2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alters the mRNA expression of critical genes associated...

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Toxicology and Applied Pharm

2,3,7,8-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 Fletchera, David Wahlstrfma, Rebecca Lundberga, Charlotte B. Nilssonb,

Kerstin C. Nilssonb, Kenneth Stocklingb, Heike Hellmoldb, Helen H3kanssona,*

aInstitute of Environmental Medicine, Karolinska Institutet, Nobels vag 13, P.O. Box 210, SE-171 77 Stockholm, SwedenbSafety Assessment, Astra Zeneca R&D Sodertalje, SE-151 85 Sodertalje, Sweden

Received 15 October 2004; accepted 3 December 2004

Available online 19 February 2005

Abstract

2,3,7,8-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 Sprague–Dawley rats were administered single doses of 0.4 Ag/kg bw or 40 Ag/kg 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 (UGT1A6/7), and metallothionein 1.

However, 0.4 Ag/kg 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 and

associated 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-

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

doi:10.1016/j.taap.2004.12.003

* Corresponding author. Fax: +46 8 34 38 49.

E-mail address: [email protected] (H. H3kansson).

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

acology 207 (2005) 1–24

N. Fletcher et al. / Toxicology and Applied Pharmacology 207 (2005) 1–242

helix–loop-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 5V-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 (Quattrochi et al., 1994), NAD(P)H:quinone

oxidoreductase (Favreau and Pickett, 1991), CYP1B1

(Zhang et al., 1998), UGT1A1/6 (Emi et al., 1996; Munzel

et 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 2.1-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 Ag/kg 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

(0.4 Ag/kg bw) or a dose intended to elicit moderate

toxicity in Sprague–Dawley rats (40 Ag/kg 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 Sprague–Dawley out-

bred CD rats (CRL:CD[SD] IGS BR) weighing 250–300 g

were obtained from Charles River Laboratories. The animals

were singly housed in polycarbonate cages; temperatures

were maintained between 18.0 and 26.0 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 (5/dose) received singles doses of TCDD in a

corn oil vehicle (5 mL/kg) by oral gavage at 0, 0.4 or 40

Ag/kg 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 �80 8C.Blood (approximately 4 mL) samples were taken prior to

termination by puncture of the orbital sinus while under

70% CO2/30% O2 anesthesia. Approximately 1 mL of

blood was collected in serum separator tubes for clinical

chemistry analysis whereas 0.5 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) 1–24 3

(http://www.affymetrix.com/products/arrays/specific/rgu34.

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

sample.

Clinical chemistry

Clinical chemistry and pathological examination was

carried out at Gene Logic Inc. laboratories. Serum samples

(5/dose/time 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 Analyzer.

Investigated 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 Sprague–Dawley

rats (B&K Universal Ab, Solentuna, Sweden) were housed

3 per cage and received R34 diet (6000 IU vitamin A/kg

diet; Lactamin, Stockholm Sweden) during a four week

acclimatization period. Rats (6/group; 273 F 18 g)

received TCDD in corn oil (1 mL/kg bw) at doses of 0,

10, and 100 Ag/kg bw and were killed 3 days following

treatment. Anesthesia was carried out using 90 mg/kg

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

�70 8C.

Real-time PCR (Taqman) experiments

RNAwas isolated using the QIAGEN RNeasy Midi Prep

Kit according to the manufacturer’s 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 NanoQ Kit. (Agilent Technologies,

Palo N Alto, USA). The procedure was performed according

to the manufacturer’s manual, Reagent Kit Guide, RNA

6000 Nano Assay, and Edition 07/01. After quantification

the total RNA was stored at �70 8C.Total 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 manufacturer’s

protocol and using 5 ng/l 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 5.0 (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 = 3–5 per treatment

group). Data are contained within GeneLogic’s Toxexpress

database.

To look for outliers and trends in the data, principal

components analysis (PCA; Simca-P 8.1), pairwise correla-

tion analysis and hierarchical clustering (Spotfire version

6.2) were conducted. PCA revealed one outlying sample in

the 6 h 40 Ag/kg 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 Kruskal–Wallis one-way ANOVA on ranks was

http:\\www.affymetrix.com


used and significant differences were evaluated using

Dunnett’s 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 Ag/kg bw

at 7 days only (18%; P b 0.05 data not shown).

Clinical chemistry

Significant changes in clinical chemistry and hematolog-

ical parameters are shown in Table 1. At 40 Ag/kg bw, TCDDincreased serum cholesterol concentrations at the 24 h and 7-

day time points. At 6 h, there was a significant decrease in

serum 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

Table 1

Clinical chemistry and hematology parameters in the serum of rats treated

with single oral doses of TCDD at 0, 0.4, and 40 Ag/kg bw and killed at 6 h,

24 h and 7 days following treatment

Parameter Dose

Control 0.4 40

6 h

Cholesterol (mg/dL) 84.6 F 6.1 76.2 F 5.9 75.4 F 5.0*

Hemoglobin (g/dL) 14.9 F 0.5 15.5 F 0.3* 15.4 F 0.3*

24 h

Triglycerides (mg/dL) 149.4 F 27.9 118.6 F 21.4 260 F 100.8*

Cholesterol (mg/dL) 77.4 F 8.1 74 F 10.9 92.4 F 7.3*

Hemoglobin (g/dL) 14.4 F 0.6 14.6 F 0.4 15.6 F 0.5*

Absolute neutrophils

(Th/AL)4.1 F 0.5 3.0 F 0.8* 4.3 F 0.6

7 days

Triglycerides (mg/dL) 102.2 F 25.5 125.6 F 33.6 54.4 F 19.4*

Cholesterol (mg/dL) 77.8 F 15.9 86.6 F 17.0 124.8 F 34*

Hemoglobin (g/dL) 14.6 F 0.6 15.1 F 0.8 15.9 F 1.4*

Red blood cells

(mil/AL)6.7 F 0.3 6.8 F 0.4 7.6 F 0.5*

Absolute reticulocytes

(mil/AL)0.2 F 0.01 0.18 F 0.02 0.12 F 0.03*

Alanine

aminotransferase

(IU/L)

