for

hemoglobin hematocrit and red blood cells at early time

point s following TCDD exposure could be hemoconcentra-

tion (Pohjanvirta and Tuomisto 1994)

Cytoskeleton

The expression of several cytoskeletal genes was found to

be altered following high-dose TCDD treatment With the

exception of microtubule-associated proteins 1A1B light

chain 3 (MPL3) changes were only observed at the 7-day

time point A marked upregulation of microtubule-associated

protein (1A1B) already at 6h (33-fold compared to control)

suggests that this gene may be directly regulated by the AhR-

TCDD complex To date however the function of this gene

has not been well characterized in the liver and its relevance

to TCDD-induced liver toxicity remains unknown


A total of 10 probesets representing nine genes coding

for proteins that function as transcription factors showed

altered expression following TCDD treatment (Table 2)Among these nrf2 was upregulated at 6 h at both the low

and high dose as well as at the high-dose at latter time

points Previously in cell studies nrf2 has been suggested to

function as a mediator of TCDD-induced Nqo1 and super-

oxide dismutase induction (Ma et al 2004 Park and Rho

2002) These results indicating early- and low-dose induc-

tion of nrf2 in rat liver further suggest that nrf2 induction is

a sensitive marker of TCDD exposure Interestingly low-

dose TCDD exposure altered the expression of Onecut1

Nfix and Klf9 at the low dose at 6 h but not at other time

points or doses While altered expression of transcription

factors could be among the earliest effects of TCDD

exposure and direct subsequent signaling processes the

relevance of these changes is questionable given that affects

were observed only at the low-dose and at one time point

Membrane proteins

High-dose TCDD exposure altered the expression of avariety of genes which code for membrane-bound proteins

associated with cellular transport exocytosis and cell

adhesion categorized collectively herein as membrane bound

proteins (Table 2) ATPase Na+K + transporting alpha 1

(Atp1a1) was downregulated on two probesets at the high-

dose at 7 days (Agrave31 Agrave27) whereas ATPase Na+K +trans-

porting beta polypeptide 3 (Atp1b3) was upregulated 29-

fold Interestingly TCDD also downregulated the expression

of FXYD domain-containing ion transport regulator 1

(FXYD1) which has been shown to associate with different

alpha and beta isoforms of NaK pumps (Geering et al

2003) and modulate their transport activities thereby it has been speculated that FXYD1 could play important roles in

maintenance of cellular volume and muscular contraction

(Crambert et al 2002) Therefore these represent novel

changes that may be associated with decreased hepatic NaK

ATPase activities and he patic plasma membrane function

observed in early studies (Matsumura et al 1984 Peterson et

al 1979)

Summary

Low-dose (04 Agkg bw) TCDD exposure elicited

altered expression of genes typically associated with phase

I and phase II metabolism However in addition the

expression of Gadd45a and Cyclin D1 were altered already

6 h after low-dose exposure suggesting that TCDD affects

genes associated with cell cycle control even following

single low-dose exposure At the high-dose widespread

changes in expression were observed for genes encoding

proteins involved in multiple cellular functions In this

study we focused primarily on genes involved with

functions in intermediary metabolism in order to further

elucidate genes and pathways associated with TCDD-

induced wasting In that respect we found broad changes

to genes coding for enzymes associated with lipid

carbohydrate and nitrogen metabolism suggesting that TCDD-induced wasting is likely a complex adaptive effect

to chemical insult Furthermore the work herein identifies

changes to genes encoding proteins critical to the metabo-

lism of cholesterol bile acid synthesis and bile acid

transport These results have the potential to explain altered

cholesterol metabolism following TCDD exposure and

suggest markedly altered synthesis and transport of bile

acids following TCDD exposure We furthermore suggest

that altered expression of genes in this pathway may be

indicative of altered bile acid mediated signaling in the liver

and thus may contribute a significant mechanism of TCDD-

induced hepatotoxicity




Acknowledgments

This study has been carried out with financial support

from the Commission of the European Communities

specific RTD projects Bonetox (EU-QLK-CT-02-02528)

and CASCADE (FOOD-CT-2003-506319) It does not

necessarily reflect its views and in no way anticipates theCommissions future policy in this area The work was also

supported by funds from the Swedish Council for Environ-

ment Agricultural Sciences and Spatial Planning (FOR-

MAS grant no 2102003-1135 Etapp2)

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Eggertsen G 1994 Molecular cloning and identification of a rat serum carboxylesterase expressed in the liver J Biol Chem 269

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Astrand M 2003 Contrast normalization of oligonucleotide arrays

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Brassil PJ Debri K Nakura H Kobayashi S Davies DS Edwards

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Brewster DW Uraih LC Birnbaum LS 1988b The acute toxicity of

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93 31ndash46

Crambert G Fuzesi M Garty H Karlish S Geering K 2002

Phospholemman (FXYD1) associates with NaK-ATPase and regu-

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Cramer CT Cooke S Ginsberg LC Kletzien RF Stapleton SR

Ulrich RG 1995 Upregulation of glucose-6-phosphate dehydrogen-

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Denison MS Fisher JM Whitlock Jr JP 1988 The DNA recognition

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Denson LA Sturm E Echevarria W Zimmerman TL Makishima

M Mangelsdorf DJ Karpen SJ 2001 The orphan nuclear receptor

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Diez-Fernandez C Sanz N Cascales M 1996 Changes in glucose-6- phosphate dehydrogenase and malic enzyme gene expression in acute

hepatic injury induced by thioacetamide Biochem Pharmacol 51

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Elshourbagy NA Near JC Kmetz PJ Sathe GM Southan C

Strickler JE Gross M Young JF Wells TN Groot PH 1990

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Emi Y Ikushiro S Iyanagi T 1996 Xenobiotic responsive element-

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Erickson SK Lear SR Deane S Dubrac S Huling SL Nguyen L

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Sehayek E Ananthanarayanan M Balasubramaniyan N Suchy FJ

Batta AK Salen G 2003 Hypercholesterolemia and changes in lipidand bile acid metabolism in male and female cyp7A1-deficient mice

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Fornace Jr AJ Nebert DW Hollander MC Luethy JD Papathana-

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Frueh FW Hayashibara KC Brown PO Whitlock Jr JP 2001 Use

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Fujisawa-Sehara A Sogawa K Yamane M Fujii-Kuriyama Y 1987Characterization of xenobiotic responsive elements upstream from the

drug-metabolizing cytochrome P-450c gene a similarity to glucocorti-

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Geering K Beguin P Garty H Karlish S Fuzesi M Horisberger

JD Crambert G 2003 FXYD proteins new tissue- and isoform-

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Gerin I Veiga-da-Cunha M Achouri Y Collet JF Van Schaftin-

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case mutated in glycogen storage disease type Ib FEBS Lett 419

235ndash238

Gibson DM Lyons RT Scott DF Muto Y 1972 Synthesis and

degradation of the lipogenic enzymes of rat liver Adv Enzyme Regul

10 187ndash204




Goldstein JA Hickman P Bergman H Vos JG 1973 Hepatic

porphyria induced by 2378-tetrachlorodibenzo-p-dioxin in the mouse

Res Commun Chem Pathol Pharmacol 6 919ndash928

Goodwin B Jones SA Price RR Watson MA McKee DD

Moore LB Galardi C Wilson JG Lewis MC Roth ME

Maloney PR Willson TM Kliewer SA 2000 A regulatory

cascade of the nuclear receptors FXR SHP-1 and LRH-1 represses bile

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organic anion transporting polypeptide 2 (oatp2) by prototypical drug-

metabolizing enzyme inducers that activate gene expression through

ligand-activated transcription factor pathways J Pharmacol Exp Ther

300 206ndash212

Harrison EH 2000 Lipases and carboxylesterases possible roles in the

hepatic utilization of vitamin A J Nutr 130 340S ndash344S

Hines RN Mathis JM Jacob CS 1988 Identification of multiple

regulatory elements on the human cytochrome P450IA1 gene Carcino-

genesis 9 1599ndash1605

Hollander MC Fornace Jr AJ 2002 Genomic instability centrosome

amplification cell cycle checkpoints and Gadd45a Oncogene 21

6228ndash6233

Hori M Kondo H Ariyoshi N Yamada H Oguri K 1997 Species-

specific alteration of hepatic glucose 6-phosphate dehydrogenaseactivity with coplanar polychlorinated biphenyl evidence for an Ah-

receptor-linked mechanism Chemosphere 35 951 ndash 958

Ishii Y Kato H Hatsumura M Ishida T Ariyoshi N Yamada H

Oguri K 2001 Effects of a highly toxic coplanar polychlorinated

biphenyl 33 V44 V5-pentachlorobiphenyl on intermediary metabolism

reduced triose phosphate content in rat liver cytosol Fukuoka Igaku

Zasshi 92 190ndash200

Iynedjian PB Jotterand D Nouspikel T Asfari M Pilot PR 1989

Transcriptional induction of glucokinase gene by insulin in cultured

liver cells and its repression by the glucagon-cAMP system J Biol

Chem 264 21824ndash 21829

Jelinek DF Andersson S Slaughter CA Russell DW 1990 Cloning

and regulation of cholesterol 7 alpha-hydroxylase the rate-limiting

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Johansson L Thomsen JS Damdimopoulos AE Spyrou GGustafsson JA Treuter E 1999 The orphan nuclear receptor SHP

inhibits agonist-dependent transcriptional activity of estrogen receptors

ERalpha and ERbeta J Biol Chem 274 345ndash353

Kakyo M Sakagami H Nishio T Nakai D Nakagomi R Tokui T

Naitoh T Matsuno S Abe T Yawo H 1999 Immunohistochem-

ical distribution and functional characterization of an organic anion

transporting polypeptide 2 (oatp2) FEBS Lett 445 343ndash346

Kelley SK Nilsson CB Green MH Green JB Hakansson H

1998 Use of model-based compartmental analysis to study effects of

2378-tetrachlorodibenzo-p-dioxin on vitamin A kinetics in rats

Toxicol Sci 44 1ndash13


2000 Mobilization of vitamin A stores in rats after administration of

2378-tetrachlorodibenzo-p-dioxin a kinetic analysis Toxicol Sci 55

478ndash484Kelling CK Christian BJ Inhorn SL Peterson RE 1985

Hypophagia-induced weight loss in mice rats and guinea pigs treated

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700ndash712

Kelling CK Menahan LA Peterson RE 1987 Hepatic indices of

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Biochem Pharmacol 36 283ndash291

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174ndash181

Klinge CM Jernigan SC Risinger KE Lee JE Tyulmenkov VV

Falkner KC Prough RA 2001 Short heterodimer partner (SHP)

orphan nuclear receptor inhibits the transcriptional activity of aryl

hydrocarbon receptor (AHR)AHR nuclear translocator (ARNT) Arch

Biochem Biophys 390 64ndash 70

Krig SR Chandraratna RA Chang MM Wu R Rice RH

2002 Gene-specific TCDD suppression of RARalpha- and RXR-

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102ndash108

Kullak-Ublick GA Stieger B Hagenbuch B Meier PJ 2000 Hepatic

transport of bile salts Semin Liver Dis 20 273ndash292Kurachi M Hashimoto S Obata A Nagai S Nagahata T Inadera H

Sone H Tohyama C Kaneko S Kobayashi K Matsushima K

2002 Identification of 2378-tetrachlorodibenzo-p-dioxin-responsive

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Biophys Res Commun 292 368 ndash377

Kwon YI Yeon JD Oh SM Chung KH 2004 Protective effects of

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239ndash247

Lakshman MR Campbell BS Chirtel SJ Ekarohita N 1988 Effects

of 2378-tetrachlorodibenzo-p-dioxin (TCDD) on de novo fatty acid

and cholesterol synthesis in the rat Lipids 23 904ndash906

Lakshman MR Chirtel SJ Chambers LL Coutlakis PJ 1989

Effects of 2378-tetrachlorodibenzo-p-dioxin on lipid synthesis and

lipogenic enzymes in the rat J Pharmacol Exp Ther 248 62ndash66Lakshman MR Ghosh P Chirtel SJ 1991 Mechanism of action of

2378-tetrachlorodibenzo-p-dioxin on intermediary metabolism in the

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Lentnek M Griffith OW Rifkind AB 1991 2378-Tetrachlorodi-

benzo-p-dioxin increases reliance on fats as a fuel source independently

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dioxin lethality Biochem Biophys Res Commun 174 1267 ndash 1271

Lin B Annabi B Hiraiwa H Pan CJ Chou JY 1998 Cloning

and characterization of cDNAs encoding a candidate glycogen

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31656ndash31660

Louet JF Hayhurst G Gonzalez FJ Girard J Decaux JF 2002

The coactivator PGC-1 is involved in the regulation of the liver

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Lu TT Makishima M Repa JJ Schoonjans K Kerr TA Auwerx J

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bile acid synthesis by nuclear receptors Mol Cell 6 507ndash515

Ma Q Kinneer K Bi Y Chan JY Kan YW 2004 Induction of

murine NAD(P)Hquinone oxidoreductase by 2378-tetrachlorodi-

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Makishima M Okamoto AY Repa JJ Tu H Learned RM Luk

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Identification of a nuclear receptor for bile acids Science 284

1362ndash1365

Mannaerts GP Van Veldhoven PP Casteels M 2000 Peroxisomallipid degradation via beta- and alpha-oxidation in mammals Cell

Biochem Biophys 32 73 ndash 87 (Spring)

Masuda N Yasumo H Tamura T Hashiguchi N Furusawa T

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several nuclear receptors Biochim Biophys Acta 1350 27ndash32

