Patterns of dioxin-altered mRNA expression in livers of dioxin-sensitive versus dioxin-resistant rats

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MOLECULAR TOXICOLOGY

Patterns of dioxin-altered mRNA expression in liversof dioxin-sensitive versus dioxin-resistant rats

Monique A. Franc Æ Ivy D. Moffat Æ Paul C. Boutros ÆJouni T. Tuomisto Æ Jouko Tuomisto Æ Raimo Pohjanvirta ÆAllan B. Okey

Received: 25 January 2008 / Accepted: 2 April 2008 / Published online: 9 May 2008

� Springer-Verlag 2008

Abstract Dioxins exert their major toxicologic effects by

binding to the aryl hydrocarbon receptor (AHR) and

altering gene transcription. Numerous dioxin-responsive

genes previously were identified both by conventional

biochemical and molecular techniques and by recent

mRNA expression microarray studies. However, of the

large set of dioxin-responsive genes the specific genes

whose dysregulation leads to death remain unknown. To

identify specific genes that may be involved in dioxin

lethality we compared changes in liver mRNA levels fol-

lowing exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin

(TCDD) in three strains/lines of dioxin-sensitive rats with

changes in three dioxin-resistant rat strains/lines. The three

dioxin-resistant strains/lines all harbor a large deletion in

the transactivation domain of the aryl hydrocarbon receptor

(AHR). Despite this deletion, many genes exhibited a

‘‘Type-I’’ response—that is, their responses were similar in

dioxin-sensitive and dioxin-resistant rats. Several genes

that previously were well established as being dioxin-

responsive or under AHR regulation emerged as Type-I

responses (e.g. CYP1A1, CYP1A2, CYP1B1 and Gsta3).

In contrast, a relatively small number of genes exhibited a

Type-II response—defined as a difference in responsive-

ness between dioxin-sensitive and dioxin-resistant rat

strains. Type-II genes include: malic enzyme 1, ubiquitin

C, cathepsin L, S-adenosylhomocysteine hydrolase and

ferritin light chain 1. In silico searches revealed that AH

response elements are conserved in the 50-flanking regions

of several genes that respond to TCDD in both the Type-I

and Type-II categories. The vast majority of changes in

mRNA levels in response to 100 lg/kg TCDD were strain-

specific; over 75% of the dioxin-responsive clones were

affected in only one of the six strains/lines. Selected genes

were assessed by quantitative RT-PCR in dose-response

and time-course experiments and responses of some genes

were assessed in Ahr-null mice to determine if their

response was AHR-dependent. Type-II genes may lie in

pathways that are central to the difference in susceptibility

to TCDD lethality in this animal model.

Keywords 2,3,7,8-Tetrachlorodibenzo-p-dioxin �TCDD � Aryl hydrocarbon receptor �mRNA expression microarray � Resistant rat model

M.A. Franc and I.D. Moffat contributed equally to this project.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00204-008-0303-0) contains supplementarymaterial, which is available to authorized users.

M. A. Franc � I. D. Moffat � P. C. Boutros � A. B. Okey (&)

Department of Pharmacology and Toxicology,

Medical Sciences Building, University of Toronto,

Toronto, ON, Canada M5S 1A8

e-mail: [email protected]

Present Address:

M. A. Franc

Department of Pharmacogenomics, Johnson & Johnson

Pharmaceutical Research and Development,

1000 Route 202 South, P.O. Box 300,

Raritan, NJ 08869, USA

J. T. Tuomisto � J. Tuomisto

Department of Environmental Health,

Centre for Environmental Health Risk Analysis,

National Public Health Institute, 70701 Kuopio, Finland

R. Pohjanvirta

Department of Food and Environmental Hygiene,

Faculty of Veterinary Medicine, University of Helsinki,

00014 Helsinki, Finland

123

Arch Toxicol (2008) 82:809–830

DOI 10.1007/s00204-008-0303-0

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Introduction

Dioxins, especially the most potent congener 2,3,7,8-tet-

rachlorodibenzo-p-dioxin (TCDD), produce a broad

spectrum of toxic outcomes in laboratory and feral animals.