58 F 7.4 45.2 F 5.8* 46.2 F 8.2*

Glucose (mg/dL) 141 F 12.9 128.6 F 18.2 108.8 F 4.1*

Total protein (g/dL) 6.6 F 0.2 6.5 F 0.1 7.16 F 0.3*

Globulin (g/dL) 2.3 F 0.2 2.4 F 0.2 2.7 F 0.3*

* P b 0.05 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 Kruskal–

Wallis one-way ANOVA on ranks and significant differences were

evaluated using DunnettTs test for multiple comparisons.

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 2/5 rats given 40 Ag/kg bw TCDD. On day 7, centrilobular hypertrophy was

observed in 4/5 rats given 40 Ag/kg 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 0.01. Applying this criteria, a

total of 288 probesets were altered in the liver of male

Sprague–Dawley rats by single oral TCDD exposure at 6 h,

24 h and/or 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 G2/M

cell 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 I/X (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 2

Probesets altered z 2-fold ( P b 0.01) compared to control in the liver of rats given TCDD by oral gavage at 0, 0.4 and 40 Ag/kg 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

symbol0.4 40 0.4 40 0.4 40

Detoxification/stress

E00778cds_s_at Cytochrome P450, 1a1 CYP1A1 2.8 313.3 997.3 345.8 690.3 170.9 701.7

K03241cds_s_at Cytochrome P450, 1a2 CYP1A2 388.3 7.1 11.0 8.7 11.2 9.1 18.5

E01184cds_s_at Cytochrome P450, 1a2 CYP1A2 744.8 5.8 8.4 8.0 10.0 8.6 14.1

M26127_s_at Cytochrome P450, 1a2 CYP1A2 767.3 3.6 5.3 5.5 7.4 5.7 9.5

rc_AI176856_at Cytochrome P450, subfamily 1B,

polypeptide 1

CYP1B1 1.3 299.2 25.0 1355.9

U09540_at Cytochrome P450, subfamily 1B,

polypeptide 1

CYP1B1 8.0 65.7 385.6

U09540_g_at Cytochrome P450, subfamily 1B,

polypeptide 1

CYP1B1 7.6 75.0 8.0 483.8

X83867cds_s_at Cytochrome P450, subfamily 1B,

polypeptide 1

CYP1B1 9.4 11.2

E00717UTR#1_s_at cDNA encoding cytochrome P-450

from rat liver

No symbol 26.9 180.7 294.5 215.3 304.8 94.4 262.0

J02679_s_at NAD(P)H dehydrogenase, quinone 1 Nqo1 109.3 2.3 14.7 3.1 10.5 2.0 12.6

M58495mRNA_at NAD(P)H dehydrogenase, quinone 1 Nqo1 4.1 20.5 17.4 11.1

D38061exon_s_at UDP glycosyltransferase 1 family,

polypeptide A6, arylsulfatase B

Arsb,

UGT1A6

30.3 2.6 10.5 8.6 17.3 5.2 19.3

S56936_s_at UDP glycosyltransferase 1 family,

polypeptide A6, arylsulfatase B

Arsb,

UGT1A6

29.3 2.3 6.5 6.6 17.4 4.2 18.3

S56937_s_at UDP glycosyltransferase 1 family,

polypeptide A6, UDP

glycosyltransferase 1 family,

polypeptide A7

UGT1A6,

UGT1A7

492.6 2.7 2.9 5.5

D83796_s_at UDP glycosyltransferase 1 family,

polypeptide A6, UDP

glycosyltransferase 1 family,

polypeptide A7

UGT1A6,

UGT1A7

1050.8 2.5 2.7 4.8

D38062exon_s_at UDP glycosyltransferase 1

family, polypeptide A7

UGT1A7 18.6 7.8 6.0 30.5 2.0 34.8

AF039212mRNA_s_at UDP glycosyltransferase 1


UGT1A7 34.8 5.2 3.1 10.6 22.7

J02612mRNA_s_at UDP glycosyltransferase 1


UGT1A7 1041.0 2.6 2.6 3.7

J05132_s_at UDP glycosyltransferase 1


UGT1A7 1747.3 2.1 2.7 3.7

J03637_at Aldehyde dehydrogenase family 3,

member A1

Aldh3a1 20.8 10.3 56.9 105.8

D38065exon_s_at UDP glycosyltransferase 1 family,

polypeptide A1

UGT1A1 177.7 �2.5

K00136mRNA_at Glutathione S-transferase, alpha

type 2

GSTA2 1814.0 2.1 2.6 3.7

S72506_s_at Glutathione S-transferase, alpha

type 2

GSTA2 11.3 8.7 3.4

S82820mRNA_s_at GSTA5 = glutathione S-transferase

Yc2 subunit [rats, Morris hepatoma

cell line, mRNA, 1274 nt]

Yc2 subunit;