Matsumura F Brewster DW Madhukar BV Bombick DW 1984

Alteration of rat hepatic plasma membrane functions by 2378-

tetrachlorodibenzo-p-dioxin (TCDD) Arch Environ Contam Toxicol

13 509ndash515

McGarry JD Brown NF 1997 The mitochondrial carnitine palmitoyl-

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Biochem 244 1ndash14

McKim Jr JM Marien K Schaup HW Selivonchick DP 1991




Alterations of hepatic acetyl-CoA carboxylase by 2378-tetrachlorodi-

benzo-p-dioxin Lipids 26 521ndash525

Meier PJ Eckhardt U Schroeder A Hagenbuch B Stieger B 1997

Substrate specificity of sinusoidal bile acid and organic anion uptake

systems in rat and human liver Hepatology 26 1667ndash 1677

Munzel PA Lehmkoster T Bruck M Ritter JK Bock KW 1998

Aryl hydrocarbon receptor-inducible or constitutive expression of

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Muzi G Gorski JR Rozman K 1989 Mode of metabolism is altered in

2378-tetrachlorodibenzo-p-dioxin (TCDD)-treated rats Toxicol Lett

47 77ndash86

Narkewicz MR Iynedjian PB Ferre P Girard J 1990 Insulin and tri-

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Nilsson CB Hakansson H 2002 The retinoid signaling systemmdashA

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Nilsson CB Hanberg A Trossvik C H3 kansson H 1996 2378-

tetrachlorodibenzo-p-dioxin affects retinol esterification in hepatic

stellate cells and kidney Environ Toxicol Pharmacol 2 17ndash23

Nilsson CB Hoegberg P Trossvik C Azais-Braesco V Blaner WS

Fex G Harrison EH Nau H Schmidt CK van Bennekum AM

Hakansson H 2000 2378-tetrachlorodibenzo-p-dioxin increasesserum and kidney retinoic acid levels and kidney retinol esterification

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Noe B Hagenbuch B Stieger B Meier PJ 1997 Isolation of a

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Pan CJ Lei KJ Annabi B Hemrika W Chou JY 1998

Transmembrane topology of glucose-6-phosphatase J Biol Chem

273 6144ndash6148

Park EY Rho HM 2002 The transcriptional activation of the human

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240 47ndash55

Parks DJ Blanchard SG Bledsoe RK Chandra G Consler TG

Kliewer SA Stimmel JB Willson TM Zavacki AM MooreDD Lehmann JM 1999 Bile acids natural ligands for an orphan

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Paulson KE Darnell Jr JE Rushmore T Pickett CB 1990 Analysis

of the upstream elements of the xenobiotic compound-inducible and

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Biol 10 1841ndash1852

Pearce NJ Yates JW Berkhout TA Jackson B Tew D Boyd H

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role of ATP citrate-lyase in the metabolic regulation of plasma lipids

Hypolipidaemic effects of SB-204990 a lactone prodrug of the potent

ATP citrate-lyaseinhibitor SB-201076 Biochem J 334(Pt 1) 113ndash 119

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Depression of adenosine triphosphatase activities in isolated liver

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Peterson RE Seefeld MD Christian BJ Potter CL Kelling CK

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Poland A Knutson JC 1982 2378-tetrachlorodibenzo-p-dioxin and

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Potter CL Sipes IG Russell DH 1983 Hypothyroxinemia and

hypothermia in rats in response to 2378-tetrachlorodibenzo-p-dioxin

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Potter CLMenahan LAPeterson RE1986 Relationship of alterations

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Puga A Maier A Medvedovic M 2000 The transcriptional signature

of dioxin in human hepatoma HepG2 cells Biochem Pharmacol 60

1129ndash1142

Pullinger CR Eng C Salen G Shefer S Batta AK Erickson SK

Verhagen A Rivera CR Mulvihill SJ Malloy MJ Kane JP2002 Human cholestrol 7alpha-hydroxylase (CYP7A1) deficiency has

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Quattrochi LC Vu T Tukey RH 1994 The human CYP1A2 gene and

induction by 3-methylcholanthrene A region of DNA that supports

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Reichel C Gao B Van Montfoort J Cattori V Rahner C Hagenbuch

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Roth W Voorman R Aust SD 1988 Activity of thyroid hormone-

inducible enzymes following treatment with 2378-tetrachlorodibenzo-

p-dioxin Toxicol Appl Pharmacol 92 65ndash74

Rushmore TH King RG Paulson KE Pickett CB 1990 Regulation

of glutathione S-transferase Ya subunit gene expression identificationof a unique xenobiotic-responsive element controlling inducible

expression by planar aromatic compounds Proc Natl Acad Sci

USA 87 3826ndash3830

Russell DW Setchell KD 1992 Bile acid biosynthesis Biochemistry

31 4737ndash4749

Sanghani SP Davis WI Dumaual NG Mahrenholz A Bosron WF

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Santostefano MJ Ross DG Savas U Jefcoate CR Birnbaum LS

1997 Differential time-course and dosendashresponse relationships of

TCDD-induced CYP1B1 CYP1A1 and CYP1A2 proteins in rats

Biochem Biophys Res Commun 233 20 ndash 24

Schmidt JV Bradfield CA 1996 Ah receptor signaling pathways

Annu Rev Cell Dev Biol 12 55ndash89

Schmidt CK Hoegberg P Fletcher N Nilsson CB Trossvik CHakansson H Nau H 2003 2378-tetrachlorodibenzo-p-dioxin

(TCDD) alters the endogenous metabolism of all-trans-retinoic acid in

the rat Arch Toxicol 77 371ndash383

Schuur AG Boekhorst FM Brouwer A Visser TJ 1997 Extra-

thyroidal effects of 2378-tetrachlorodibenzo-p-dioxin on thyroid

hormone turnover in male SpraguendashDawley rats Endocrinology 138

3727ndash3734

Seefeld MD Corbett SW Keesey RE Peterson RE 1984

Characterization of the wasting syndrome in rats treated with 2378-

tetrachlorodibenzo-p-dioxin Toxicol Appl Pharmacol 73 311ndash 322

Seol W Choi HS Moore DD 1996 An orphan nuclear hormone

receptor that lacks a DNA binding domain and heterodimerizes with

other receptors Science 272 1336ndash1339

Seol W Hanstein B Brown M Moore DD 1998 Inhibition of

estrogen receptor action by the orphan receptor SHP (short heterodimer partner) Mol Endocrinol 12 1551ndash1557

Serra D Casals N Asins G Royo T Ciudad CJ Hegardt FG

1993 Regulation of mitochondrial 3-hydroxy-3-methylglutaryl-coen-

zyme A synthase protein by starvation fat feeding and diabetes Arch


Sheikh MS Hollander MC Fornance Jr AJ 2000 Role of Gadd45 in

apoptosis Biochem Pharmacol 59 43ndash45

Sinal CJ Tohkin M Miyata M Ward JM Lambert G Gonzalez

FJ 2000 Targeted disruption of the nuclear receptor FXRBAR

impairs bile acid and lipid homeostasis Cell 102 731ndash744

Stahl BU Beer DG Weber LW Rozman K 1993 Reduction of

hepatic phosphoenolpyruvate carboxykinase (PEPCK) activity by

2378-tetrachlorodibenzo-p-dioxin (TCDD) is due to decreased mRNA

levels Toxicology 79 81ndash95




Stumpo DJ Kletzien RF 1985 The effect of ethanol alone and in

combination with the glucocorticoids and insulin on glucose-6-

phosphate dehydrogenase synthesis and mRNA in primary cultures of

hepatocytes Biochem J 226 123ndash 130

Sullivan AC Triscari J Hamilton JG Miller ON Wheatley VR

1974 Effect of (Agrave)-hydroxycitrate upon the accumulation of lipid in the

rat I Lipogenesis Lipids 9 121ndash128

Sun G Alexson SE Harrison EH 1997 Purification and character-ization of a neutral bile salt-independent retinyl ester hydrolase from rat

liver microsomes Relationship to rat carboxylesterase ES-2 J Biol

Chem 272 24488ndash 24493

Sweatlock JA Gasiewicz TA 1985 The effect of TCDD exposure on

the concentration of ketone bodies in the rat Toxicologist 5 64

Takimoto K Lindahl R Dunn TJ Pitot HC 1994 Structure of the 5 V

flanking region of class 3 aldehyde dehydrogenase in the rat Arch

Biochem Biophys 312 539ndash546

Tomaszewski KE Montgomery CA Melnick RL 1988 Modulation

of 2378-tetrachlorodibenzo-p-dioxin toxicity in F344 rats by di(2-

ethylhexyl)phthalate Chem-Biol Interact 65 205ndash 222

Unkila M Ruotsalainen M PohjanvirtaR Viluksela M MacDonald E

Tuomisto JT Rozman K Tuomisto J 1995 Effect of 2378-

tetrachlorodibenzo-p-dioxin (TCDD) on tryptophan and glucose homeo-

stasis in the most TCDD-susceptible and the most TCDD-resistant species guinea pigs and hamsters Arch Toxicol 69 677 ndash 683

Vanden Heuvel JP Clark GC Kohn MC Tritscher AM Greenlee

WF Lucier GW Bell DA 1994 Dioxin-responsive genes

examination of dosendashresponse relationships using quantitative reverse

transcriptase-polymerase chain reaction Cancer Res 54 62ndash68

Viluksela M Stahl BU Rozman KK 1995 Tissue-specific effects of

2378-tetrachlorodibenzo-p-dioxin (TCDD) on the activity of phos-

phoenolpyruvate carboxykinase (PEPCK) in rats Toxicol Appl

Pharmacol 135 308ndash 315

Viluksela M Unkila M Pohjanvirta R Tuomisto JT Stahl BU

Rozman KK Tuomisto J 1999 Effects of 2378-tetrachlorodi-

benzo-p-dioxin (TCDD) on liver phosphoenolpyruvate carboxykinase

(PEPCK) activity glucose homeostasis and plasma amino acid

concentrations in the most TCDD-susceptible and the most TCDD-

resistant rat strains Arch Toxicol 73 323ndash 336Walker NJ Crofts FG Li Y Lax SF Hayes CL Strickland PT

Lucier GW Sutter TR 1998 Induction and localization of

cytochrome P450 1B1 (CYP1B1 protein in the livers of TCDD-treated

rats detection using polyclonal antibodies raised to histidine-tagged

fusion proteins produced and purified from bacteria Carcinogenesis 19

395ndash402

Walker NJ Portier CJ Lax SF Crofts FG Li Y Lucier GW

Sutter TR 1999 Characterization of the dose-response of CYP1B1

CYP1A1 and CYP1A2 in the liver of female SpraguendashDawley rats

following chronic exposure to 2378-tetrachlorodibenzo-p-dioxin

Toxicol Appl Pharmacol 154 279ndash286

Wang H Chen J Hollister K Sowers LC Forman BM 1999a

Endogenous bile acids are ligands for the nuclear receptor FXRBAR

Mol Cell 3 543ndash553

Wang XW Zhan Q Coursen JD Khan MA Kontny HU Yu L

Hollander MC OrsquoConnor PM Fornace Jr AJ Harris CC

1999b GADD45 induction of a G2M cell cycle checkpoint Proc Natl

Acad Sci USA 96 3706ndash3711

Wang X Sperkova Z Napoli JL 2001 Analysis of mouse retinaldehydrogenase type 2 promoter and expression Genomics 74

245ndash250

Weber LW Lebofsky M Greim H Rozman K 1991a Key enzymes of

gluconeogenesis are dose-dependently reduced in 2378-tetrachlorodi-

benzo-p-dioxin (TCDD)-treated rats Arch Toxicol 65 119 ndash 123

Weber LW Lebofsky M Stahl BU Gorski JR Muzi G Rozman

K 1991b Reduced activities of key enzymes of gluconeogenesis as

possible cause of acute toxicity of 2378-tetrachlorodibenzo-p-dioxin

(TCDD) in rats Toxicology 66 133ndash144

Weber LW Lebofsky M Stahl BU Smith S Rozman KK 1995

Correlation between toxicity and effects on intermediary metabolism in

2378-tetrachlorodibenzo-p-dioxin-treated male C57BL6J and DBA

2J mice Toxicol Appl Pharmacol 131 155ndash162

Whitlock Jr JP 1999 Induction of cytochrome P4501A1 Annu Rev

Pharmacol Toxicol 39 103ndash125Whitlock Jr JP Okino ST Dong L Ko HP Clarke-Katzenberg R