These include hepatotoxicity, immunotoxicity, teratoge-

nicity, cancer, wasting and death (Pohjanvirta and

Tuomisto 1994). It is firmly established that the essential

first step in dioxin toxicity is the binding of TCDD and

related dioxins to the aryl hydrocarbon receptor (AHR)

(reviewed in Okey 2007) although not all compounds that

bind the AHR with high affinity cause dioxin-like toxicity

(Fried et al. 2007). The most conclusive evidence that the

AHR mediates dioxin toxicity arises from experiments in

mice whose AHR has been genetically knocked out; these

Ahr-null mice are extraordinarily resistant to TCDD tox-

icities (Fernandez-Salguero et al. 1995, 1996; Lin et al.

2001; Mimura et al. 1997; Thurmond et al. 1999). Dioxin

toxicity also is dramatically reduced in mice whose AHR

has been genetically altered to create an AHR that has an

impaired nuclear translocation/gene-transactivation

domain (Bunger et al. 2003) and in mice that are hypo-

morphic for the AHR’s dimerization partner, ARNT

(Walisser et al. 2004).

Dioxin toxicity appears to be fundamentally a conse-

quence of dysregulation of gene expression mediated by

the AHR (Okey et al. 2005). Previous studies using con-

ventional biochemical and molecular methods along with

more recent mRNA expression array methods have

revealed numerous genes that are regulated by the AHR or

that exhibit altered expression following TCDD exposure

in cell culture (Frericks et al. 2006; Frueh et al. 2001;

Karyala et al. 2004; Lee et al. 2006; Martinez et al. 2002;

Puga et al. 2000; Schwanekamp et al. 2006; Zeytun et al.

2002) or in vivo (Boverhof et al. 2005, 2006; Fletcher et al.

2005; Hayes et al. 2005, 2007; Ovando et al. 2006; Slatter

et al. 2006; Tijet et al. 2006; Vezina et al. 2004). A few

dioxin-responsive genes have been identified that may be

central to thymic toxicity (Boverhof et al. 2004; Frericks

et al. 2006; Kolluri et al. 1999) or to hepatotoxicity

(Fletcher et al. 2005; Pande et al. 2005). However, the key

genes whose dysregulation by dioxins underlie the lethal

effects of dioxin remain elusive despite rapid progress in

recent studies in this field.

To identify the genes relevant to susceptibility to TCDD

lethality, we coupled mRNA expression microarray anal-

ysis with an in vivo genetic model of dioxin resistance.

Specifically, we contrasted mRNA expression profiles in

rats that are highly resistant to lethality from TCDD with

expression profiles in rats that are sensitive to TCDD. Our

rationale was that since the AHR is a transcription factor

and also is the primary mediator of dioxin toxicity, those

genes that are mechanistically responsible for major dioxin

toxicities should respond differently to TCDD in dioxin-

resistant rats than in dioxin-sensitive rats.

The Han/Wistar (Kuopio) (H/W) strain is the prototype

resistant rat strain. H/W rats are remarkably resistant to

lethal effects of TCDD with an LD50 of [9,600 lg/kg

compared with LD50 values of 10–50 lg/kg in most

standard laboratory rat strains (Pohjanvirta and Tuomisto

1994). However, for other endpoints (e.g., thymic atrophy,

fetotoxicity, hypercholesterolemia) H/W rats exhibit sus-

ceptibility similar to that of most standard laboratory rat

strains. This prototype rat model is useful for examining

the key mechanisms of specific TCDD toxicities. Important

leads towards understanding the cause of death from

TCDD have emerged from this model including: a poten-

tial role for the CNS in TCDD susceptibility (Pohjanvirta

et al. 1989), identification of a mutant form of the AHR

which is a major determinant of the lethality response

(Pohjanvirta et al. 1999) and evidence of a modifier gene,

gene ‘‘B’’ (of unknown identity), as a subsidiary contrib-

utor to lethality (Tuomisto et al. 1999). Nevertheless, the

events that are key to lethality and associated toxicities

have evaded discovery. We contrasted mRNA expression

between dioxin-resistant rat strains and lines that express

the H/W form of AHR with that in dioxin-sensitive strains

and lines that express wildtype AHR. The resistant rat

constitutes a powerful model system for several reasons:

(1) it is a within-species model; (2) the magnitude of dif-

ference in TCDD response phenotypes between H/W rats

and a substrain of Long–Evans (Turku/AB) (L-E) rats is

very large; (3) only some TCDD response phenotypes

differ between strains while others are conserved. Where

similarities exist between the two strains, these may help to

identify mechanisms of toxicity or adaptive responses that

are common to both sensitive and resistant animals.