GSTA5

89.0 4.0 5.4 3.2

X62660mRNA_at RRGTS8 R.rattus mRNA for

glutathione transferase subunit

GSTA4 195.2 2.3 4.2

X62660mRNA_g_at RRGTS8 R.rattus mRNA for

glutathione transferase subunit

GSTA4 277.0 2.9 4.8

rc_AI102562_at Metallothionein Mt1a 6951.4 9.6 9.2

M11794cds#2_f_at Metallothionein Mt1a 4851.2 9.1 2.5 9.1

rc_AI234950_at Acid phosphatase 2 Acp2 171.5 2.0 2.9

AF045464_s_at Aflatoxin B1 aldehyde reductase Afar 260.2 2.9

J03786_s_at Cytochrome P450 15-beta gene CYP2c12 152.8 6.3

J00728cds_f_at Rat cytochrome P-450e

(phenobarbital-inducible)

gene, exon 9

No symbol 389.2 �2.0 �2.5

(continued on next page)


Table 2 (continued)


symbol0.4 40 0.4 40 0.4 40

Detoxification/stress

L00320cds_f_at RATCYPB9 Rat

cytochrome P-450b

(phenobarbital-inducible)

gene, exon 9

Rat CYP2B9 80.2 �2.7

M13234cds_f_at RATCYPEZ78 Rat cytochrome

P-450e gene, exons 7 and 8

No symbol 302.7 �2.1

U40004_s_at cytochrome P450 pseudogene

(CYP2J3P2)

CYP2J3P2 243.8 �2.0

U46118_at cytochrome P450 3A9 CYP3A9 195.3 �10.8

M18363cds_s_at Cytochrome P450, subfamily IIC

(mephenytoin 4-hydroxylase)

CYP2C 2338.4 �2.9

X79081mRNA_f_at Cytochrome P450, subfamily IIC

(mephenytoin 4-hydroxylase)

CYP2C 628.6 �4.9

U70825_at 5-oxoprolinase Oplah 82.1 �2.8

S48325_s_at Cytochrome P450, subfamily 2E,

polypeptide 1

CYP2e1 4958.0 �2.7

M20131cds_s_at Cytochrome P450, subfamily 2E,

polypeptide 1

CYP2e1 5661.5 �2.3

AF056333_s_at Cytochrome P450, subfamily 2E,

polypeptide 1

CYP2e1 2872.3 �2.7

M58041_s_at Cytochrome P450 2c22 CYP2c22 1510.4 �2.3

M84719_at Flavin-containing

monooxygenase 1

FMO1 239.0 �3.4

U63923_at Thioredoxin reductase 1 Txnrd1 127.7 2.5

rc_AA891286_at Thioredoxin reductase 1 Txnrd1 268.2 2.3

rc_AI172247_at Xanthine dehydrogenase Xdh 195.6 2.0

AF037072_at Carbonic anhydrase 3 Ca3 540.9 �4.9 �20.7

L32591mRNA_at Growth arrest and

DNA-damage-inducible

45 alpha

Gadd45a 38.2 2.0 3.3 4.0 6.5

L32591mRNA_g_at Growth arrest and


45 alpha

Gadd45a 79.8 2.6 2.5 3.5

rc_AI070295_g_at Growth arrest and


45 alpha

Gadd45a 39.1 4.9

AF025670_g_at Caspase 6 Casp6 81.0 2.1

Lipid metabolism

J05210_at ATP citrate-lyase Acly 389.0 �3.0 �2.9

J05210_g_at ATP citrate-lyase Acly 1087.5 �2.4

L07736_at Carnitine palmitoyltransferase 1 CPT1 846.3 3.6

J02749_at Acetyl-CoA acyltransferase 1,

3-oxo acyl-CoA thiolase A

Acaa1 107.9 3.4 2.5 5.3

M76767_s_at Fatty acid synthase Fasn 181.4 �2.4

S69874_s_at Fatty acid binding protein 5,

epidermal

Fabp 106.5 4.2

rc_AA799779_g_at Acyl-CoA:

dihydroxyacetonephosphate

acyltransferase

Gnpat 48.8 2.1

U10357_at Pyruvate dehydrogenase kinase 2 Pdk2 326.3 �3.3

U10357_g_at Pyruvate dehydrogenase kinase 2 Pdk2 443.4 �2.0

S81497_s_at Lipase A, lysosomal acid Lipa 131.1 �2.6

M33648_at 3-Hydroxy-3-methylglutaryl-CoA

synthase 2, mitochondrial precursor

Hmgcs2 2929.0 �2.0

rc_AA817846_at 3-hydroxybutyrate

dehydrogenase

(heart, mitochondrial)

Bdh 482.5 �2.1

AF003835_at Isopentenyl-diphosphate

delta isomerase

Idi1 172.7 �2.5


(continued on next page

Table 2 (continued)


symbol0.4 40 0.4 40 0.4 40

Lipid metabolism

M89945mRNA_at Farensyl diphosphate synthase Fdps 1023.9 �2.3

M00002_at Apolipoprotein A-IV Apoa4 703.8 �3.5

J05460_s_at Cytochrome P450, 7a1 CYP7A1 437.5 �9.7 �8.0

U18374_at Farnesoid X receptor Nr1h4 (FXR) 156.3 �2.3 �2.0

D86580_at Short heterodimer partner SHP (nr0b2) 139.0 �3.6 �3.6

D86745cds_s_at Short heterodimer partner SHP (nr0b2) 171.2 �4.3 �4.0

M77479_at Solute carrier family 10 (sodium/

bile acid cotransporter family),

member 1

Slc10a1

(Ntcp)