Ma Q Li H 1996 Cytochromes P450 5 induction of cytochrome

P4501A1 a model for analyzing mammalian gene transcription

FASEB J 10 809ndash818

Woods JS 1973 Studies of the effects of 2378-tetrachlorodibenzo-p-

dioxin on mammalian hepatic delta-aminolevulinic acid synthetase

Environ Health Perspect 5 221ndash 225

Yang KH Croft WA Peterson RE 1977 Effects of 2378-

tetrachlorodibenzo-p-dioxin on plasma disappearance and biliary

excretion of foreign compounds in rats Toxicol Appl Pharmacol

40 485ndash496

Yang KH Yoo BS Choe SY 1983 Effects of halogenated dibenzo-p-

dioxins on plasma disappearance and biliary excretion of ouabain in

rats Toxicol Lett 15 259ndash264

Zhan Q Fan S Smith ML Bae I Yu K Alamo Jr I OrsquoConnorPM Fornace Jr AJ 1996 Abrogation of p53 function affects gadd

gene responses to DNA base-damaging agents and starvation DNA

Cell Biol 15 805ndash815

Zhan Q Antinore MJ Wang XW Carrier F Smith ML Harris

CC Fornace Jr AJ 1999 Association with Cdc2 and inhibition of

Cdc2Cyclin B1 kinase activity by the p53-regulated protein Gadd45

Oncogene 18 2892ndash2900

Zhang L Savas U Alexander DL Jefcoate CR 1998 Character-

ization of the mouse Cyp1B1 gene Identification of an enhancer region

that directs aryl hydrocarbon receptor-mediated constitutive and

induced expression J Biol Chem 273 5174ndash5183




helixndashloop-helix protein that binds TCDD in the cyto-

plasm and following release of its chaperone proteins

translocates to the nucleus where it associates with

enhancer elements in the 5 V-flanking region of the

CYP1A1 gene known as dioxin-responsive elements

(DRE reviewed in Whitlock 1999 Whitlock et al

1996) The CYP1A1 gene contains multiple copies of the DRE sequence which have been shown to be required

for inducer-dependent transcription in DNA transfection

experiments (Denison et al 1988 1989 Fujisawa-Sehara

et al 1987 Hines et al 1988) Furthermore DRE

elements were well conserved with respect to location

within the CYP1A1 gene for mice rats and humans

(Denison et al 1988 1989 Fujisawa-Sehara et al 1987

Hines et al 1988) DREs have also been found for the

well known battery of AhR responsive genes including

CYP1A2 (Quattr ochi et al 1994) NAD(P)Hquinone

oxidoreductase (Favreau and Pickett 1991) CYP1B1

(Zhang et al 1998) UGT1A16 (Emi et al 1996 Munzelet al 1998) aldehyde dehydrogenase class 3 (Takimoto et

al 1994) and glutathione S-transferase Ya (Paulson et al

1990 Rushmore et al 1990)

More recently global expression studies have been

carried out to investigate other novel genes affected by

TCDD exposure Puga et al (2000) investigated the

transcriptome of human HepG2 cells using commercial

cDNA arrays Exposure to 10 nM TCDD for 8 h altered the

expression of 310 known genes and a similar number of

expressed sequence tags more than 21-fold Of these 310

genes 30 were upregulated and 78 downregulated regard-

less of cycloheximide treatment In another study in HepG2

cells Frueh et al (2001) found that TCDD up or down-

regulated 112 genes two-fold or more It is however

important to consider that these studies were conducted in

vitro in immortalized cell lines and may not necessarily

reflect transcriptional changes occurring in the liver follow-

ing in vivo exposure To that end using serial analyses of

gene expression (SAGE) Kurachi et al (2002) investigated

gene expression changes in mouse liver 7 days after

treatment with a dose of 20 Agkg bw TCDD Together

these studies confirmed the complicated nature of the action

of TCDD on liver cells

If one accepts that TCDD evokes a change in the

transcription of early response genes which subsequently propagate changes in cellular signaling pathways it

should be of importance to identify those genes that

are involved in the initial response Of similar interest is

to identify changes that occur after low-dose exposures

and equally those that are observed after relatively high-

dose exposure In this way it may be possible to

distinguish between adaptive changes to TCDD exposure

or the transcriptional response in a low stress state and

that associated with overt toxicity Therefore in this

study rats were exposed to a low single dose of TCDD

(04 Agkg bw) or a dose intended to elicit moderate

toxicity in SpraguendashDawley rats (40 Agkg bw) Changes

in gene expression were investigated 6 h 24 h and 7

days after TCDD exposure using the Affymetrix U34A

chip Selected novel gene changes were confirmed by

RT-PCR analyses Clinical chemistry and pathological

analyses were also conducted in support of the global

gene expression analyses

Materials and methods

Chemicals

TCDD was obtained from Cambridge Isotope Labs

(ED-901-C)

Animals

Animal experiments were conducted according to GLP at

Gene Logic Inc laboratories Male SpraguendashDawley out- bred CD rats (CRLCD[SD] IGS BR) weighing 250ndash300 g

were obtained from Charles River Laboratories The animals

were singly housed in polycarbonate cages temperatures

were maintained between 180 and 260 8C with a relative

humidity between 30 and 70 Rats were supplied with

feed (2018 Teklad Certified Global diet) and tap water

(routinely analyzed for contaminants and microbes) ad

libitum during the study During a 7-day acclimatization

period rats were observed for general health and suitability

for inclusion in the study

Experimental design

Rats (5dose) received singles doses of TCDD in a

corn oil vehicle (5 mLkg) by oral gavage at 0 04 or 40

Agkg bw on Day 1 Doses were determined in a

preliminary dose-ranging study the high-dose was

designed to elicit moderate toxicity Animals were killed

by decapitation 6 h 24 h and 7 days following

treatment livers were removed snap frozen within

approximately 2 min of death and stored at Agrave80 8C

Blood (approximately 4 mL) samples were taken prior to

termination by puncture of the orbital sinus while under

70 CO230 O2 anesthesia Approximately 1 mL of

blood was collected in serum separator tubes for clinicalchemistry analysis whereas 05 mL of blood was

collected into EDTA tubes for hematological analyses

Microarray experiments

Sample preparation processing and hybridization to

the Rat Genome U34A chip was performed by Gene

Logic Inc as described in the GeneChip Expression

Analysis Technical Manual (Affymetrix Santa Clara CA)

Information on the Rat Genome U34A chip which

analyzes approximately 7000 full-length sequences and

approximately 1000 EST clusters is available on the internet






sample

Clinical chemistry




















microscopically
















Agrave70 8C
















holm Sweden)









were normalized

Data analysis








database




































Clinical chemistry















Pathology












































Table 1




Parameter Dose

Control 04 40



24 h





(Th AL)

41 F 05 30 F 08 43 F 06

7 days





67 F 03 68 F 04 76 F 05


(mil AL)

02 F 001 018 F 002 012 F 003

Alanine

aminotransferase

(IUL)

58 F 74 452 F 58 462 F 82

Glucose (mgdL) 141 F 129 1286 F 182 1088 F 41


Globulin (gdL) 23 F 02 24 F 02 27 F 03









Table 2




symbol04 40 04 40 04 40






polypeptide 1

CYP1B1 13 2992 250 13559


polypeptide 1

CYP1B1 80 657 3856


polypeptide 1

CYP1B1 76 750 80 4838


polypeptide 1

CYP1B1 94 112


from rat liver

No symbol 269 1807 2945 2153 3048 944 2620




Arsb

UGT1A6

303 26 105 86 173 52 193



Arsb

UGT1A6

293 23 65 66 174 42 183


polypeptide A6 UDP


polypeptide A7

UGT1A6

UGT1A7

4926 27 29 55


polypeptide A6 UDP


polypeptide A7

UGT1A6

UGT1A7

10508 25 27 48



UGT1A7 186 78 60 305 20 348


UGT1A7 348 52 31 106 227



UGT1A7 10410 26 26 37



UGT1A7 17473 21 27 37


member A1

Aldh3a1 208 103 569 1058


polypeptide A1



type 2

GSTA2 18140 21 26 37


type 2

GSTA2 113 87 34



Yc2 subunit

GSTA5

890 40 54 32



GSTA4 1952 23 42



GSTA4 2770 29 48








gene exon 9








04 40 04 40 04 40



cytochrome P-450b







(CYP2J3P2)








CYP2C 6286 Agrave49



polypeptide 1



polypeptide 1



polypeptide 1




monooxygenase 1

FMO1 2390 Agrave34







45 alpha

Gadd45a 382 20 33 40 65



45 alpha

Gadd45a 798 26 25 35



45 alpha

Gadd45a 391 49


Lipid metabolism






Acaa1 1079 34 25 53



epidermal

Fabp 1065 42


acyltransferase

Gnpat 488 21








dehydrogenase


Bdh 4825 Agrave21


delta isomerase

Idi1 1727 Agrave25







04 40 04 40 04 40

Lipid metabolism




97Agrave






member 1

Slc10a1

(Ntcp)

11044 Agrave21


(organic anion


Slc21a5

oatp2





N-methyltransferase

PEMT 6315 Agrave27



Enpp2 2917 27 41


pyrophosphatase

phosphodiesterase 2

Enpp2 1615 37 34






transport protein



transport protein



dehydrogenase

G6pd 728 33 35







carboxykinase




Nitrogen metabolism





24Agrave



















04 40 04 40 04 40

Nitrogen metabolism




Bhmt 22602 20



(active)

Dhfr1 2389 22


nucleotidase

Bpnt1 577 31




subunit Va

Cox5a 9753 31

Retinoid metabolism






CES3 2496 Agrave64




Steroid metabolism









gene 2

Sth2 18559 Agrave37


gene 2

Sth2 29775 Agrave36


gene 2

Sth2 11993 Agrave31



complete cds



dehydrogenase

3 beta- and steroid

delta-isomerase

Hsd3b 7019 Agrave24


type 1

Dio 1 826 Agrave43



Hsd17b2 15271 20


(hepatic steroid


CYP2A1 13452 36

L24207 _ i _ at (Ad) L24207 Rattus


6-beta-hydroxylase

(CYP3A1) mRNA

CYP3A1 1659 24


6-beta-hydroxylase

(CYP3A1) mRNA

Cyp3A1 1068 27


(PNCN inducible)


3A poypeptide 3

Cyp3A1

Cyp3a3

6993 25







04 40 04 40 04 40

Kinases


2-epimerase


Uae1 2653 Agrave23

Circadian rhythm




member 4

Slc4a4 493 70



member 1

Slc17a1 915 24 30



member 1

Slc17a1 428 36 56



transporting beta

polypeptide 3

Atp1b3 968 29












member 7




member 1








type 2)



member 1






(YWK-II)

LOC64312 3213 21



carcinoembryonic


adhesion molecule 1

Ceacam1 322 676



adhesion molecule 1

Ceacam1 787 23

U32575 _ at Sec6 Sec6 181 44 62

U32575 _ g _ at Sec6 Sec6 342 20 45 94




23 kDa

Snap23 205 34






04 40 04 40 04 40

Cell cycle




33Agrave

24

RNA processing


Cell signaling




receptor type D



receptor type D

Ptprd 835 Agrave21








Tnfrsf1a 1906 20






Ngfrap1 308



Ralb 282 20












partial cds

HNF3-G 631 Agrave27






Cited2 or

MRG1

864 Agrave36







Heme synthesis
















04 40 04 40 04 40

Immune




Psmb8 3232 Agrave21


antigen gene







s subcomponent

C1s 6862 29







31







3 end of exon 6









protein 3



Igfbp1 516 25 47 29 49

Cytoskeleton


non-muscle

Myh9 1052 Agrave38



isoform of actin





1A1B light chain 3

MPL3 3218 33 26















member 10

Serpina 10

Rasp-1

5175 22






04 40 04 40 04 40




No symbol 661 29


No symbol 888 22






CP-2 804 23 30



induced mRNA





of RGS16

Mir16 1508 21


of RGS16

Mir16 2135 20


monophosphatase 2

Impa2 385 32






No symbol 362 2


norvegicus cDNA



No symbol 323 4




[Rattus norvegicus]





No symbol 113 5





3 gb = AA955983 Ag =

Rn7854len = 542

No symbol 5035 2

U47312 _ s _ at U47312 RNU47312 Rat R2




[Rattus norvegicus]





3 gb = AA875171gi =

2980119 Ag = Rn2814

len = 458

No symbol 863 2





AA817987gi = 2887867 Ag =

Rn23920len = 373








04 40 04 40 04 40


rc _ AA859899 _ at rc _ AA859899 UI-R-E0-




AA859899gi = 2949419 Ag =

Rn810len = 353






No symbol 99 54

ESTs


norvegicus cDNA

643 28


norvegicus cDNA

722 22 20 20


1300 24


norvegicus cDNA

10889 22 26


norvegicus cDNA

268 28


norvegicus cDNA

42918 116 110


norvegicus cDNA

21687 22


norvegicus cDNA

421 22


norvegicus cDNA

2863 34


norvegicus cDNA

1472 20


norvegicus cDNA

553 20


norvegicus cDNA

808 22


norvegicus cDNA

2143 20


norvegicus cDNA

2686 20 41


norvegicus cDNA

550 21


norvegicus cDNA

291 20


norvegicus cDNA

1054 25


479 23


norvegicus cDNA

1380 Agrave22


norvegicus cDNA

1628 Agrave24


norvegicus cDNA

788 21


norvegicus cDNA

875 Agrave23


norvegicus cDNA

593 Agrave35


norvegicus cDNA


































thesis pathway

Detoxification































metabolism









04 40 04 40 04 40

ESTs


norvegicus cDNA

2262 Agrave21


3426Agrave

22


norvegicus cDNA

1340 Agrave21


norvegicus cDNA

6360 Agrave24


norvegicus cDNA

438 23


norvegicus cDNA

391 Agrave39







































































acetyl-CoA














































































































































































exposure (Table 1)














































































treated rats







































maintained




Nitrogen metabolism

















































































































(Ahuja et al 2003)

Heme synthesis
















Cytoskeleton





























Membrane proteins


















al 1979)

Summary






























Acknowledgments









References






17118ndash17124




12 191ndash204












231ndash246







2813ndash2824



10035ndash10038



34280ndash34286







93 31ndash46




11476ndash 11481











39124ndash39132













1159ndash1163


















4556ndash4561














388ndash394




235ndash238



10 187ndash204















300 206ndash212








6228ndash6233












Chem 264 21824ndash 21829





















700ndash712







174ndash181









102ndash108










239ndash247
















31656ndash31660

















1362ndash1365










13 509ndash515


















47 77ndash86


















273 6144ndash6148





240 47ndash55



































1129ndash1142







269 6949ndash6954













31 4737ndash4749
















3727ndash3734


































Chem 272 24488ndash 24493































395ndash402














245ndash250























40 485ndash496




















sample

Clinical chemistry




















microscopically
















Agrave70 8C
















holm Sweden)









were normalized

Data analysis








database




































Clinical chemistry















Pathology












































Table 1




Parameter Dose

Control 04 40



24 h





(Th AL)