Resistance to TCDD lethality in H/W rats is associated

with a genetic variation in the AHR that results in deletion

of either 38 or 43 amino acids from the transactivation

domain of the receptor (Moffat et al. 2007; Okey et al.

2005; Pohjanvirta et al. 1998; Tuomisto et al. 1999).

Because the AHR transactivation domain is crucial to

dioxin toxicity in mice (Bunger et al. 2003), we hypothe-

sized (Okey et al. 2005) that the deletion in the AHR

transactivation domain of H/W rats alters a region of the

AHR protein that is required for dysregulation of the par-

ticular dioxin-responsive genes that are important

determinants of susceptibility to TCDD lethality.

Numerous phenotypic responses to TCDD are similar

between L–E and H/W rats; these are termed ‘‘Type-I’’

responses. In contrast, there are many responses, including

lethality, that differ markedly between rats with wildtype

AHR and rats that carry variant H/W forms of AHR; these

are termed ‘‘Type-II’’ responses (Simanainen et al. 2002,

2003). Our main goal was to identify sets of genes that

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exhibit Type-I responses and sets of genes that exhibit

Type-II responses to TCDD using mRNA expression

arrays. Type-I response genes may be relevant to toxicity

endpoints that are widespread among rat strains or that

represent common adaptive mechanisms and may lead to

insight into new aspects of AHR function. Type-II response

genes are potentially central components in the mecha-

nism(s) that determine susceptibility to dioxin lethality.

Knowledge of the mechanisms by which dioxins cause

toxicity in animal models may facilitate assessment of

dioxin risk to humans (Tuomisto 2005).

Materials and methods

Overview of experiments

Table 1 provides a summary of animal treatments and

mRNA expression analyses.

Chemicals

TCDD was purchased from the UFA-Oil Institute (Ufa,

Russia) and was [99% pure as determined by gas chro-

matography–mass spectrometry (Vartiainen et al. 1995).

TCDD was dissolved in ether and added to corn oil; the

ether was subsequently evaporated off.

Resistant rat model

Responses of mRNA levels to TCDD were compared

among three dioxin-sensitive rat strains and lines versus

three dioxin-resistant rat strains and lines. TCDD-sensi-

tive rats (‘‘sensitive collective’’) comprised L–E, Line-C

(LnC) and Sprague Dawley (SD) rats; TCDD-resistant

rats (‘‘resistant collective’’) comprised H/W, Line-A

(LnA) and F1 offspring of a L–E 9 H/W cross. LnA and

LnC rats originally were developed by multiple crosses

between H/W and L–E rat strains combined with phe-

notyping for susceptibility to TCDD lethality (Tuomisto

et al. 1999). LnA rats are similar to H/W rats in their

resistance to TCDD lethality (LD50 for males

[10,000 lg/kg) and LnC rats are similar to L–E rats in

their sensitivity (LD50 for males *40 lg/kg) (Tuomisto

et al. 1999). The main determinant of sensitivity or

resistance in these lines is the AHR isoform (Tuomisto

et al. 1999). LnA differs from LnC at the AH locus but

these lines have fewer differences at other genetic loci

than do the prototype L–E and H/W strains. Therefore,

LnA, LnC and F1 rats were included in the study to

reduce the impact of confounding loci to help to distin-

guish those genes that are toxicologically relevant from

those that are strain-specific. Sprague Dawley rats were

included as an independent sensitive strain

(LD50 *50 lg/kg). L–E, LnC and SD are homozygous

for the wildtype form of the AHR. H/W and LnA are

homozygous for the variant AHR that has a deletion in

the transactivation domain. F1 rats are heterozygous.