1104.4 �2.1

U88036_at Solute carrier family 21

(organic anion

transporter), member 5

Slc21a5;

oatp2

463.0 �3.2 �2.9

D10262_at Choline kinase Chk 72.6 2.4 2.9 2.3

E04239cds_s_at Choline kinase Chk 12.9 3.1

L14441_at Phosphatidylethanolamine

N-methyltransferase

PEMT 631.5 �2.7

D28560_at Ectonucleotide

pyrophosphatase/

phosphodiesterase 2

Enpp2 291.7 2.7 4.1

D28560_g_at Ectonucleotide

pyrophosphatase/

phosphodiesterase 2

Enpp2 161.5 3.7 3.4

D78588_at Diacylglycerol kinase zeta Dgkz 53.4 �2.3

AB009372_at Lysophospholipase LOC246266 94.3 �4.8 �15.6

Carbohydrate metabolism

X53588_at Glucokinase Gck 74.8 �3.2 �3.0

AF080468_at Glucose-6-phosphatase

transport protein

G6pt1 664.5 �2.6 �2.6

AF080468_g_at Glucose-6-phosphatase

transport protein

G6pt1 813.4 �2.3 �2.4

X07467_at Glucose-6-phosphate

dehydrogenase

G6pd 72.8 3.3 3.5

rc_AI008020_at Malic enzyme 1 Me1 27.5 2.6 4.3 2.0

rc_AI171506_g_at Malic enzyme 1 Me1 75.2 4.3 4.5

M26594_at Malic enzyme 1 Me1 43.0 4.1 3.6

rc_AI171506_at Malic enzyme 1 Me1 38.8 5.1 4.4

rc_AI059508_s_at Transketolase Tkt 116.4 �2.5

K03243mRNA_s_at Phosphoenolpyruvate

carboxykinase

PEPCK 2417.9 �3.2 �4.2

U32314_at Pyruvate carboxylase Pc 351.5 �2.2 �2.2

U32314_g_at Pyruvate carboxylase Pc 311.1 �2.0

Nitrogen metabolism

AB003400_at d-Amino acid oxidase Dao1 123.9 �7.6

X12459_at Arginosuccinate synthetase Ass 2611.8 �2.1 �3.1

rc_AI179613_at Glutamate dehydrogenase 1 Glud1 1740.5 �2.4

rc_AI233216_at Glutamate dehydrogenase 1 Glud1 690.5 �2.4 �2.1

rc_AA852004_s_at Glutamine synthetase Glul 90.2 �3.1 �3.1

M91652complete_seq_at Glutamine synthetase Glul 257.0 �2.4 �2.1

rc_AI232783_s_at Glutamine synthetase Glul 655.2 �2.3

J05499_at Liver mitochondrial glutaminase Ga 203.0 �3.9

M58308_at Histidine ammonia lyase Hal 343.6 �4.4

D10354_s_at Alanine aminotransferase Alat 257.0 �3.2

D13667cds_s_at Serine pyruvate aminotransferase Spat 96.7 �2.7

X06357cds_s_at Serine pyruvate aminotransferase Spat 446.5 �2.2

X13119cds_s_at Serine dehydratase Sds 27.1 10.5

X06150cds_at Glycine methyltransferase Gnmt 235.9 �2.0

E03229cds_s_at Cytolosic cysteine dioxygenase Cdo1 2481.9 �3.3 �2.9

AF056031_at Kynurenine 3-hydroxylase Kmo 268.3 �2.2

Z50144_at Kynurenine aminotransferase 2 Kat2 116.4 �2.5


)

Table 2 (continued)


symbol0.4 40 0.4 40 0.4 40

Nitrogen metabolism

Z50144_g_at Kynurenine aminotransferase 2 Kat2 245.8 �2.2

J04171_at Aspartate aminotransferase Asat 168.7 2.5 2.2

AF038870_at Betaine-homocysteine

methyltransferase

Bhmt 2260.2 2.0

J03959_g_at Urate oxidase Uox 62.2 2.2

rc_AA900413_at Dihydrofolate reductase 1

(active)

Dhfr1 238.9 2.2

AJ000347_g_at 3(2),5-bisphosphate

nucleotidase

Bpnt1 57.7 3.1

D90404_at cathepsin C Ctsc 1175.7 �2.4

Mitochondrial electron transport chain

X15030_at Cytochrome c oxidase,

subunit Va

Cox5a 975.3 3.1

Retinoid metabolism

X65296cds_s_at Carboxylesterase 3

(carboxylesterase ES10)

CES3 471.9 �2.1 �8.1

L46791_at Carboxylesterase 3

(carboxylesterase ES10)