41 F 05 30 F 08 43 F 06

7 days





67 F 03 68 F 04 76 F 05


(mil AL)

02 F 001 018 F 002 012 F 003

Alanine

aminotransferase

(IUL)

58 F 74 452 F 58 462 F 82

Glucose (mgdL) 141 F 129 1286 F 182 1088 F 41


Globulin (gdL) 23 F 02 24 F 02 27 F 03









Table 2




symbol04 40 04 40 04 40






polypeptide 1

CYP1B1 13 2992 250 13559


polypeptide 1

CYP1B1 80 657 3856


polypeptide 1

CYP1B1 76 750 80 4838


polypeptide 1

CYP1B1 94 112


from rat liver

No symbol 269 1807 2945 2153 3048 944 2620




Arsb

UGT1A6

303 26 105 86 173 52 193



Arsb

UGT1A6

293 23 65 66 174 42 183


polypeptide A6 UDP


polypeptide A7

UGT1A6

UGT1A7

4926 27 29 55


polypeptide A6 UDP


polypeptide A7

UGT1A6

UGT1A7

10508 25 27 48



UGT1A7 186 78 60 305 20 348


UGT1A7 348 52 31 106 227



UGT1A7 10410 26 26 37



UGT1A7 17473 21 27 37


member A1

Aldh3a1 208 103 569 1058


polypeptide A1



type 2

GSTA2 18140 21 26 37


type 2

GSTA2 113 87 34



Yc2 subunit

GSTA5

890 40 54 32



GSTA4 1952 23 42



GSTA4 2770 29 48








gene exon 9








04 40 04 40 04 40



cytochrome P-450b







(CYP2J3P2)








CYP2C 6286 Agrave49



polypeptide 1



polypeptide 1



polypeptide 1




monooxygenase 1

FMO1 2390 Agrave34







45 alpha

Gadd45a 382 20 33 40 65



45 alpha

Gadd45a 798 26 25 35



45 alpha

Gadd45a 391 49


Lipid metabolism






Acaa1 1079 34 25 53



epidermal

Fabp 1065 42


acyltransferase

Gnpat 488 21








dehydrogenase


Bdh 4825 Agrave21


delta isomerase

Idi1 1727 Agrave25







04 40 04 40 04 40

Lipid metabolism




97Agrave






member 1

Slc10a1

(Ntcp)

11044 Agrave21


(organic anion


Slc21a5

oatp2





N-methyltransferase

PEMT 6315 Agrave27



Enpp2 2917 27 41


pyrophosphatase

phosphodiesterase 2

Enpp2 1615 37 34






transport protein



transport protein



dehydrogenase

G6pd 728 33 35







carboxykinase




Nitrogen metabolism





24Agrave



















04 40 04 40 04 40

Nitrogen metabolism




Bhmt 22602 20



(active)

Dhfr1 2389 22


nucleotidase

Bpnt1 577 31




subunit Va

Cox5a 9753 31

Retinoid metabolism






CES3 2496 Agrave64




Steroid metabolism









gene 2

Sth2 18559 Agrave37


gene 2

Sth2 29775 Agrave36


gene 2

Sth2 11993 Agrave31



complete cds



dehydrogenase

3 beta- and steroid

delta-isomerase

Hsd3b 7019 Agrave24


type 1

Dio 1 826 Agrave43



Hsd17b2 15271 20


(hepatic steroid


CYP2A1 13452 36

L24207 _ i _ at (Ad) L24207 Rattus


6-beta-hydroxylase

(CYP3A1) mRNA

CYP3A1 1659 24


6-beta-hydroxylase

(CYP3A1) mRNA

Cyp3A1 1068 27


(PNCN inducible)


3A poypeptide 3

Cyp3A1

Cyp3a3

6993 25







04 40 04 40 04 40

Kinases


2-epimerase


Uae1 2653 Agrave23

Circadian rhythm




member 4

Slc4a4 493 70



member 1

Slc17a1 915 24 30



member 1

Slc17a1 428 36 56



transporting beta

polypeptide 3

Atp1b3 968 29












member 7




member 1








type 2)



member 1






(YWK-II)

LOC64312 3213 21



carcinoembryonic


adhesion molecule 1

Ceacam1 322 676



adhesion molecule 1

Ceacam1 787 23

U32575 _ at Sec6 Sec6 181 44 62

U32575 _ g _ at Sec6 Sec6 342 20 45 94




23 kDa

Snap23 205 34






04 40 04 40 04 40

Cell cycle




33Agrave

24

RNA processing


Cell signaling




receptor type D



receptor type D

Ptprd 835 Agrave21








Tnfrsf1a 1906 20






Ngfrap1 308



Ralb 282 20












partial cds

HNF3-G 631 Agrave27






Cited2 or

MRG1

864 Agrave36







Heme synthesis
















04 40 04 40 04 40

Immune




Psmb8 3232 Agrave21


antigen gene







s subcomponent

C1s 6862 29







31







3 end of exon 6









protein 3



Igfbp1 516 25 47 29 49

Cytoskeleton


non-muscle

Myh9 1052 Agrave38



isoform of actin





1A1B light chain 3

MPL3 3218 33 26















member 10

Serpina 10

Rasp-1

5175 22






04 40 04 40 04 40




No symbol 661 29


No symbol 888 22






CP-2 804 23 30



induced mRNA





of RGS16

Mir16 1508 21


of RGS16

Mir16 2135 20


monophosphatase 2

Impa2 385 32






No symbol 362 2


norvegicus cDNA



No symbol 323 4




[Rattus norvegicus]





No symbol 113 5





3 gb = AA955983 Ag =

Rn7854len = 542

No symbol 5035 2

U47312 _ s _ at U47312 RNU47312 Rat R2




[Rattus norvegicus]





3 gb = AA875171gi =

2980119 Ag = Rn2814

len = 458

No symbol 863 2





AA817987gi = 2887867 Ag =

Rn23920len = 373








04 40 04 40 04 40


rc _ AA859899 _ at rc _ AA859899 UI-R-E0-




AA859899gi = 2949419 Ag =

Rn810len = 353






No symbol 99 54

ESTs


norvegicus cDNA

643 28


norvegicus cDNA

722 22 20 20


1300 24


norvegicus cDNA

10889 22 26


norvegicus cDNA

268 28


norvegicus cDNA

42918 116 110


norvegicus cDNA

21687 22


norvegicus cDNA

421 22


norvegicus cDNA

2863 34


norvegicus cDNA

1472 20


norvegicus cDNA

553 20


norvegicus cDNA

808 22


norvegicus cDNA

2143 20


norvegicus cDNA

2686 20 41


norvegicus cDNA

550 21


norvegicus cDNA

291 20


norvegicus cDNA

1054 25


479 23


norvegicus cDNA

1380 Agrave22


norvegicus cDNA

1628 Agrave24


norvegicus cDNA

788 21


norvegicus cDNA

875 Agrave23


norvegicus cDNA

593 Agrave35


norvegicus cDNA


































thesis pathway

Detoxification































metabolism









04 40 04 40 04 40

ESTs


norvegicus cDNA

2262 Agrave21


3426Agrave

22


norvegicus cDNA

1340 Agrave21


norvegicus cDNA

6360 Agrave24


norvegicus cDNA

438 23


norvegicus cDNA

391 Agrave39







































































acetyl-CoA














































































































































































exposure (Table 1)














































































treated rats







































maintained




Nitrogen metabolism

















































































































(Ahuja et al 2003)

Heme synthesis
















Cytoskeleton





























Membrane proteins


















al 1979)

Summary






























Acknowledgments









References






17118ndash17124




12 191ndash204












231ndash246







2813ndash2824



10035ndash10038



34280ndash34286







93 31ndash46




11476ndash 11481











39124ndash39132













1159ndash1163


















4556ndash4561














388ndash394




235ndash238



10 187ndash204















300 206ndash212








6228ndash6233












Chem 264 21824ndash 21829





















700ndash712







174ndash181









102ndash108










239ndash247
















31656ndash31660

















1362ndash1365










13 509ndash515


















47 77ndash86


















273 6144ndash6148





240 47ndash55



































1129ndash1142







269 6949ndash6954













31 4737ndash4749
















3727ndash3734


































Chem 272 24488ndash 24493































395ndash402














245ndash250























40 485ndash496


























Clinical chemistry















Pathology












































Table 1




Parameter Dose

Control 04 40



24 h





(Th AL)

41 F 05 30 F 08 43 F 06

7 days





67 F 03 68 F 04 76 F 05


(mil AL)

02 F 001 018 F 002 012 F 003

Alanine

aminotransferase

(IUL)

58 F 74 452 F 58 462 F 82

Glucose (mgdL) 141 F 129 1286 F 182 1088 F 41


Globulin (gdL) 23 F 02 24 F 02 27 F 03









Table 2




symbol04 40 04 40 04 40






polypeptide 1

CYP1B1 13 2992 250 13559


polypeptide 1

CYP1B1 80 657 3856


polypeptide 1

CYP1B1 76 750 80 4838


polypeptide 1

CYP1B1 94 112


from rat liver

No symbol 269 1807 2945 2153 3048 944 2620




Arsb

UGT1A6

303 26 105 86 173 52 193



Arsb

UGT1A6

293 23 65 66 174 42 183


polypeptide A6 UDP


polypeptide A7

UGT1A6

UGT1A7

4926 27 29 55


polypeptide A6 UDP


polypeptide A7

UGT1A6

UGT1A7

10508 25 27 48



UGT1A7 186 78 60 305 20 348


UGT1A7 348 52 31 106 227



UGT1A7 10410 26 26 37



UGT1A7 17473 21 27 37


member A1

Aldh3a1 208 103 569 1058


polypeptide A1



type 2

GSTA2 18140 21 26 37


type 2

GSTA2 113 87 34



Yc2 subunit

GSTA5

890 40 54 32



GSTA4 1952 23 42



GSTA4 2770 29 48








gene exon 9








04 40 04 40 04 40



cytochrome P-450b







(CYP2J3P2)








CYP2C 6286 Agrave49



polypeptide 1



polypeptide 1



polypeptide 1




monooxygenase 1

FMO1 2390 Agrave34







45 alpha

Gadd45a 382 20 33 40 65



45 alpha

Gadd45a 798 26 25 35



45 alpha

Gadd45a 391 49


Lipid metabolism






Acaa1 1079 34 25 53



epidermal

Fabp 1065 42


acyltransferase

Gnpat 488 21








dehydrogenase


Bdh 4825 Agrave21


delta isomerase

Idi1 1727 Agrave25







04 40 04 40 04 40

Lipid metabolism




97Agrave






member 1

Slc10a1

(Ntcp)

11044 Agrave21


(organic anion


Slc21a5

oatp2





N-methyltransferase

PEMT 6315 Agrave27



Enpp2 2917 27 41


pyrophosphatase

phosphodiesterase 2

Enpp2 1615 37 34






transport protein



transport protein



dehydrogenase

G6pd 728 33 35







carboxykinase




Nitrogen metabolism





24Agrave



















04 40 04 40 04 40

Nitrogen metabolism




Bhmt 22602 20



(active)

Dhfr1 2389 22


nucleotidase

Bpnt1 577 31




subunit Va

Cox5a 9753 31

Retinoid metabolism






CES3 2496 Agrave64




Steroid metabolism









gene 2

Sth2 18559 Agrave37


gene 2

Sth2 29775 Agrave36


gene 2

Sth2 11993 Agrave31



complete cds



dehydrogenase

3 beta- and steroid

delta-isomerase

Hsd3b 7019 Agrave24


type 1

Dio 1 826 Agrave43



Hsd17b2 15271 20


(hepatic steroid


CYP2A1 13452 36

L24207 _ i _ at (Ad) L24207 Rattus


6-beta-hydroxylase

(CYP3A1) mRNA

CYP3A1 1659 24


6-beta-hydroxylase

(CYP3A1) mRNA

Cyp3A1 1068 27


(PNCN inducible)


3A poypeptide 3

Cyp3A1

Cyp3a3

6993 25







04 40 04 40 04 40

Kinases


2-epimerase


Uae1 2653 Agrave23

Circadian rhythm




member 4

Slc4a4 493 70



member 1

Slc17a1 915 24 30



member 1

Slc17a1 428 36 56



transporting beta

polypeptide 3

Atp1b3 968 29












member 7




member 1








type 2)



member 1






(YWK-II)

LOC64312 3213 21



carcinoembryonic


adhesion molecule 1

Ceacam1 322 676



adhesion molecule 1

Ceacam1 787 23

U32575 _ at Sec6 Sec6 181 44 62

U32575 _ g _ at Sec6 Sec6 342 20 45 94




23 kDa

Snap23 205 34






04 40 04 40 04 40

Cell cycle




33Agrave

24

RNA processing


Cell signaling




receptor type D



receptor type D

Ptprd 835 Agrave21








Tnfrsf1a 1906 20






Ngfrap1 308



Ralb 282 20












partial cds

HNF3-G 631 Agrave27






Cited2 or

MRG1

864 Agrave36







Heme synthesis
















04 40 04 40 04 40

Immune




Psmb8 3232 Agrave21


antigen gene







s subcomponent

C1s 6862 29







31







3 end of exon 6









protein 3



Igfbp1 516 25 47 29 49

Cytoskeleton


non-muscle

Myh9 1052 Agrave38



isoform of actin





1A1B light chain 3

MPL3 3218 33 26















member 10

Serpina 10

Rasp-1

5175 22






04 40 04 40 04 40




No symbol 661 29


No symbol 888 22






CP-2 804 23 30



induced mRNA





of RGS16

Mir16 1508 21


of RGS16

Mir16 2135 20


monophosphatase 2

Impa2 385 32






No symbol 362 2


norvegicus cDNA



No symbol 323 4




[Rattus norvegicus]