Initially, 50 lg/kg TCDD was used in the Clontech 1.2 k

experiments. Subsequently, the dose was increased to

100 lg/kg TCDD to ensure that the LD50 for all three

sensitive strains was exceeded. The duration of treatment

in all experiments was selected to allow detection of both

up-regulation and down-regulation of mRNA levels. The

duration of TCDD exposure was shortened slightly from

the initial 24 h (Clontech 1.2 k experiments) to 19 h (all

other experiments) for operational convenience. The liver

was selected for study since the liver is a major target for

TCDD toxicity and is differentially susceptible to TCDD

toxicity in the rat model (mild hepatotoxicity in H/W;

Table 1 Overview of the experimental design

Experiment

Clontech 1.2 k Clontech and

OCI 15 k

RT-PCR dose–response RT-PCR

time-course

Ahr-null mice

Strain/line L–E and H/W L–E and H/W L–E and H/W, LnC and

LnA (100 lg/kg

TCDD only)

L–E and H/W C57BL/6 versus Ahr-null

Treatment TCDD 50 lg/kg TCDD 100 lg/kg TCDD 0.001, 0.01, 0.1,

1, 10, 100, 1,000 and

3,000 (H/W only)

lg/kg

TCDD 100 lg/kg TCDD 1,000 lg/kg

Age of males (weeks) 10–12 10–12 10–12 10–12 10–15

Time of liver harvest (h) 24 19 h 19 h 3, 6, 10 or 19 h 19 h

Method of RNA

isolation

Absolutely RNA

(Stratagene)

Absolutely RNA

(Stratagene)

RNeasy (Qiagen) RNeasy (Qiagen) RNeasy (Qiagen)

Arch Toxicol (2008) 82:809–830 811

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severe hepatotoxicity in L–E). The tissue responsible for

animal death following TCDD exposure is unknown.

Male rats, 10–12 weeks of age, were obtained from

breeding colonies at the National Public Health Institute,

Division of Environmental Health, Kuopio, Finland. The

rats were housed in stainless steel wire-mesh cages with

unlimited access to standard animal feed (R36, Ewos,

Sodertalje, Sweden) and tap water. Lights were on between

7:00 and 19:00. The ambient room temperature and

humidity were maintained at 21.5 ± 1�C and 55 ± 10%,

respectively.

mRNA expression array studies

Three independent expression array platforms were used as

hypothesis-generating screening tools and did not consti-

tute a comprehensive investigation of the entire known rat

transcriptome. Subsequently, mRNA levels for selected

genes were measured by real-time quantitative RT-PCR.

Clontech 1.2 Rat Arrays

Initially, the effect of TCDD on mRNA expression was

examined in L–E and H/W rats only, using Clontech

Atlas� Rat 1.2 nylon membrane array. This array contained

1,185 cDNA fragments (200–600 bp in length) represent-

ing rat genes of known identity. The rats were given a

single dose of 50 lg/kg TCDD or the corn oil vehicle by

gavage, and then euthanized by decapitation after 24 h. To

minimize the impact of biological variability, the livers

from three rats were pooled within each treatment group

and total RNA was isolated from this pool using the

Stratagene (La Jolla, CA, USA) Absolutely RNA� Mini-

prep Kit with on-column DNase treatment according to the

manufacturer’s instructions. The RNA yield was quantified

by UV spectrophotometry (A260/280 in TE buffer[1.9) and

RNA integrity was verified using an Agilent 2100 Bioan-

alyzer with an electropherogram peak integration ratio

C1.7 for the 18S and 28S ribosomal RNA constituents. The

mRNA enriched from 50 lg of total RNA was reverse-

transcribed to incorporate [a-32P]dATP with a cocktail of

primers specific to only those genes spotted on the array

using the Atlas� Pure Total RNA Labeling System

(Clontech, Laboratories Inc., Palo Alto, CA, USA). Radi-

olabeled cDNAs from each strain (L–E or H/W) and

treatment group (control or TCDD-treated) were hybrid-

ized to separate membranes. Hybridization and washing

were performed according to the manufacturer’s instruc-

tions. Radioactive emissions were captured with a single

phosphor screen (48 h) and digitized using a Storm Phos-

phorimager (Molecular Dynamics, Sunnyvale, CA, USA).

Image intensities were quantitated with Atlas Image 2.0

software. Quantitation parameters were: signal

threshold = 200% of background; global normalization

(sum method); ratio threshold = 1.5 9 background; dif-

ference threshold = 2 9 average background; background =

mean signal intensity of non-spotted area of the array. Fold

changes between TCDD-treated and control animals of a

given strain were calculated, except when the signal for the

control animal was below the detection limit specified by

Clontech software. In these cases the relative differences

were calculated. Since there was only one measurement for

each strain with Clontech 1.2 k arrays, no tests of statistical

significance could be performed.