CES3 249.6 �6.4

D00362_s_at Esterase 2 ES2 1729.6 �5.2

M20629_s_at Esterase 2 ES2 2035.0 �2.8

AF016387_at Retinoid X receptor, gamma Rxrg 41.0 2.1

Steroid metabolism

S81448_s_at Steroid 5 alpha-reductase 1 Srd5a1 328.8 �33.2

J05035_g_at Steroid 5 alpha-reductase 1 Srd5a1 883.5 �17.7

J05035_at Steroid 5 alpha-reductase 1 Srd5a1 456.0 �13.1

M31363mRNA_f_at (A.d.) M31363mRNA

RATHSST Rat hydroxysteroid

sulfotransferase mRNA

No symbol 2996.6 �4.5

rc_AA818122_f_at Sulfotransferase hydroxysteroid

gene 2

Sth2 1855.9 �3.7

D14988_f_at Sulfotransferase hydroxysteroid

gene 2

Sth2 2977.5 �3.6

D14987_f_at Sulfotransferase hydroxysteroid

gene 2

Sth2 1199.3 �3.1

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 701.9 �2.4

X57999cds_at Deiodinase, iodothyronine,

type 1

Dio 1 82.6 �4.3

X91234_at 17-beta hydroxysteroid

dehydrogenase type 2

Hsd17b2 1527.1 2.0

M33312cds_s_at Cytochrome P450 IIA1

(hepatic steroid

hydroxylase IIA1) gene

CYP2A1 1345.2 3.6

L24207_i_at (A.d.) L24207 Rattus

norvegicus testosterone

6-beta-hydroxylase

(CYP3A1) mRNA,

CYP3A1 165.9 2.4

L24207_r_at Rattus norvegicus testosterone

6-beta-hydroxylase

(CYP3A1) mRNA

Cyp3A1 106.8 2.7

D13912_s_at Cytochrome P-450PCN

(PNCN inducible),

cytochrome P450, subfamily

3A, poypeptide 3

Cyp3A1,

Cyp3a3

699.3 2.5



Table 2 (continued)


symbol0.4 40 0.4 40 0.4 40

Kinases

rc_AI145931_at UDP-N-acetylglucosamine-

2-epimerase/

N-acetylmannosamine kinase

Uae1 265.3 �2.3

Circadian rhythm

AB016532_at Period homolog 2 Per2 6.5 4.4

Membrane bound proteins

AF004017_at Solute carrier family 4,

member 4

Slc4a4 49.3 7.0


(vesicular glutamate transporter),

member 1

Slc17a1 91.5 2.4 3.0

U28504_g_at Solute carrier family 17

(vesicular glutamate transporter),

member 1

Slc17a1 42.8 3.6 5.6

AB015433_s_at Solute carrier family 3, member 2 Slc3a2 157.7 2.1 4.0

X89225cds_s_at Solute carrier family 3, member 2 Slc3a2 104.6 3.0

D84450_at ATPase, Na+K+

transporting, beta

polypeptide 3

Atp1b3 96.8 2.9

M74494_g_at ATPase, Na+K+

transporting, alpha 1

Atp1a1 244.4 �3.1

M28647_g_at ATPase, Na+K+

transporting, alpha 1

Atp1a1 491.1 �2.7

rc_AA799645_g_at FXYD domain-containing

ion transport regulator 1

Fxyd1 130.7 �2.0 �2.9

L27651_at Solute carrier family 22

(organic anion transporter),

member 7

Slc22a7 317.4 �2.1


(iron-regulated transporter),

member 1

Slc39a1 69.6 �2.0

rc_AI145680_s_at Solute carrier 16

(monocarboxylic acid

transporter), member 1

Slc16a1 173.6 �2.3

L28135_at Solute carrier family 2

A2 (glucose transporter,

type 2)

Slc2a2 465.3 �2.3

U76379_s_at Solute carrier family 22,

member 1

Slc22a1 418.2 �2.1

AJ011656cds_s_at Claudin 3 Cldn3 353.3 �2.5

S61865_s_at Syndecan Synd1 206.1 �2.0

X60651mRNA_s_at Syndecan Synd1 93.7 �2.9

M31322_g_at Sperm membrane protein

(YWK-II)

LOC64312 321.3 2.1

AF097593_at Cadherin 2 Cdh2 95.0 �2.4

U23056_at C-CAM4 protein LOC287009 24.0 2.5 54.4

U23055cds_s_at Partial cds: C-CAM4 protein,

carcinoembryonic

antigen-related cell

adhesion molecule 1

Ceacam1 32.2 67.6

J04963_at Carcinoembryonic

antigen-related cell

adhesion molecule 1

Ceacam1 78.7 2.3

U32575_at Sec6 Sec6 18.1 4.4 6.2

U32575_g_at Sec6 Sec6 34.2 2.0 4.5 9.4

rc_AA926292_s_at Trans-Golgi network protein 1 Ttgn1 91.9 2.0 2.6

rc_AA859954_at Vacuole membrane protein 1 Vmp1 147.2 2.6

rc_AA892759_at Synaptosomal-associated protein,

23 kDa

Snap23 20.5 3.4


Table 2 (continued)


symbol0.4 40 0.4 40 0.4 40

Cell cycle

X75207_s_at Cyclin D1 Ccnd1 69.6 �2.0 �2.4

D14014_g_at Cyclin D1 Ccnd1 130.7 �3.6

D14014_at Cyclin D1 Ccnd1 123.8 �3.3 �2.4

RNA processing

AF041066_at Ribonuclease, RNase A family 4 Rnase4 1518.3 �2.3

Cell signaling

X52140_at Integrin, alpha 1 Itga1 106.9 �2.1

M83680_at GTPase Rab14 Rab14 73.5 �2.3

L19180_g_at Protein tyrosine phosphatase,

receptor type, D

Ptprd 93.5 �5.9 �8.1

L19933_s_at Protein tyrosine phosphatase,

receptor type, D

Ptprd 83.5 �2.1

K03249_at G protein-coupled receptor

37-like 1, enoyl-Coenzyme A,

hydratase/3-hydroxyacyl

Coenzyme A dehydrogenase

Ehhadh 221.9 �3.6

M63122_at Tumor necrosis factor receptor

super family, member 1a

Tnfrsf1a 190.6 2.0

rc_AA892251_at Arginine vasopressin receptor 1A Avpr1a 206.4 2.5 2.7

D85435_g_at PKC-delta binding protein Prkcdbp 428.0 2.8 2.4

rc_AA900505_at RhoB gene Arhb 31.0 4.0

rc_AA874794_g_at Nerve growth factor receptor

(TNFRSF16) associated protein 1

Ngfrap1 30.8 2.5

L19699_g_at V-ral simian leukemia viral

oncogene homolog B (ras related)