No symbol 113 5





3 gb = AA955983 Ag =

Rn7854len = 542

No symbol 5035 2

U47312 _ s _ at U47312 RNU47312 Rat R2




[Rattus norvegicus]





3 gb = AA875171gi =

2980119 Ag = Rn2814

len = 458

No symbol 863 2





AA817987gi = 2887867 Ag =

Rn23920len = 373








04 40 04 40 04 40


rc _ AA859899 _ at rc _ AA859899 UI-R-E0-




AA859899gi = 2949419 Ag =

Rn810len = 353






No symbol 99 54

ESTs


norvegicus cDNA

643 28


norvegicus cDNA

722 22 20 20


1300 24


norvegicus cDNA

10889 22 26


norvegicus cDNA

268 28


norvegicus cDNA

42918 116 110


norvegicus cDNA

21687 22


norvegicus cDNA

421 22


norvegicus cDNA

2863 34


norvegicus cDNA

1472 20


norvegicus cDNA

553 20


norvegicus cDNA

808 22


norvegicus cDNA

2143 20


norvegicus cDNA

2686 20 41


norvegicus cDNA

550 21


norvegicus cDNA

291 20


norvegicus cDNA

1054 25


479 23


norvegicus cDNA

1380 Agrave22


norvegicus cDNA

1628 Agrave24


norvegicus cDNA

788 21


norvegicus cDNA

875 Agrave23


norvegicus cDNA

593 Agrave35


norvegicus cDNA


































thesis pathway

Detoxification































metabolism









04 40 04 40 04 40

ESTs


norvegicus cDNA

2262 Agrave21


3426Agrave

22


norvegicus cDNA

1340 Agrave21


norvegicus cDNA

6360 Agrave24


norvegicus cDNA

438 23


norvegicus cDNA

391 Agrave39







































































acetyl-CoA














































































































































































exposure (Table 1)














































































treated rats







































maintained




Nitrogen metabolism

















































































































(Ahuja et al 2003)

Heme synthesis
















Cytoskeleton





























Membrane proteins


















al 1979)

Summary






























Acknowledgments









References






17118ndash17124




12 191ndash204












231ndash246







2813ndash2824



10035ndash10038



34280ndash34286







93 31ndash46




11476ndash 11481











39124ndash39132













1159ndash1163


















4556ndash4561














388ndash394




235ndash238



10 187ndash204















300 206ndash212








6228ndash6233












Chem 264 21824ndash 21829





















700ndash712







174ndash181









102ndash108










239ndash247
















31656ndash31660

















1362ndash1365










13 509ndash515


















47 77ndash86


















273 6144ndash6148





240 47ndash55



































1129ndash1142







269 6949ndash6954













31 4737ndash4749
















3727ndash3734


































Chem 272 24488ndash 24493































395ndash402














245ndash250























40 485ndash496


















Table 2




symbol04 40 04 40 04 40






polypeptide 1

CYP1B1 13 2992 250 13559


polypeptide 1

CYP1B1 80 657 3856


polypeptide 1

CYP1B1 76 750 80 4838


polypeptide 1

CYP1B1 94 112


from rat liver

No symbol 269 1807 2945 2153 3048 944 2620




Arsb

UGT1A6

303 26 105 86 173 52 193



Arsb

UGT1A6

293 23 65 66 174 42 183


polypeptide A6 UDP


polypeptide A7

UGT1A6

UGT1A7

4926 27 29 55


polypeptide A6 UDP


polypeptide A7

UGT1A6

UGT1A7

10508 25 27 48



UGT1A7 186 78 60 305 20 348


UGT1A7 348 52 31 106 227



UGT1A7 10410 26 26 37



UGT1A7 17473 21 27 37


member A1

Aldh3a1 208 103 569 1058


polypeptide A1



type 2

GSTA2 18140 21 26 37


type 2

GSTA2 113 87 34



Yc2 subunit

GSTA5

890 40 54 32



GSTA4 1952 23 42



GSTA4 2770 29 48








gene exon 9








04 40 04 40 04 40



cytochrome P-450b







(CYP2J3P2)








CYP2C 6286 Agrave49



polypeptide 1



polypeptide 1



polypeptide 1




monooxygenase 1

FMO1 2390 Agrave34







45 alpha

Gadd45a 382 20 33 40 65



45 alpha

Gadd45a 798 26 25 35



45 alpha

Gadd45a 391 49


Lipid metabolism






Acaa1 1079 34 25 53



epidermal

Fabp 1065 42


acyltransferase

Gnpat 488 21








dehydrogenase


Bdh 4825 Agrave21


delta isomerase

Idi1 1727 Agrave25







04 40 04 40 04 40

Lipid metabolism




97Agrave






member 1

Slc10a1

(Ntcp)

11044 Agrave21


(organic anion


Slc21a5

oatp2





N-methyltransferase

PEMT 6315 Agrave27



Enpp2 2917 27 41


pyrophosphatase

phosphodiesterase 2

Enpp2 1615 37 34






transport protein



transport protein



dehydrogenase

G6pd 728 33 35







carboxykinase




Nitrogen metabolism





24Agrave



















04 40 04 40 04 40

Nitrogen metabolism




Bhmt 22602 20



(active)

Dhfr1 2389 22


nucleotidase

Bpnt1 577 31




subunit Va

Cox5a 9753 31

Retinoid metabolism






CES3 2496 Agrave64




Steroid metabolism









gene 2

Sth2 18559 Agrave37


gene 2

Sth2 29775 Agrave36


gene 2

Sth2 11993 Agrave31



complete cds



dehydrogenase

3 beta- and steroid

delta-isomerase

Hsd3b 7019 Agrave24


type 1

Dio 1 826 Agrave43



Hsd17b2 15271 20


(hepatic steroid


CYP2A1 13452 36

L24207 _ i _ at (Ad) L24207 Rattus


6-beta-hydroxylase

(CYP3A1) mRNA

CYP3A1 1659 24


6-beta-hydroxylase

(CYP3A1) mRNA

Cyp3A1 1068 27


(PNCN inducible)


3A poypeptide 3

Cyp3A1

Cyp3a3

6993 25







04 40 04 40 04 40

Kinases


2-epimerase


Uae1 2653 Agrave23

Circadian rhythm




member 4

Slc4a4 493 70



member 1

Slc17a1 915 24 30



member 1

Slc17a1 428 36 56



transporting beta

polypeptide 3

Atp1b3 968 29












member 7




member 1








type 2)



member 1






(YWK-II)

LOC64312 3213 21



carcinoembryonic


adhesion molecule 1

Ceacam1 322 676



adhesion molecule 1

Ceacam1 787 23

U32575 _ at Sec6 Sec6 181 44 62

U32575 _ g _ at Sec6 Sec6 342 20 45 94




23 kDa

Snap23 205 34






04 40 04 40 04 40

Cell cycle




33Agrave

24

RNA processing


Cell signaling




receptor type D



receptor type D

Ptprd 835 Agrave21








Tnfrsf1a 1906 20






Ngfrap1 308



Ralb 282 20












partial cds

HNF3-G 631 Agrave27






Cited2 or

MRG1

864 Agrave36







Heme synthesis
















04 40 04 40 04 40

Immune




Psmb8 3232 Agrave21


antigen gene







s subcomponent

C1s 6862 29







31







3 end of exon 6









protein 3



Igfbp1 516 25 47 29 49

Cytoskeleton


non-muscle

Myh9 1052 Agrave38



isoform of actin





1A1B light chain 3

MPL3 3218 33 26















member 10

Serpina 10

Rasp-1

5175 22






04 40 04 40 04 40




No symbol 661 29


No symbol 888 22






CP-2 804 23 30



induced mRNA





of RGS16

Mir16 1508 21


of RGS16

Mir16 2135 20


monophosphatase 2

Impa2 385 32






No symbol 362 2


norvegicus cDNA



No symbol 323 4




[Rattus norvegicus]





No symbol 113 5





3 gb = AA955983 Ag =

Rn7854len = 542

No symbol 5035 2

U47312 _ s _ at U47312 RNU47312 Rat R2




[Rattus norvegicus]





3 gb = AA875171gi =

2980119 Ag = Rn2814

len = 458

No symbol 863 2





AA817987gi = 2887867 Ag =

Rn23920len = 373








04 40 04 40 04 40


rc _ AA859899 _ at rc _ AA859899 UI-R-E0-




AA859899gi = 2949419 Ag =

Rn810len = 353






No symbol 99 54

ESTs


norvegicus cDNA

643 28


norvegicus cDNA

722 22 20 20


1300 24


norvegicus cDNA

10889 22 26


norvegicus cDNA

268 28


norvegicus cDNA

42918 116 110


norvegicus cDNA

21687 22


norvegicus cDNA

421 22


norvegicus cDNA

2863 34


norvegicus cDNA

1472 20


norvegicus cDNA

553 20


norvegicus cDNA

808 22


norvegicus cDNA

2143 20


norvegicus cDNA

2686 20 41


norvegicus cDNA

550 21


norvegicus cDNA

291 20


norvegicus cDNA

1054 25


479 23


norvegicus cDNA

1380 Agrave22


norvegicus cDNA

1628 Agrave24


norvegicus cDNA

788 21


norvegicus cDNA

875 Agrave23


norvegicus cDNA

593 Agrave35


norvegicus cDNA


































thesis pathway

Detoxification































metabolism









04 40 04 40 04 40

ESTs


norvegicus cDNA

2262 Agrave21


3426Agrave

22


norvegicus cDNA

1340 Agrave21


norvegicus cDNA

6360 Agrave24


norvegicus cDNA

438 23


norvegicus cDNA

391 Agrave39







































































acetyl-CoA














































































































































































exposure (Table 1)














































































treated rats







































maintained




Nitrogen metabolism

















































































































(Ahuja et al 2003)

Heme synthesis
















Cytoskeleton





























Membrane proteins


















al 1979)

Summary






























Acknowledgments









References






17118ndash17124




12 191ndash204












231ndash246







2813ndash2824



10035ndash10038



34280ndash34286







93 31ndash46




11476ndash 11481











39124ndash39132













1159ndash1163


















4556ndash4561














388ndash394




235ndash238



10 187ndash204















300 206ndash212








6228ndash6233












Chem 264 21824ndash 21829





















700ndash712







174ndash181









102ndash108










239ndash247
















31656ndash31660

















1362ndash1365










13 509ndash515


















47 77ndash86


















273 6144ndash6148





240 47ndash55



































1129ndash1142







269 6949ndash6954













31 4737ndash4749
















3727ndash3734


































Chem 272 24488ndash 24493































395ndash402














245ndash250























40 485ndash496




















04 40 04 40 04 40



cytochrome P-450b







(CYP2J3P2)








CYP2C 6286 Agrave49



polypeptide 1



polypeptide 1



polypeptide 1




monooxygenase 1

FMO1 2390 Agrave34







45 alpha

Gadd45a 382 20 33 40 65



45 alpha

Gadd45a 798 26 25 35



45 alpha

Gadd45a 391 49


Lipid metabolism






Acaa1 1079 34 25 53



epidermal

Fabp 1065 42


acyltransferase

Gnpat 488 21








dehydrogenase


Bdh 4825 Agrave21


delta isomerase

Idi1 1727 Agrave25







04 40 04 40 04 40

Lipid metabolism




97Agrave






member 1

Slc10a1

(Ntcp)

11044 Agrave21


(organic anion


Slc21a5

oatp2





N-methyltransferase

PEMT 6315 Agrave27



Enpp2 2917 27 41


pyrophosphatase

phosphodiesterase 2

Enpp2 1615 37 34






transport protein



transport protein



dehydrogenase

G6pd 728 33 35







carboxykinase




Nitrogen metabolism





24Agrave



















04 40 04 40 04 40

Nitrogen metabolism




Bhmt 22602 20



(active)

Dhfr1 2389 22


nucleotidase

Bpnt1 577 31




subunit Va

Cox5a 9753 31

Retinoid metabolism






CES3 2496 Agrave64




Steroid metabolism









gene 2

Sth2 18559 Agrave37


gene 2

Sth2 29775 Agrave36


gene 2

Sth2 11993 Agrave31



complete cds



dehydrogenase

3 beta- and steroid

delta-isomerase

Hsd3b 7019 Agrave24


type 1

Dio 1 826 Agrave43



Hsd17b2 15271 20


(hepatic steroid


CYP2A1 13452 36

L24207 _ i _ at (Ad) L24207 Rattus


6-beta-hydroxylase

(CYP3A1) mRNA

CYP3A1 1659 24


6-beta-hydroxylase

(CYP3A1) mRNA

Cyp3A1 1068 27


(PNCN inducible)


3A poypeptide 3

Cyp3A1

Cyp3a3

6993 25







04 40 04 40 04 40

Kinases


2-epimerase


Uae1 2653 Agrave23

Circadian rhythm




member 4

Slc4a4 493 70



member 1

Slc17a1 915 24 30



member 1

Slc17a1 428 36 56



transporting beta

polypeptide 3

Atp1b3 968 29












member 7




member 1








type 2)



member 1






(YWK-II)