Clontech 4 k Rat Arrays

In a second experiment we examined mRNA expression

using Clontech Atlas� 4 k Rat Plastic Arrays which con-

tained 80 bp oligonucleotides from 4,000 rat genes. The

L–E and H/W rats were given a single dose of 100 lg/kg

TCDD or corn oil vehicle by gavage; after 19 h, RNA was

isolated from approximately 150 mg frozen liver from

individual rats using Stratagene’s Absolutely RNA� Mini-

prep Kit (with on-column DNase treatment), according to

the manufacturer’s instructions. The RNA quality was

measured as described above. Ten micrograms of total RNA

from one TCDD-treated rat or from a pool of six control rats

from each rat strain (L–E or H/W), was reverse-transcribed

to incorporate [a-33P]dATP with a cocktail of random

primers using the Atlas� Pure Total RNA Labeling System

(Clontech, Laboratories Inc.). Radiolabeled cDNAs from

each strain (L–E or H/W) and treatment group (control or

TCDD-treated) were hybridized to separate membranes.

Hybridization, washing, imaging and data analysis were as

described above for the Clontech 1.2 k arrays.

OCI 15 k mouse cDNA arrays

In a third experiment, we examined mRNA expression

using cDNA-spotted glass arrays manufactured by the

Ontario Cancer Institute (OCI, Toronto, Canada) that con-

tain *15,000 cDNA sequences, each spotted in adjacent

duplicates. Source clones for the OCI cDNA arrays were

from the National Institute on Aging (NIH, USA) (Tanaka

et al. 2000). Spotted products represent clones derived from

mouse mRNAs that are expressed in pre- and peri-implan-

tation embryos, E12.5. Clones had an average insert size of

*200 bp. A mouse array was used since a comparable rat

array was not available at the time of the investigation. The

RNA samples from L–E and H/W rats used in the Clontech

4 k experiments and RNA samples from the additional four

rat strains/lines (RNA extracted by the same method) were

analyzed using the OCI cDNA arrays.

There were six rats per treatment group (control or

TCDD-treated). For each sample, 15 lg total RNA was

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reverse-transcribed with SuperScript� II RT enzyme

(Invitrogen Life Technologies, Burlington, ON, Canada) to

incorporate either Cyanine3-dCTP (Cy3) or Cyanine5-

dCTP (Cy5). Reverse transcription, sample purification,

hybridization and washing were according to the UHN

Microarray Centre’s direct labeling protocol as described at

http://www.microarrays.ca/support/PDFs/Directlabeling%

20protocol_GFX_Oct2004.pdf.

We used an indirect reference experimental design

(Yang and Speed 2002) with aliquots from all six control

animals of a given strain pooled to generate a strain-spe-

cific reference sample (Fig. 1). Labeling of the reference

with Cy3 was balanced equally with Cy5 labeling. Spe-

cifically, three animals were labeled with Cy5 and three

with Cy3 (‘‘fluor flips’’) in each treatment group. Fluores-

cence from the hybridized arrays was captured with an

Axon GenePix� 4000A scanner (Axon Instruments, Foster

City, CA, USA) and the resulting 32-bit TIFF images were

quantitated with GenePix Pro 3.0 (Axon Instruments) with

photomultiplier tube voltages adjusted for optimal signal-

to-background, maximization of the dynamic range and

balance between the mean overall intensities of the two

channels (ratio = 1 ± 0.1). Output files were then parsed

with Perl scripts and loaded into a custom-built Oracle

relational database. We employed Bayesian background

correction (Kooperberg et al. 2002) with quantile

smoothing (Bolstad et al. 2003) and robust smoothing

splines for normalization (Workman et al. 2002). General

linear models were fitted to the normalized data, using the

limma package (v1.7.4) of BioConductor in the R language

(v1.9.1). From the linear models contrasts, the TCDD-

effect (treatment vs. control) and the dye-effect were

extracted. To these values, an empirical Bayes moderation

of the standard error along with a false discovery rate

adjustment (Storey and Tibshirani 2003) were applied. For

each spot, a P value and fold-change were calculated

(Smyth 2003). An adjusted P value of 0.05 was set as the

threshold for significance. To classify genes as Type-I or

Type-II responses, each gene was assigned two scores: the

Type-I score was calculated as the absolute value of the

sum of the signs of the statistically significant changes

across all six strains/lines in log space; the Type-II score

was calculated as the absolute value of the difference

between the sum of the signs of the statistically significant

changes between the two collectives in log space according

to the equations:

Type I Score ¼X

strains

sgn ðFoldChangeÞjj

Type II Score ¼ jX

resistant

sgn ðFoldChangeÞ

�X

sensitive

sgn ðFoldChangeÞj

Type-I responses were identified as genes exhibiting a

Type-I score C4 along with a Type-II score B1 (indicating

significant change in at least four strains/lines). Type-II

responses were identified as genes with a Type-I score B2

along with a Type-II score C2 (indicating significant

change in two strains of one collective, and no strain in the

other collective).

To link a putative gene-identity to each cDNA clone on

the array we employed a cluster-assignment algorithm

using UniGene Build Rn.124. Briefly, each cDNA was

BLASTed against dbEST and the top-ranked hits were

matched to specific UniGene builds.

In silico search for AH response elements

near dioxin-responsive genes

Genes for which genomic sequences were available in the

public domain (Gibbs et al. 2004) were scanned for the

presence of AH response elements (AHREs) in the 50-flanking region; the specific sequences and search method are

as described previously (Boutros et al. 2004; Tijet et al.

Individualanimals

Referencesample

Vehicle Control TCDD - Single dose 100 µg/kg

C1 C2 C3 C4 C5 C6

3 ratios 3 ratios

Control values ( n = 6 )

Pool of C1 to C6

labeled with Cy3Pool of C1 to C6

labeled with Cy5

label with Cy5label with Cy3

T1 T2 T3 T4 T5 T6

3 ratios 3 ratios

Treated values ( n = 6 )

Pool of C1 to C6

labeled with Cy3Pool of C1 to C6

labeled with Cy5

label with Cy5label with Cy3

Fig. 1 Experimental design for

the OCI mRNA expression

array experiment. Within each

strain, RNA aliquots from all six

control animals (C) were pooled

to generate a strain-specific

reference sample. The reference

sample was co-hybridized with

labeled cDNA from each

individual animal in control and

in treated groups (T). Three

animals were labeled with Cy5

and three with Cy3 (reciprocal

fluor labeling) within each

treatment group

Arch Toxicol (2008) 82:809–830 813

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2006). Subsequently, a PhyloHMM conservation score was

associated with each nucleotide base within each identified

AHRE-motif; then the average PhyloHMM score for each

motif was calculated (Siepel and Haussler 2004). Phyl-

oHMM scores provide a measure of conservation that

accounts for phylogenetic relationships across different

species. Scores can range from 0 (minimal conservation) to 1

(strong conservation). A high degree of phylogenetic con-

servation across multiple mammalian species signifies motifs

that are most likely to constitute functional regulatory sites.

Real-time RT-PCR

We used two real-time RT-PCR methods to quantitate the

levels of selected mRNAs. We selected both Type-I and

Type-II candidate responses for further study. Please see

Supplementary material for primer sequences (Table S1)

and details of the RT-PCR procedure.

Dose–response and time-course analysis of mRNA

expression

To determine the relationship of TCDD dose to the

response of selected genes, four rats per group were ga-

vaged with corn oil (vehicle control) or with doses of

0.001, 0.01, 0.1, 1, 10, 100 and 1,000 lg/kg TCDD for the

dioxin-sensitive L–E rats. For the dioxin-resistant H/W rats

the range was expanded to 3,000 lg/kg TCDD, the maxi-

mal dose that could be given by the method used. Rats

were euthanized by decapitation 19 h after dosing.

For the time-course analysis, rats were treated by gavage

with corn oil or with 100 lg/kg TCDD. Four animals were

treated per time-point and dose-group. Rats were euthanized

by decapitation at 3, 6, 10 or 19 h after dosing. Total RNA

was isolated from approximately 150 mg frozen rat liver

using RNeasy kits (Qiagen, with on-column DNase treat-

ment) according to manufacturer’s instructions. Total RNA

yield was quantified by UV spectrophotometry and RNA

integrity was verified using an Agilent 2100 Bioanalyzer.