Ralb 28.2 2.0

AJ010828_at Chemokine orphan receptor 1 Rdc1 4.9 13.3

AF017437_g_at Integrin-associated protein Cd47 18.7 2.5

Transcription factors

Y14933mRNA_s_at One cut domain, family member 1

alternative name: hepatocyte

nuclear factor 6 beta

Onecut1 108.4 �7.3

AB012234_g_at Nuclear factor I/X Nfix 73.2 �4.5

D12769_at Kruppel-like factor 9 Klf9 188.6 �2.0

AB017044exon_at AB017044exon Rattus

norvegicus gene for hepatocyte

nuclear factor 3 gamma,

partial cds.

HNF3-G 63.1 �2.7

X84210complete_seq_s_at Nuclear factor I/A Nfia 75.2 �2.4

rc_AI234146_at Cysteine rich protein 1 Csrp1 139.7 �2.7 �6.3

rc_AI014091_at Cbp/p300-interacting

transactivator, with Glu/Asp-rich

carboxy-terminal domain, 2

Cited2 or

MRG1

86.4 �3.6

L25785_at Transforming growth factor beta

1 induced transcript 4

(stimulated clone 22 homologue)

Tgfb1i4/� 473.4 �2.8 �3.6 �3.7

rc_AI177161_g_at NF-E2-related factor 2 Nfe2l2/nrf2 40.6 2.6 4.1 4.7 5.3

rc_AI177161_at NF-E2-related factor 2 Nfe2l2/nrf2 64.8 2.5 3.3 3.7 5.9

Heme synthesis

J03190_at Aminolevulinic acid synthase 1 Alas1 300.5 �4.3

J03190_g_at Aminolevulinic acid synthase 1 Alas1 192.4 �2.3

D86297_at Aminolevulinic acid synthase 2 Alas2 112.2 �2.7

rc_AI178971_at Hemoglobin, alpha 1 Hba1 61.0 �5.2

X56325mRNA_s_at Hemoglobin, alpha 1 Hba1 4004.1 �2.8

M94918mRNA_f_at Hemoglobin, beta Hbb 2895.4 �2.9

M94919mRNA_f_at mRNA RATBETGLOY Rat

beta-globin gene, exons 1–3

No symbol 1654.7 �2.6


(continued on next page

Table 2 (continued)


symbol0.4 40 0.4 40 0.4 40

Immune

D10729_s_at Proteasome (prosome,

macropain) subunit, beta type,

8 (low molecular

mass polypeptide 7)

Psmb8 323.2 �2.1

M64795_f_at M64795 Rat MHC class I

antigen gene


M33025_s_at Parathymosin Ptms 472.7 �3.0

rc_AI136977_g_at FK506 binding protein 4 59kDa Fkbp4 90.7 �2.3

rc_AI136977_at FK506 binding protein 4 59kDa Fkbp4 47.3 �13.1

M86564_at Prothymosin alpha Ptma 185.8 �2.1

D88250_at Complement component 1,

s subcomponent

C1s 686.2 2.9

M31038_at RT1 class Ib gene RT1Aw2 43.8 �2.7

rc_AA945608_at Serum amyloid P-component Sap 1415.1 �2.5

Cell differentiation

rc_AI231292_at Cystatin C Cst3 223.5 �2.0

rc_AA858673_at Pancreatic secretory trypsin

inhibitor type II (PSTI-II)

LOC266602 1458.8 �3.1

M15481_g_at Insulin-like growth factor 1 Igf1 3034.7 �2.6

M15481_at Insulin-like growth factor 1 Igf1 454.8 �2.6

X06107_i_at Insulin-like growth factor 1 Igf1 187.1 �2.3

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 253.5 �2.4

S46785_at Insulin-like growth factor binding

protein, acid labile subunit

Igfals 752.6 �2.4

M31837_at Insulin-like growth factor binding

protein 3

Igfbp3 132.5 �2.6

M58634_at Insulin-like growth factor binding

protein 1

Igfbp1 51.6 2.5 4.7 2.9 4.9

Cytoskeleton

U31463_at Myosin, heavy polypeptide 9,

non-muscle

Myh9 105.2 �3.8

X52815cds_f_at X52815cds RRGAMACT Rat

mRNA for cytoplasmic-gamma

isoform of actin


rc_AI179012_s_at Actin, beta Actb 2586.8 �3.2

X70706cds_at Plastin 3 (T-isoform) Pls3 103.3 �2.0

U05784_s_at Microtubule-associated proteins

1A/1B light chain 3

MPL3 321.8 3.3 2.6

rc_AA944422_at Calponin 3, acidic Cnn3 79.4 2.2

rc_AA892814_s_at Calpain, small subunit Capns1 339.5 �2.2

L24776_at tropomyosin 3, gamma Tpm3 44.6 2.0

Poorly characterized and/or unknown function in liver

X12355_s_at Glucose regulated protein, 58 kDa Grp58 472.5 �2.9

rc_AI234604_s_at Heat shock cognate protein 70 Hsc70 1018.9 �2.2

D30649mRNA_s_at Alkaline phosphodiesterase LOC54410 78.9 �2.1 �3.5

U62897_at Carboxypeptidase D Cpd 88.0 �2.4

rc_AA859837_g_at Guanine deaminase Gda 295.6 �2.1

J00738_s_at Alpha-2u globulin PGCL4 LOC259247 992.6 �117.6

AB000199_at CCA2 protein Cca2 379.3 �2.3

U55765_at Serine (or cysteine) proteinase

inhibitor, clade A (alpha-1

antiproteinase, antitrypsin),

member 10

Serpina 10

Rasp-1

517.5 2.2


)