LOC64312 3213 21



carcinoembryonic


adhesion molecule 1

Ceacam1 322 676



adhesion molecule 1

Ceacam1 787 23

U32575 _ at Sec6 Sec6 181 44 62

U32575 _ g _ at Sec6 Sec6 342 20 45 94




23 kDa

Snap23 205 34






04 40 04 40 04 40

Cell cycle




33Agrave

24

RNA processing


Cell signaling




receptor type D



receptor type D

Ptprd 835 Agrave21








Tnfrsf1a 1906 20






Ngfrap1 308



Ralb 282 20












partial cds

HNF3-G 631 Agrave27






Cited2 or

MRG1

864 Agrave36







Heme synthesis
















04 40 04 40 04 40

Immune




Psmb8 3232 Agrave21


antigen gene







s subcomponent

C1s 6862 29







31







3 end of exon 6









protein 3



Igfbp1 516 25 47 29 49

Cytoskeleton


non-muscle

Myh9 1052 Agrave38



isoform of actin





1A1B light chain 3

MPL3 3218 33 26















member 10

Serpina 10

Rasp-1

5175 22






04 40 04 40 04 40




No symbol 661 29


No symbol 888 22






CP-2 804 23 30



induced mRNA





of RGS16

Mir16 1508 21


of RGS16

Mir16 2135 20


monophosphatase 2

Impa2 385 32






No symbol 362 2


norvegicus cDNA



No symbol 323 4




[Rattus norvegicus]





No symbol 113 5





3 gb = AA955983 Ag =

Rn7854len = 542

No symbol 5035 2

U47312 _ s _ at U47312 RNU47312 Rat R2




[Rattus norvegicus]





3 gb = AA875171gi =

2980119 Ag = Rn2814

len = 458

No symbol 863 2





AA817987gi = 2887867 Ag =

Rn23920len = 373








04 40 04 40 04 40


rc _ AA859899 _ at rc _ AA859899 UI-R-E0-




AA859899gi = 2949419 Ag =

Rn810len = 353






No symbol 99 54

ESTs


norvegicus cDNA

643 28


norvegicus cDNA

722 22 20 20


1300 24


norvegicus cDNA

10889 22 26


norvegicus cDNA

268 28


norvegicus cDNA

42918 116 110


norvegicus cDNA

21687 22


norvegicus cDNA

421 22


norvegicus cDNA

2863 34


norvegicus cDNA

1472 20


norvegicus cDNA

553 20


norvegicus cDNA

808 22


norvegicus cDNA

2143 20


norvegicus cDNA

2686 20 41


norvegicus cDNA

550 21


norvegicus cDNA

291 20


norvegicus cDNA

1054 25


479 23


norvegicus cDNA

1380 Agrave22


norvegicus cDNA

1628 Agrave24


norvegicus cDNA

788 21


norvegicus cDNA

875 Agrave23


norvegicus cDNA

593 Agrave35


norvegicus cDNA


































thesis pathway

Detoxification































metabolism









04 40 04 40 04 40

ESTs


norvegicus cDNA

2262 Agrave21


3426Agrave

22


norvegicus cDNA

1340 Agrave21


norvegicus cDNA

6360 Agrave24


norvegicus cDNA

438 23


norvegicus cDNA

391 Agrave39







































































acetyl-CoA














































































































































































exposure (Table 1)














































































treated rats







































maintained




Nitrogen metabolism

















































































































(Ahuja et al 2003)

Heme synthesis
















Cytoskeleton





























Membrane proteins


















al 1979)

Summary






























Acknowledgments









References






17118ndash17124




12 191ndash204












231ndash246







2813ndash2824



10035ndash10038



34280ndash34286







93 31ndash46




11476ndash 11481











39124ndash39132













1159ndash1163


















4556ndash4561














388ndash394




235ndash238



10 187ndash204















300 206ndash212








6228ndash6233












Chem 264 21824ndash 21829





















700ndash712







174ndash181









102ndash108










239ndash247
















31656ndash31660

















1362ndash1365










13 509ndash515


















47 77ndash86


















273 6144ndash6148





240 47ndash55



































1129ndash1142







269 6949ndash6954













31 4737ndash4749
















3727ndash3734


































Chem 272 24488ndash 24493































395ndash402














245ndash250























40 485ndash496





















04 40 04 40 04 40

Lipid metabolism




97Agrave






member 1

Slc10a1

(Ntcp)

11044 Agrave21


(organic anion


Slc21a5

oatp2





N-methyltransferase

PEMT 6315 Agrave27



Enpp2 2917 27 41


pyrophosphatase

phosphodiesterase 2

Enpp2 1615 37 34






transport protein



transport protein



dehydrogenase

G6pd 728 33 35







carboxykinase




Nitrogen metabolism





24Agrave



















04 40 04 40 04 40

Nitrogen metabolism




Bhmt 22602 20



(active)

Dhfr1 2389 22


nucleotidase

Bpnt1 577 31




subunit Va

Cox5a 9753 31

Retinoid metabolism






CES3 2496 Agrave64




Steroid metabolism









gene 2

Sth2 18559 Agrave37


gene 2

Sth2 29775 Agrave36


gene 2

Sth2 11993 Agrave31



complete cds



dehydrogenase

3 beta- and steroid

delta-isomerase

Hsd3b 7019 Agrave24


type 1

Dio 1 826 Agrave43



Hsd17b2 15271 20


(hepatic steroid


CYP2A1 13452 36

L24207 _ i _ at (Ad) L24207 Rattus


6-beta-hydroxylase

(CYP3A1) mRNA

CYP3A1 1659 24


6-beta-hydroxylase

(CYP3A1) mRNA

Cyp3A1 1068 27


(PNCN inducible)


3A poypeptide 3

Cyp3A1

Cyp3a3

6993 25







04 40 04 40 04 40

Kinases


2-epimerase


Uae1 2653 Agrave23

Circadian rhythm




member 4

Slc4a4 493 70



member 1

Slc17a1 915 24 30



member 1

Slc17a1 428 36 56



transporting beta

polypeptide 3

Atp1b3 968 29












member 7




member 1








type 2)



member 1






(YWK-II)

LOC64312 3213 21



carcinoembryonic


adhesion molecule 1

Ceacam1 322 676



adhesion molecule 1

Ceacam1 787 23

U32575 _ at Sec6 Sec6 181 44 62

U32575 _ g _ at Sec6 Sec6 342 20 45 94




23 kDa

Snap23 205 34






04 40 04 40 04 40

Cell cycle




33Agrave

24

RNA processing


Cell signaling




receptor type D



receptor type D

Ptprd 835 Agrave21








Tnfrsf1a 1906 20






Ngfrap1 308



Ralb 282 20












partial cds

HNF3-G 631 Agrave27






Cited2 or

MRG1

864 Agrave36







Heme synthesis
















04 40 04 40 04 40

Immune




Psmb8 3232 Agrave21


antigen gene







s subcomponent

C1s 6862 29







31







3 end of exon 6









protein 3



Igfbp1 516 25 47 29 49

Cytoskeleton


non-muscle

Myh9 1052 Agrave38



isoform of actin





1A1B light chain 3

MPL3 3218 33 26















member 10

Serpina 10

Rasp-1

5175 22






04 40 04 40 04 40




No symbol 661 29


No symbol 888 22






CP-2 804 23 30



induced mRNA





of RGS16

Mir16 1508 21


of RGS16

Mir16 2135 20


monophosphatase 2

Impa2 385 32






No symbol 362 2


norvegicus cDNA



No symbol 323 4




[Rattus norvegicus]





No symbol 113 5





3 gb = AA955983 Ag =

Rn7854len = 542

No symbol 5035 2

U47312 _ s _ at U47312 RNU47312 Rat R2




[Rattus norvegicus]





3 gb = AA875171gi =

2980119 Ag = Rn2814

len = 458

No symbol 863 2





AA817987gi = 2887867 Ag =

Rn23920len = 373








04 40 04 40 04 40


rc _ AA859899 _ at rc _ AA859899 UI-R-E0-




AA859899gi = 2949419 Ag =

Rn810len = 353






No symbol 99 54

ESTs


norvegicus cDNA

643 28


norvegicus cDNA

722 22 20 20


1300 24


norvegicus cDNA

10889 22 26


norvegicus cDNA

268 28


norvegicus cDNA

42918 116 110


norvegicus cDNA

21687 22


norvegicus cDNA

421 22


norvegicus cDNA

2863 34


norvegicus cDNA

1472 20


norvegicus cDNA

553 20


norvegicus cDNA

808 22


norvegicus cDNA

2143 20


norvegicus cDNA

2686 20 41


norvegicus cDNA

550 21


norvegicus cDNA

291 20


norvegicus cDNA

1054 25


479 23


norvegicus cDNA

1380 Agrave22


norvegicus cDNA

1628 Agrave24


norvegicus cDNA

788 21


norvegicus cDNA

875 Agrave23


norvegicus cDNA

593 Agrave35


norvegicus cDNA


































thesis pathway

Detoxification































metabolism









04 40 04 40 04 40

ESTs


norvegicus cDNA

2262 Agrave21


3426Agrave

22


norvegicus cDNA

1340 Agrave21


norvegicus cDNA

6360 Agrave24


norvegicus cDNA

438 23


norvegicus cDNA

391 Agrave39







































































acetyl-CoA














































































































































































exposure (Table 1)














































































treated rats







































maintained




Nitrogen metabolism

















































































































(Ahuja et al 2003)

Heme synthesis
















Cytoskeleton





























Membrane proteins


















al 1979)

Summary






























Acknowledgments









References






17118ndash17124




12 191ndash204












231ndash246







2813ndash2824



10035ndash10038



34280ndash34286







93 31ndash46




11476ndash 11481











39124ndash39132













1159ndash1163


















4556ndash4561














388ndash394




235ndash238



10 187ndash204















300 206ndash212








6228ndash6233












Chem 264 21824ndash 21829





















700ndash712







174ndash181









102ndash108










239ndash247
















31656ndash31660

















1362ndash1365










13 509ndash515


















47 77ndash86


















273 6144ndash6148





240 47ndash55



































1129ndash1142







269 6949ndash6954













31 4737ndash4749
















3727ndash3734


































Chem 272 24488ndash 24493































395ndash402














245ndash250























40 485ndash496




















04 40 04 40 04 40

Nitrogen metabolism




Bhmt 22602 20



(active)

Dhfr1 2389 22


nucleotidase

Bpnt1 577 31




subunit Va

Cox5a 9753 31

Retinoid metabolism






CES3 2496 Agrave64




Steroid metabolism









gene 2

Sth2 18559 Agrave37


gene 2

Sth2 29775 Agrave36


gene 2

Sth2 11993 Agrave31



complete cds



dehydrogenase

3 beta- and steroid

delta-isomerase

Hsd3b 7019 Agrave24


type 1

Dio 1 826 Agrave43



Hsd17b2 15271 20


(hepatic steroid


CYP2A1 13452 36

L24207 _ i _ at (Ad) L24207 Rattus


6-beta-hydroxylase

(CYP3A1) mRNA

CYP3A1 1659 24


6-beta-hydroxylase

(CYP3A1) mRNA

Cyp3A1 1068 27


(PNCN inducible)


3A poypeptide 3

Cyp3A1

Cyp3a3

6993 25







04 40 04 40 04 40

Kinases


2-epimerase


Uae1 2653 Agrave23

Circadian rhythm




member 4

Slc4a4 493 70



member 1

Slc17a1 915 24 30



member 1

Slc17a1 428 36 56



transporting beta

polypeptide 3

Atp1b3 968 29












member 7




member 1








type 2)



member 1






(YWK-II)

LOC64312 3213 21



carcinoembryonic


adhesion molecule 1

Ceacam1 322 676



adhesion molecule 1

Ceacam1 787 23

U32575 _ at Sec6 Sec6 181 44 62

U32575 _ g _ at Sec6 Sec6 342 20 45 94




23 kDa

Snap23 205 34






04 40 04 40 04 40

Cell cycle




33Agrave

24

RNA processing


Cell signaling




receptor type D



receptor type D

Ptprd 835 Agrave21








Tnfrsf1a 1906 20






Ngfrap1 308



Ralb 282 20












partial cds

HNF3-G 631 Agrave27






Cited2 or

MRG1

864 Agrave36







Heme synthesis
















04 40 04 40 04 40

Immune




Psmb8 3232 Agrave21


antigen gene







s subcomponent

C1s 6862 29







31







3 end of exon 6









protein 3



Igfbp1 516 25 47 29 49

Cytoskeleton


non-muscle

Myh9 1052 Agrave38



isoform of actin





1A1B light chain 3

MPL3 3218 33 26















member 10

Serpina 10

Rasp-1

5175 22






04 40 04 40 04 40




No symbol 661 29


No symbol 888 22






CP-2 804 23 30



induced mRNA





of RGS16

Mir16 1508 21


of RGS16

Mir16 2135 20


monophosphatase 2

Impa2 385 32






No symbol 362 2


norvegicus cDNA



No symbol 323 4




[Rattus norvegicus]





No symbol 113 5





3 gb = AA955983 Ag =

Rn7854len = 542

No symbol 5035 2

U47312 _ s _ at U47312 RNU47312 Rat R2




[Rattus norvegicus]





3 gb = AA875171gi =

2980119 Ag = Rn2814

len = 458

No symbol 863 2





AA817987gi = 2887867 Ag =

Rn23920len = 373








04 40 04 40 04 40


rc _ AA859899 _ at rc _ AA859899 UI-R-E0-




AA859899gi = 2949419 Ag =

Rn810len = 353






No symbol 99 54

ESTs


norvegicus cDNA

643 28


norvegicus cDNA

722 22 20 20


1300 24


norvegicus cDNA

10889 22 26


norvegicus cDNA

268 28


norvegicus cDNA

42918 116 110


norvegicus cDNA

21687 22


norvegicus cDNA

421 22


norvegicus cDNA

2863 34


norvegicus cDNA

1472 20


norvegicus cDNA

553 20


norvegicus cDNA

808 22


norvegicus cDNA

2143 20


norvegicus cDNA

2686 20 41


norvegicus cDNA

550 21


norvegicus cDNA

291 20


norvegicus cDNA

1054 25


479 23


norvegicus cDNA

1380 Agrave22


norvegicus cDNA

1628 Agrave24


norvegicus cDNA

788 21


norvegicus cDNA

875 Agrave23


norvegicus cDNA

593 Agrave35


norvegicus cDNA


































thesis pathway

Detoxification































metabolism









04 40 04 40 04 40

ESTs


norvegicus cDNA

2262 Agrave21


3426Agrave

22


norvegicus cDNA

1340 Agrave21


norvegicus cDNA

6360 Agrave24


norvegicus cDNA

438 23


norvegicus cDNA

391 Agrave39







































































acetyl-CoA














































































































































































exposure (Table 1)














































































treated rats







































maintained




Nitrogen metabolism

















































































































(Ahuja et al 2003)