RT-PCR was performed as described in Supplementary

material using the 50fluorogenic probe method. Dose–

response curves and ED50 values for alteration of mRNA

levels were generated with Graph Pad 4.0 software using a

sigmoidal four-parameter logistic regression. The R2 values

were, CYP1A1: 0.95 for L–E and H/W; Nfe2l2: 0.58 for L–E

and 0.51 for H/W; Tgfb1i4: 0.76 for L–E and 0.82 for H/W.

Ahr-null mice

To determine if the AH receptor was essential for candidate

genes (identified by expression array studies) to respond to

TCDD, we tested the ability of TCDD to alter expression of

some genes (Cyp1a1, Nfe2l2, Tgfbp1i4 and Polr2c) in

livers of Ahr-null (Ahr-/-) mice. Details of the treatment

and RNA isolation were as reported previously (Tijet et al.

2006). Briefly, male Ahr-/- mice in a C57BL/6J back-

ground as well as wild-type (Ahr+/+) C57BL/6J mice were

given a single dose of 1,000 lg/kg TCDD or corn oil

vehicle by gavage. Liver was harvested 19 h after treat-

ment. There were three TCDD-treated and three control

mice in the Ahr-/- groups and six TCDD-treated and five

control mice in the Ahr+/+ groups. RT-PCR using the

50fluorogenic probe method was performed as described in

Supplementary material.

Results

Response of classic AHR-responsive genes detected

by Clontech arrays

Initial experiments with Clontech Atlas� Rat 1.2 Arrays

demonstrated that four classic AHR-regulated/TCDD-

responsive genes—CYP1A1, CYP1A2, CYP1B1 and

Gsta3—continue to be significantly upregulated by TCDD in

H/W rats despite loss of a large segment from the AHR

transactivation domain in this dioxin-resistant strain

(Fig. 2). The magnitudes of induction (ratio of TCDD-

treated to control) for these four well-known AH-regulated

genes were similar between the prototype dioxin-sensitive

L–E strain and the prototype dioxin-resistant H/W strain:

approximately 16-fold for CYP1A2 and threefold for Gsta3.

Fold-induction could not be computed for CYP1A1 or

CYP1B1 because the constitutive mRNA levels in control

L–E and control H/W animals were indistinguishable from

background for each of these genes. However, the spot

intensity for CYP1A1 in TCDD-treated L–E rats as well as in

H/W rats was approximately 1,000-times higher than back-

ground levels. For CYP1B1, TCDD-treated L–E and H/W

rats both exhibited signals approximately 30-times higher

than background. Only one experiment was performed on

Clontech 1.2 k arrays. A second experiment on Clontech

Atlas� 4 k Rat Arrays confirmed that CYP1A1, CYP1A2,

CYP1B1 and Gsta3 were similarly induced by TCDD

treatment in H/W rats as well as in L–E rats (data not shown).

Overall patterns of mRNA expression detected by OCI

cDNA arrays

Of the 15,297 total unique clones on the mouse-derived

OCI cDNA array (Table 2), 10,533 (69%) could be

assigned to a rat UniGene Cluster ID based on nucleotide

sequence. Since some clusters were represented by multi-

ple clones, the actual number of unique clusters examined

was 7,538; this represents the estimated number of unique

genes screened for responsiveness to TCDD in our OCI

814 Arch Toxicol (2008) 82:809–830

123

Page 7

cDNA experiments. Of the 7,538 unique UniGene clusters,

398 (5.3%) responded significantly (Padjusted \ 0.05) to

TCDD treatment in at least one rat strain or line (Fig. 3;

Table 2) of which no more than 20 would be expected to be

false-positives based on the adjusted statistical threshold

employed. However, only 13 of the 7,538 total clusters on

the array responded significantly to TCDD treatment in all

six strains/lines (Table 3).

We classified TCDD-responsive genes detected on

cDNA arrays into two categories, using the scheme pre-

viously developed for toxic endpoints by Simanainen et al.

(2002): Type-I responses are defined as responses to TCDD

that are similar between dioxin-sensitive and dioxin-resis-

tant rat strains/lines. Type-II responses are defined as

responses to TCDD that show reduced efficacy (magnitude

of effect) in dioxin-resistant rat strains/lines.