Table 2 (continued)


symbol0.4 40 0.4 40 0.4 40


X96437mRNA_g_at X96437mRNA RNPRG1

R.norvegicus PRG1 gene

No symbol 66.1 2.9

X96437mRNA_at X96437mRNA RNPRG1

R.norvegicus PRG1 gene

No symbol 88.8 2.2

S61960_s_at Cysteine conjugate beta-lyase No symbol 86.9 2.5 2.4 4.2

rc_AA893239_at 2-hydroxyphytanoyl-CoA lyase Hpcl2 309.0 �2.3

S85184_at S85184 Cyclic Protein-2 =

cathepsin L proenzyme [rats,

Sertoli cells, mRNA, 1790 nt]

CP-2 80.4 2.3 3.0

S77494_s_at Lysyl oxidase Lox 55.8 �4.3

X61381cds_s_at RRIIMRNA R. rattus interferon

induced mRNA


rc_AI172293_at Sterol-C4-methyl oxidase-like Sc4mol 685.0 �2.1

E12625cds_at Sterol-C4-methyl oxidase-like Sc4mol 367.9 �2.5

rc_AA891916_at Membrane interacting protein

of RGS16

Mir16 150.8 2.1

rc_AA891916_g_at Membrane interacting protein

of RGS16

Mir16 213.5 2.0

rc_AA859981_at Inositol (myo)-1(or 4)-

monophosphatase 2

Impa2 38.5 3.2

D17809_at Beta-4N-

acetylgalactosaminyltransferase

Galgt1 145.5 �2.3 �2.5

X14848cds#12_at MIRNXX Rattus norvegicus

mitochondrial genome

No symbol 36.2 2.9

rc_AI639029_s_at Rat mixed-tissue library Rattus

norvegicus cDNA

clone rx05067 3, mRNA

sequence [Rattus norvegicus]

No symbol 32.3 4.4

rc_AI638989_at Rat mixed-tissue library

Rattus norvegicus cDNA clone

rx01268 3, mRNA sequence

[Rattus norvegicus]



Rattus norvegicus cDNA



No symbol 11.3 5.4

rc_AA955983_at rc_AA955983 UI-R-E1-fb-e-

12-0-UI.s1 Rattus norvegicus

cDNA, 3 end/clone = UI-R-E1-

fb-e-12-0-UI/clone_end =

3 /gb = AA955983/Ag =

Rn.7854/len = 542

No symbol 503.5 2.1

U47312_s_at U47312 RNU47312 Rat R2

cerebellum DDRT-T-PCR

Rattus norvegicus cDNA clone

LIARCD-3, mRNA sequence

[Rattus norvegicus]


rc_AA875171_at rc_AA875171 UI-R-E0-ce-f-


cDNA, 3 end/clone = UI-R-E0-

ce-f-12-0-UI/clone_end =

3 /gb = AA875171/gi =

2980119/Ag = Rn.2814/

len = 458

No symbol 86.3 2.1

rc_AA817987_f_at rc_AA817987 UI-R-A0-ah-a-


cDNA, 3 end/clone = UI-R-A0-

ah-a-06-0-UI/clone_end = 3 /gb =

AA817987/gi = 2887867/Ag =

Rn.23920/len = 373




Table 2 (continued)


symbol0.4 40 0.4 40 0.4 40


rc_AA859899_at rc_AA859899 UI-R-E0-

cg-a-03-0-UI.s1 Rattus

norvegicus cDNA, 3 end/

clone = UI-R-E0-cg-a-03-

0-UI/clone_end = 3 /gb =

AA859899/gi = 2949419/Ag =

Rn.810/len = 353



Rattus norvegicus cDNA



No symbol 9.9 5.4

ESTs

rc_AI236601_at EST233163 Rattus

norvegicus cDNA

64.3 2.8

rc_AA892246_at EST196049 Rattus

norvegicus cDNA

72.2 2.2 2.0 2.0


norvegicus cDNA

130.0 2.4


norvegicus cDNA

1088.9 2.2 2.6


norvegicus cDNA,

26.8 2.8

rc_AI176456_at EST220041 Rattus

norvegicus cDNA

4291.8 11.6 11.0

rc_AA892888_g_at EST196691 Rattus

norvegicus cDNA

2168.7 2.2


norvegicus cDNA

42.1 2.2

rc_AI014135_g_at EST207690 Rattus

norvegicus cDNA

286.3 3.4


norvegicus cDNA

147.2 2.0


norvegicus cDNA

55.3 2.0


norvegicus cDNA

80.8 2.2


norvegicus cDNA

214.3 2.0


norvegicus cDNA

268.6 2.0 4.1


norvegicus cDNA

55.0 2.1


norvegicus cDNA

29.1 2.0


norvegicus cDNA

105.4 2.5


norvegicus cDNA

47.9 2.3

rc_H33001_at EST108598 Rattus

norvegicus cDNA

138.0 �2.2

rc_H31813_at EST106240 Rattus

norvegicus cDNA

162.8 �2.4


norvegicus cDNA

78.8 2.1


norvegicus cDNA

87.5 �2.3


norvegicus cDNA

59.3 �3.5


norvegicus cDNA

672.0 �2.2 �3.0


Table 2 (continued)