Heme synthesis
















Cytoskeleton





























Membrane proteins


















al 1979)

Summary






























Acknowledgments









References






17118ndash17124




12 191ndash204












231ndash246







2813ndash2824



10035ndash10038



34280ndash34286







93 31ndash46




11476ndash 11481











39124ndash39132













1159ndash1163


















4556ndash4561














388ndash394




235ndash238



10 187ndash204















300 206ndash212








6228ndash6233












Chem 264 21824ndash 21829





















700ndash712







174ndash181









102ndash108










239ndash247
















31656ndash31660

















1362ndash1365










13 509ndash515


















47 77ndash86


















273 6144ndash6148





240 47ndash55



































1129ndash1142







269 6949ndash6954













31 4737ndash4749
















3727ndash3734


































Chem 272 24488ndash 24493































395ndash402














245ndash250























40 485ndash496





















04 40 04 40 04 40

Kinases


2-epimerase


Uae1 2653 Agrave23

Circadian rhythm




member 4

Slc4a4 493 70



member 1

Slc17a1 915 24 30



member 1

Slc17a1 428 36 56



transporting beta

polypeptide 3

Atp1b3 968 29












member 7




member 1








type 2)



member 1






(YWK-II)

LOC64312 3213 21



carcinoembryonic


adhesion molecule 1

Ceacam1 322 676



adhesion molecule 1

Ceacam1 787 23

U32575 _ at Sec6 Sec6 181 44 62

U32575 _ g _ at Sec6 Sec6 342 20 45 94




23 kDa

Snap23 205 34






04 40 04 40 04 40

Cell cycle




33Agrave

24

RNA processing


Cell signaling




receptor type D



receptor type D

Ptprd 835 Agrave21








Tnfrsf1a 1906 20






Ngfrap1 308



Ralb 282 20












partial cds

HNF3-G 631 Agrave27






Cited2 or

MRG1

864 Agrave36







Heme synthesis
















04 40 04 40 04 40

Immune




Psmb8 3232 Agrave21


antigen gene







s subcomponent

C1s 6862 29







31







3 end of exon 6









protein 3



Igfbp1 516 25 47 29 49

Cytoskeleton


non-muscle

Myh9 1052 Agrave38



isoform of actin





1A1B light chain 3

MPL3 3218 33 26















member 10

Serpina 10

Rasp-1

5175 22






04 40 04 40 04 40




No symbol 661 29


No symbol 888 22






CP-2 804 23 30



induced mRNA





of RGS16

Mir16 1508 21


of RGS16

Mir16 2135 20


monophosphatase 2

Impa2 385 32






No symbol 362 2


norvegicus cDNA



No symbol 323 4




[Rattus norvegicus]





No symbol 113 5





3 gb = AA955983 Ag =

Rn7854len = 542

No symbol 5035 2

U47312 _ s _ at U47312 RNU47312 Rat R2




[Rattus norvegicus]





3 gb = AA875171gi =

2980119 Ag = Rn2814

len = 458

No symbol 863 2





AA817987gi = 2887867 Ag =

Rn23920len = 373








04 40 04 40 04 40


rc _ AA859899 _ at rc _ AA859899 UI-R-E0-




AA859899gi = 2949419 Ag =

Rn810len = 353






No symbol 99 54

ESTs


norvegicus cDNA

643 28


norvegicus cDNA

722 22 20 20


1300 24


norvegicus cDNA

10889 22 26


norvegicus cDNA

268 28


norvegicus cDNA

42918 116 110


norvegicus cDNA

21687 22


norvegicus cDNA

421 22


norvegicus cDNA

2863 34


norvegicus cDNA

1472 20


norvegicus cDNA

553 20


norvegicus cDNA

808 22


norvegicus cDNA

2143 20


norvegicus cDNA

2686 20 41


norvegicus cDNA

550 21


norvegicus cDNA

291 20


norvegicus cDNA

1054 25


479 23


norvegicus cDNA

1380 Agrave22


norvegicus cDNA

1628 Agrave24


norvegicus cDNA

788 21


norvegicus cDNA

875 Agrave23


norvegicus cDNA

593 Agrave35


norvegicus cDNA


































thesis pathway

Detoxification































metabolism









04 40 04 40 04 40

ESTs


norvegicus cDNA

2262 Agrave21


3426Agrave

22


norvegicus cDNA

1340 Agrave21


norvegicus cDNA

6360 Agrave24


norvegicus cDNA

438 23


norvegicus cDNA

391 Agrave39







































































acetyl-CoA














































































































































































exposure (Table 1)














































































treated rats







































maintained




Nitrogen metabolism

















































































































(Ahuja et al 2003)

Heme synthesis
















Cytoskeleton





























Membrane proteins


















al 1979)

Summary






























Acknowledgments









References






17118ndash17124




12 191ndash204












231ndash246







2813ndash2824



10035ndash10038



34280ndash34286







93 31ndash46




11476ndash 11481











39124ndash39132













1159ndash1163


















4556ndash4561














388ndash394




235ndash238



10 187ndash204















300 206ndash212








6228ndash6233












Chem 264 21824ndash 21829





















700ndash712







174ndash181









102ndash108










239ndash247
















31656ndash31660

















1362ndash1365










13 509ndash515


















47 77ndash86


















273 6144ndash6148





240 47ndash55



































1129ndash1142







269 6949ndash6954













31 4737ndash4749
















3727ndash3734


































Chem 272 24488ndash 24493































395ndash402














245ndash250























40 485ndash496




















04 40 04 40 04 40

Cell cycle




33Agrave

24

RNA processing


Cell signaling




receptor type D



receptor type D

Ptprd 835 Agrave21








Tnfrsf1a 1906 20






Ngfrap1 308



Ralb 282 20












partial cds

HNF3-G 631 Agrave27






Cited2 or

MRG1

864 Agrave36







Heme synthesis
















04 40 04 40 04 40

Immune




Psmb8 3232 Agrave21


antigen gene







s subcomponent

C1s 6862 29







31







3 end of exon 6









protein 3



Igfbp1 516 25 47 29 49

Cytoskeleton


non-muscle

Myh9 1052 Agrave38



isoform of actin





1A1B light chain 3

MPL3 3218 33 26















member 10

Serpina 10

Rasp-1

5175 22






04 40 04 40 04 40




No symbol 661 29


No symbol 888 22






CP-2 804 23 30



induced mRNA





of RGS16

Mir16 1508 21


of RGS16

Mir16 2135 20


monophosphatase 2

Impa2 385 32






No symbol 362 2


norvegicus cDNA



No symbol 323 4




[Rattus norvegicus]





No symbol 113 5





3 gb = AA955983 Ag =

Rn7854len = 542

No symbol 5035 2

U47312 _ s _ at U47312 RNU47312 Rat R2




[Rattus norvegicus]





3 gb = AA875171gi =

2980119 Ag = Rn2814

len = 458

No symbol 863 2





AA817987gi = 2887867 Ag =

Rn23920len = 373








04 40 04 40 04 40


rc _ AA859899 _ at rc _ AA859899 UI-R-E0-




AA859899gi = 2949419 Ag =

Rn810len = 353






No symbol 99 54

ESTs


norvegicus cDNA

643 28


norvegicus cDNA

722 22 20 20


1300 24


norvegicus cDNA

10889 22 26


norvegicus cDNA

268 28


norvegicus cDNA

42918 116 110


norvegicus cDNA

21687 22


norvegicus cDNA

421 22


norvegicus cDNA

2863 34


norvegicus cDNA

1472 20


norvegicus cDNA

553 20


norvegicus cDNA

808 22


norvegicus cDNA

2143 20


norvegicus cDNA

2686 20 41


norvegicus cDNA

550 21


norvegicus cDNA

291 20


norvegicus cDNA

1054 25


479 23


norvegicus cDNA

1380 Agrave22


norvegicus cDNA

1628 Agrave24


norvegicus cDNA

788 21


norvegicus cDNA

875 Agrave23


norvegicus cDNA

593 Agrave35


norvegicus cDNA


































thesis pathway

Detoxification































metabolism









04 40 04 40 04 40

ESTs


norvegicus cDNA

2262 Agrave21


3426Agrave

22


norvegicus cDNA

1340 Agrave21


norvegicus cDNA

6360 Agrave24


norvegicus cDNA

438 23


norvegicus cDNA

391 Agrave39







































































acetyl-CoA














































































































































































exposure (Table 1)














































































treated rats







































maintained




Nitrogen metabolism

















































































































(Ahuja et al 2003)

Heme synthesis
















Cytoskeleton





























Membrane proteins


















al 1979)

Summary






























Acknowledgments









References






17118ndash17124




12 191ndash204












231ndash246







2813ndash2824



10035ndash10038



34280ndash34286







93 31ndash46




11476ndash 11481











39124ndash39132













1159ndash1163


















4556ndash4561














388ndash394




235ndash238



10 187ndash204















300 206ndash212








6228ndash6233












Chem 264 21824ndash 21829





















700ndash712







174ndash181









102ndash108










239ndash247
















31656ndash31660

















1362ndash1365










13 509ndash515


















47 77ndash86


















273 6144ndash6148





240 47ndash55



































1129ndash1142







269 6949ndash6954













31 4737ndash4749
















3727ndash3734


































Chem 272 24488ndash 24493































395ndash402














245ndash250























40 485ndash496





















04 40 04 40 04 40

Immune




Psmb8 3232 Agrave21


antigen gene







s subcomponent

C1s 6862 29







31







3 end of exon 6









protein 3



Igfbp1 516 25 47 29 49

Cytoskeleton


non-muscle

Myh9 1052 Agrave38



isoform of actin





1A1B light chain 3

MPL3 3218 33 26















member 10

Serpina 10

Rasp-1

5175 22






04 40 04 40 04 40




No symbol 661 29


No symbol 888 22






CP-2 804 23 30



induced mRNA





of RGS16

Mir16 1508 21


of RGS16

Mir16 2135 20


monophosphatase 2

Impa2 385 32






No symbol 362 2


norvegicus cDNA



No symbol 323 4




[Rattus norvegicus]





No symbol 113 5





3 gb = AA955983 Ag =

Rn7854len = 542

No symbol 5035 2

U47312 _ s _ at U47312 RNU47312 Rat R2




[Rattus norvegicus]





3 gb = AA875171gi =

2980119 Ag = Rn2814

len = 458

No symbol 863 2





AA817987gi = 2887867 Ag =

Rn23920len = 373








04 40 04 40 04 40


rc _ AA859899 _ at rc _ AA859899 UI-R-E0-




AA859899gi = 2949419 Ag =

Rn810len = 353






No symbol 99 54

ESTs


norvegicus cDNA

643 28


norvegicus cDNA

722 22 20 20


1300 24


norvegicus cDNA

10889 22 26


norvegicus cDNA

268 28


norvegicus cDNA

42918 116 110


norvegicus cDNA

21687 22


norvegicus cDNA

421 22


norvegicus cDNA

2863 34


norvegicus cDNA

1472 20


norvegicus cDNA

553 20


norvegicus cDNA

808 22


norvegicus cDNA

2143 20


norvegicus cDNA

2686 20 41


norvegicus cDNA

550 21


norvegicus cDNA

291 20


norvegicus cDNA

1054 25


479 23


norvegicus cDNA

1380 Agrave22


norvegicus cDNA

1628 Agrave24


norvegicus cDNA

788 21


norvegicus cDNA

875 Agrave23


norvegicus cDNA

593 Agrave35


norvegicus cDNA


































thesis pathway

Detoxification































metabolism









04 40 04 40 04 40

ESTs


norvegicus cDNA

2262 Agrave21


3426Agrave

22


norvegicus cDNA

1340 Agrave21


norvegicus cDNA

6360 Agrave24


norvegicus cDNA

438 23


norvegicus cDNA

391 Agrave39







































































acetyl-CoA














































































































































































exposure (Table 1)














































































treated rats







































maintained




Nitrogen metabolism

















































































































(Ahuja et al 2003)

Heme synthesis
















Cytoskeleton





























Membrane proteins


















al 1979)

Summary






























Acknowledgments









References






17118ndash17124




12 191ndash204












231ndash246







2813ndash2824



10035ndash10038



34280ndash34286







93 31ndash46




11476ndash 11481











39124ndash39132













1159ndash1163


















4556ndash4561














388ndash394




235ndash238



10 187ndash204















300 206ndash212








6228ndash6233












Chem 264 21824ndash 21829





















700ndash712







174ndash181









102ndash108










239ndash247
















31656ndash31660

















1362ndash1365










13 509ndash515


















47 77ndash86


















273 6144ndash6148





240 47ndash55



































1129ndash1142







269 6949ndash6954













31 4737ndash4749
















3727ndash3734


































Chem 272 24488ndash 24493































395ndash402














245ndash250























40 485ndash496




















04 40 04 40 04 40




No symbol 661 29


No symbol 888 22






CP-2 804 23 30



induced mRNA





of RGS16

Mir16 1508 21


of RGS16

Mir16 2135 20


monophosphatase 2

Impa2 385 32






No symbol 362 2


norvegicus cDNA



No symbol 323 4




[Rattus norvegicus]