symbol0.4 40 0.4 40 0.4 40

ESTs

rc_AA892799_s_at EST196602 Rattus

norvegicus cDNA

226.2 �2.1

rc_AI169695_f_at EST215591 Rattus

norvegicus cDNA

342.6 �2.2


norvegicus cDNA

134.0 �2.1

rc_AI169735_g_at EST215634 Rattus

norvegicus cDNA

636.0 �2.4


norvegicus cDNA

43.8 2.3


norvegicus cDNA

39.1 �3.9

Values at 6 h, 24 h, and 7 days represent fold-changes compared to corresponding vehicle control values following dosing with TCDD at 0.4 and 40 Ag/kg 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 0.01; See Microarray data analysis.


2-fold in one probeset at 6 h, and subsequent changes were

seen 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 Ag/kg 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 induced

changes 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 metabolism/bile 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 3,

member 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 Ag/kg bw,

respectively. CYP1A2 induction was approximately 100-fold

less than for CYP1A1. UGT1A6/7 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). Effects

on 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.,


1985; Peterson et al., 1984; Seefeld et al., 1984). Briefly,

single doses of 5 and 15 Ag/kg bw TCDD were shown to

decrease body weight gain in Sprague–Dawley rats, whereas

doses of 25 and 50 Ag/kg 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 Ag/kg 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 40–50% 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

Ag/kg bw, 75% at 50 Ag/kg bw, and 95% at 100 Ag/kg 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 changes

in 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 body’s 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 2.4-fold at 7 days (�1.8, P b 0.01 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-carbohydrate

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 in

hepatic 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-CoA

acyltransferase 1 (Acaa1), which functions in the h-oxidationof long chain fatty acids in peroxisomes [reviewed in

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

dose at 6 h, 24 h, and 7 days (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 3.6-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 2–3 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 Ag/kg bwTCDD versus controls fed ad libitum (Tomaszewski et al.,

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

given 20 Ag/kg 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 3.3-fold (depend-

ent on probe set) at the high dose at 7 days (1.9-fold, P b

0.01 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 (�1.8; P b 0.01)

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

intake/starvation would constitute an inappropriate response

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 (�5.1 fold, n.s. P = 0.012), and 7

days following exposure to 40 Ag/kg bw TCDD (Table 2).

Subsequently, RT-PCR on independent samples showed a

dose-dependent and approximate 6-fold decrease in

CYP7A1 expression following a dose of 100 Ag/kg 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 transcription

of 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 2.2-fold (n.s) at 6 h

and 3.6-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 Ag/kg bw and

slightly, but significantly, at 10 Ag/kg bw (Fig. 1b).

Likewise, SHP mRNA expression was decreased approx-

imately 10-fold at 100 Ag/kg bw (Fig. 1c). SHP is an orphan

nuclear 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

RAR:RXR 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 1.7- 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 Ag/kg bw, 3 days after TCDD exposure (Fig. 1d). It

is interesting that Ntcp, which is inducible by retinoids,

should 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 (�3.2 and �2.9 fold, respectively). RT-PCR

analysis confirmed down-regulation of oatp2 at 10 and 100

Ag/kg bw, the change at the high-dose approximately 8-fold

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

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

Ag/kg bw TCDD (n = 6). Statistical analyses were as described in Materials and methods. * Indicates significantly different from controls at P b 0.05. 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).


hepatocytes and is predominately expressed in perivenous/

pericentral hepatocytes (Kakyo et al., 1999; Reichel et al.,

1999). This membrane transport protein carries a wide

variety 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 3.9 Ag/kg 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 and

transport. Decreased CYP7A1 expression and/or 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


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 al.,

1977, 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 phosphoenolpyruvate

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 (i.e.,

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.

Carbohydrate metabolism

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 2.5-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 altered

expression of G6pt1 could influence glucose-6-phosphatase

activity, which has also previously been shown to be

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 3.3-fold and 3.5-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 2.5 and 1.8 times

(n.s., 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-phosphate,

which 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 Ag/kg bw altered the expres-

sion of several genes associated with amino acid metabo-

lism/nitrogen 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 metabolizing

enzymes 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 been

directed towards the inhibition of PEPCK, the expression

and/or 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 Ag/kg 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 to

TCDD-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 0.05 (data not

shown). Likewise, esterase 2 (ES2) expression was

decreased up to 5.2-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

there is no direct 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 CYP1A1/2 or perhaps UGT1A6/7

enzymes. CYP1A1 has been shown to metabolize various

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

UGT1A6/7 on retinoid species, but increased retinoid

glucuronidation 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 RXRg at 6 h following high-dose

TCDD exposure. A second probeset also showed significant

increases in RXRg 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 RXRg

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

receptors 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 clinical

chemistry 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

points 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 1A/1B light

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

time point. A marked upregulation of microtubule-associated

protein (1A/1B) already at 6h (3.3-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.

Transcription factors

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 a

variety 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 (�3.1; �2.7), whereas ATPase, Na+K+trans-

porting, beta polypeptide 3 (Atp1b3) was upregulated 2.9-

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 Na/K 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 Na/K

ATPase activities and hepatic plasma membrane function

observed in early studies (Matsumura et al., 1984; Peterson et

al., 1979).

Summary

Low-dose (0.4 Ag/kg 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 the

Commissions 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. 21.0/2003-1135 Etapp2).

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