No symbol 113 5





3 gb = AA955983 Ag =

Rn7854len = 542

No symbol 5035 2

U47312 _ s _ at U47312 RNU47312 Rat R2




[Rattus norvegicus]





3 gb = AA875171gi =

2980119 Ag = Rn2814

len = 458

No symbol 863 2





AA817987gi = 2887867 Ag =

Rn23920len = 373








04 40 04 40 04 40


rc _ AA859899 _ at rc _ AA859899 UI-R-E0-




AA859899gi = 2949419 Ag =

Rn810len = 353






No symbol 99 54

ESTs


norvegicus cDNA

643 28


norvegicus cDNA

722 22 20 20


1300 24


norvegicus cDNA

10889 22 26


norvegicus cDNA

268 28


norvegicus cDNA

42918 116 110


norvegicus cDNA

21687 22


norvegicus cDNA

421 22


norvegicus cDNA

2863 34


norvegicus cDNA

1472 20


norvegicus cDNA

553 20


norvegicus cDNA

808 22


norvegicus cDNA

2143 20


norvegicus cDNA

2686 20 41


norvegicus cDNA

550 21


norvegicus cDNA

291 20


norvegicus cDNA

1054 25


479 23


norvegicus cDNA

1380 Agrave22


norvegicus cDNA

1628 Agrave24


norvegicus cDNA

788 21


norvegicus cDNA

875 Agrave23


norvegicus cDNA

593 Agrave35


norvegicus cDNA


































thesis pathway

Detoxification































metabolism









04 40 04 40 04 40

ESTs


norvegicus cDNA

2262 Agrave21


3426Agrave

22


norvegicus cDNA

1340 Agrave21


norvegicus cDNA

6360 Agrave24


norvegicus cDNA

438 23


norvegicus cDNA

391 Agrave39







































































acetyl-CoA














































































































































































exposure (Table 1)














































































treated rats







































maintained




Nitrogen metabolism

















































































































(Ahuja et al 2003)

Heme synthesis
















Cytoskeleton





























Membrane proteins


















al 1979)

Summary






























Acknowledgments









References






17118ndash17124




12 191ndash204












231ndash246







2813ndash2824



10035ndash10038



34280ndash34286







93 31ndash46




11476ndash 11481











39124ndash39132













1159ndash1163


















4556ndash4561














388ndash394




235ndash238



10 187ndash204















300 206ndash212








6228ndash6233












Chem 264 21824ndash 21829





















700ndash712







174ndash181









102ndash108










239ndash247
















31656ndash31660

















1362ndash1365










13 509ndash515


















47 77ndash86


















273 6144ndash6148





240 47ndash55



































1129ndash1142







269 6949ndash6954













31 4737ndash4749
















3727ndash3734


































Chem 272 24488ndash 24493































395ndash402














245ndash250























40 485ndash496





















04 40 04 40 04 40


rc _ AA859899 _ at rc _ AA859899 UI-R-E0-




AA859899gi = 2949419 Ag =

Rn810len = 353






No symbol 99 54

ESTs


norvegicus cDNA

643 28


norvegicus cDNA

722 22 20 20


1300 24


norvegicus cDNA

10889 22 26


norvegicus cDNA

268 28


norvegicus cDNA

42918 116 110


norvegicus cDNA

21687 22


norvegicus cDNA

421 22


norvegicus cDNA

2863 34


norvegicus cDNA

1472 20


norvegicus cDNA

553 20


norvegicus cDNA

808 22


norvegicus cDNA

2143 20


norvegicus cDNA

2686 20 41


norvegicus cDNA

550 21


norvegicus cDNA

291 20


norvegicus cDNA

1054 25


479 23


norvegicus cDNA

1380 Agrave22


norvegicus cDNA

1628 Agrave24


norvegicus cDNA

788 21


norvegicus cDNA

875 Agrave23


norvegicus cDNA

593 Agrave35


norvegicus cDNA


































thesis pathway

Detoxification































metabolism









04 40 04 40 04 40

ESTs


norvegicus cDNA

2262 Agrave21


3426Agrave

22


norvegicus cDNA

1340 Agrave21


norvegicus cDNA

6360 Agrave24


norvegicus cDNA

438 23


norvegicus cDNA

391 Agrave39







































































acetyl-CoA














































































































































































exposure (Table 1)














































































treated rats







































maintained




Nitrogen metabolism

















































































































(Ahuja et al 2003)

Heme synthesis
















Cytoskeleton





























Membrane proteins


















al 1979)

Summary






























Acknowledgments









References






17118ndash17124




12 191ndash204












231ndash246







2813ndash2824



10035ndash10038



34280ndash34286







93 31ndash46




11476ndash 11481











39124ndash39132













1159ndash1163


















4556ndash4561














388ndash394




235ndash238



10 187ndash204















300 206ndash212








6228ndash6233












Chem 264 21824ndash 21829





















700ndash712







174ndash181









102ndash108










239ndash247
















31656ndash31660

















1362ndash1365










13 509ndash515


















47 77ndash86


















273 6144ndash6148





240 47ndash55



































1129ndash1142







269 6949ndash6954













31 4737ndash4749
















3727ndash3734


































Chem 272 24488ndash 24493































395ndash402














245ndash250























40 485ndash496















































thesis pathway

Detoxification































metabolism









04 40 04 40 04 40

ESTs


norvegicus cDNA

2262 Agrave21


3426Agrave

22


norvegicus cDNA

1340 Agrave21


norvegicus cDNA

6360 Agrave24


norvegicus cDNA

438 23


norvegicus cDNA

391 Agrave39







































































acetyl-CoA














































































































































































exposure (Table 1)














































































treated rats







































maintained




Nitrogen metabolism

















































































































(Ahuja et al 2003)

Heme synthesis
















Cytoskeleton





























Membrane proteins


















al 1979)

Summary






























Acknowledgments









References






17118ndash17124




12 191ndash204












231ndash246







2813ndash2824



10035ndash10038



34280ndash34286







93 31ndash46




11476ndash 11481











39124ndash39132













1159ndash1163


















4556ndash4561














388ndash394




235ndash238



10 187ndash204















300 206ndash212








6228ndash6233












Chem 264 21824ndash 21829





















700ndash712







174ndash181









102ndash108










239ndash247
















31656ndash31660

















1362ndash1365










13 509ndash515


















47 77ndash86


















273 6144ndash6148





240 47ndash55



































1129ndash1142







269 6949ndash6954













31 4737ndash4749
















3727ndash3734


































Chem 272 24488ndash 24493































395ndash402














245ndash250























40 485ndash496


















































































acetyl-CoA














































































































































































exposure (Table 1)














































































treated rats







































maintained




Nitrogen metabolism

















































































































(Ahuja et al 2003)

Heme synthesis
















Cytoskeleton





























Membrane proteins


















al 1979)

Summary






























Acknowledgments









References






17118ndash17124




12 191ndash204












231ndash246







2813ndash2824



10035ndash10038



34280ndash34286







93 31ndash46




11476ndash 11481











39124ndash39132













1159ndash1163


















4556ndash4561














388ndash394




235ndash238



10 187ndash204















300 206ndash212








6228ndash6233












Chem 264 21824ndash 21829





















700ndash712







174ndash181









102ndash108










239ndash247
















31656ndash31660

















1362ndash1365










13 509ndash515


















47 77ndash86


















273 6144ndash6148





240 47ndash55



































1129ndash1142







269 6949ndash6954













31 4737ndash4749
















3727ndash3734


































Chem 272 24488ndash 24493































395ndash402














245ndash250























40 485ndash496





















































































































































exposure (Table 1)














































































treated rats







































maintained




Nitrogen metabolism

















































































































(Ahuja et al 2003)

Heme synthesis
















Cytoskeleton





























Membrane proteins


















al 1979)

Summary






























Acknowledgments









References






17118ndash17124




12 191ndash204












231ndash246







2813ndash2824



10035ndash10038



34280ndash34286







93 31ndash46




11476ndash 11481











39124ndash39132













1159ndash1163


















4556ndash4561














388ndash394




235ndash238



10 187ndash204















300 206ndash212








6228ndash6233












Chem 264 21824ndash 21829





















700ndash712







174ndash181









102ndash108










239ndash247
















31656ndash31660

















1362ndash1365










13 509ndash515


















47 77ndash86


















273 6144ndash6148





240 47ndash55



































1129ndash1142







269 6949ndash6954













31 4737ndash4749
















3727ndash3734


































Chem 272 24488ndash 24493































395ndash402














245ndash250























40 485ndash496









































exposure (Table 1)














































































treated rats







































maintained




Nitrogen metabolism

















































































































(Ahuja et al 2003)

Heme synthesis
















Cytoskeleton





























Membrane proteins


















al 1979)

Summary






























Acknowledgments









References






17118ndash17124




12 191ndash204












231ndash246







2813ndash2824



10035ndash10038



34280ndash34286







93 31ndash46




11476ndash 11481











39124ndash39132













1159ndash1163


















4556ndash4561














388ndash394




235ndash238



10 187ndash204















300 206ndash212








6228ndash6233












Chem 264 21824ndash 21829





















700ndash712







174ndash181









102ndash108










239ndash247
















31656ndash31660

















1362ndash1365










13 509ndash515


















47 77ndash86


















273 6144ndash6148





240 47ndash55



































1129ndash1142







269 6949ndash6954













31 4737ndash4749
















3727ndash3734


































Chem 272 24488ndash 24493































395ndash402














245ndash250























40 485ndash496



















































































treated rats







































maintained




Nitrogen metabolism

















































































































(Ahuja et al 2003)

Heme synthesis
















Cytoskeleton





























Membrane proteins


















al 1979)

Summary






























Acknowledgments









References






17118ndash17124




12 191ndash204












231ndash246







2813ndash2824



10035ndash10038



34280ndash34286







93 31ndash46




11476ndash 11481











39124ndash39132













1159ndash1163


















4556ndash4561














388ndash394




235ndash238



10 187ndash204















300 206ndash212








6228ndash6233












Chem 264 21824ndash 21829





















700ndash712







174ndash181









102ndash108










239ndash247
















31656ndash31660

















1362ndash1365










13 509ndash515


















47 77ndash86


















273 6144ndash6148





240 47ndash55



































1129ndash1142







269 6949ndash6954













31 4737ndash4749
















3727ndash3734


































Chem 272 24488ndash 24493































395ndash402














245ndash250























40 485ndash496


















Nitrogen metabolism

















































































































(Ahuja et al 2003)

Heme synthesis
















Cytoskeleton





























Membrane proteins


















al 1979)

Summary






























Acknowledgments









References






17118ndash17124




12 191ndash204












231ndash246







2813ndash2824



10035ndash10038



34280ndash34286







93 31ndash46




11476ndash 11481











39124ndash39132













1159ndash1163


















4556ndash4561














388ndash394




235ndash238



10 187ndash204















300 206ndash212








6228ndash6233












Chem 264 21824ndash 21829





















700ndash712







174ndash181









102ndash108










239ndash247
















31656ndash31660

















1362ndash1365










13 509ndash515


















47 77ndash86


















273 6144ndash6148





240 47ndash55



































1129ndash1142







269 6949ndash6954













31 4737ndash4749
















3727ndash3734


































Chem 272 24488ndash 24493































395ndash402














245ndash250























40 485ndash496























(Ahuja et al 2003)

Heme synthesis
















Cytoskeleton





























Membrane proteins


















al 1979)

Summary






























Acknowledgments









References






17118ndash17124




12 191ndash204












231ndash246







2813ndash2824



10035ndash10038



34280ndash34286







93 31ndash46




11476ndash 11481











39124ndash39132













1159ndash1163


















4556ndash4561














388ndash394




235ndash238



10 187ndash204















300 206ndash212








6228ndash6233












Chem 264 21824ndash 21829





















700ndash712







174ndash181









102ndash108










239ndash247
















31656ndash31660

















1362ndash1365










13 509ndash515


















47 77ndash86


















273 6144ndash6148





240 47ndash55



































1129ndash1142







269 6949ndash6954













31 4737ndash4749
















3727ndash3734


































Chem 272 24488ndash 24493































395ndash402














245ndash250























40 485ndash496


















Acknowledgments









References






17118ndash17124




12 191ndash204












231ndash246







2813ndash2824



10035ndash10038



34280ndash34286







93 31ndash46




11476ndash 11481











39124ndash39132













1159ndash1163


















4556ndash4561














388ndash394




235ndash238



10 187ndash204















300 206ndash212








6228ndash6233












Chem 264 21824ndash 21829





















700ndash712







174ndash181









102ndash108










239ndash247
















31656ndash31660

















1362ndash1365










13 509ndash515


















47 77ndash86


















273 6144ndash6148





240 47ndash55



































1129ndash1142







269 6949ndash6954













31 4737ndash4749
















3727ndash3734


































Chem 272 24488ndash 24493































395ndash402














245ndash250























40 485ndash496





























300 206ndash212








6228ndash6233












Chem 264 21824ndash 21829





















700ndash712







174ndash181









102ndash108










239ndash247
















31656ndash31660

















1362ndash1365










13 509ndash515


















47 77ndash86


















273 6144ndash6148





240 47ndash55



































1129ndash1142







269 6949ndash6954













31 4737ndash4749
















3727ndash3734


































Chem 272 24488ndash 24493































395ndash402














245ndash250























40 485ndash496




























47 77ndash86


















273 6144ndash6148





240 47ndash55



































1129ndash1142







269 6949ndash6954













31 4737ndash4749
















3727ndash3734


































Chem 272 24488ndash 24493































395ndash402














245ndash250























40 485ndash496



























Chem 272 24488ndash 24493































395ndash402














245ndash250























40 485ndash496
















Documents

2005 Micro Array Study