As shown in Table 2, the majority of genes that

responded to TCDD could not be assigned to a specific

Type-I or Type-II category and were classified as

‘‘Ambiguous’’ (i.e., Type-I Score = 4; Type-II Score = 2).

Of those genes that could be classified, there were more

Type-I responders than Type-II responders (Table 3).

For some genes, multiple clones representing different

regions of the transcript were present on the array; the

similarity of response for multiple independent clones

gives added confidence in the validity of the TCDD effect

for these genes. In particular, three clones representing

ferritin light chain 1 (Ftl1), three clones representing S-

adenosylhomocysteine hydrolase (Ahcy) and two clones

representing a cluster similar to ribosomal protein S18,

cytosolic-rat showed induction by TCDD. Two clones

representing glutamate dehydrogenase 1 (Glud1) and two

clones representing transforming growth factor beta 1

induced transcript 4 (Tgfb1i4) showed repression

(Table 3).

Type-I response genes

In total, 38 clones responded significantly to TCDD in

dioxin-sensitive as well as in dioxin-resistant collectives on

OCI cDNA arrays (upper portion of Table 3); i.e., repre-

sent Type-I responses. These clones represent 32 unique

UniGene clusters and 18 RGD (Rat Genome Database)

genes (Table 2). As was seen with Clontech arrays, the

OCI cDNA arrays revealed that several genes that are well

known to be upregulated by AHR agonists were induced

independently of the dioxin-sensitivity phenotype. These

include (Table 3) CYP1B1 (Zhang et al. 2003), Nfe2l2

(Miao et al. 2005; Tijet et al. 2006) and UGT1A6 (Bock

and Kohle 2005b). Clearly the AHR transactivation-

domain deletion in H/W and LnA rats does not prevent

induction by TCDD of these standard AHR-regulated

genes.

Fig. 2 Response in dioxin-sensitive L–E rats and dioxin-resistant H/

W rats of genes that are well known to be AHR-regulated and TCDD-

responsive. Rats were treated with a single TCDD dose of 50 lg/kg or

the corn oil vehicle as a control. Liver was harvested 24 h after

TCDD and RNA was pooled from three animals within each

treatment and strain. mRNA levels were determined with Clontech

1.2 k arrays as described in ‘‘Materials and methods’’. The figures

show only a sub-section from each array that contains four classic

AHR-regulated/dioxin-responsive genes: CYP1A1, CYP1A2, CYP1B1and Gsta3. The mRNA level for each of these genes is represented by

the intensity of the spots encircled by dotted lines

Table 2 Summary of the gene

clusters on OCI cDNA arrays

and their responsiveness to

TCDD

Number of

clones/clusters

Percent of all

assignable clones

Percent of

unique clusters

Total unique clones per array 15,297 100 N/A

Total clones assignable to UniGene clusters 10,533 68.9 N/A

Total unique UniGene clusters (i.e., number

of genes screened)

7,538 71.6 100

UniGene clusters that significantly respond to

TCDD in at least one strain (P \ 0.05)

398 3.8 5.3

Type-I responsive named genes 18 0.17 0.24

Type-II responsive named genes 10 0.10 0.13

Arch Toxicol (2008) 82:809–830 815

123

Page 8

In addition to detecting upregulation of classic AHR-

mediated genes, the OCI cDNA arrays also identified

multiple novel genes, not previously known to be affected

by TCDD, whose expression was altered by TCDD in at

least four of the six rat lines/strains tested. For genes that

exhibited this Type-I response, approximately half were

upregulated by TCDD and half were downregulated

(Table 3).

Type-II response genes

Genes that responded differently between dioxin-sensitive

and dioxin-resistant collectives on OCI cDNA arrays are

listed in the lower portion of Table 3. These 22 clones

represent 15 unique UniGene clusters and 10 RGD genes

(Table 2). The magnitude of the TCDD effect on mRNA

levels generally was much lower for Type-II response genes

than for Type-I response genes. No gene showed a striking

difference in magnitude of response between sensitive and

resistant collectives. Consequently, no Type-II genes from

the OCI cDNA arrays were selected for further follow-up by

RT-PCR. However, it cannot be excluded that some genes

in the Type-II group may be involved in susceptibility to the

lethal effects of TCDD since the magnitude of change in

mRNA expression does not necessarily reflect the biologi-

cal impact of that change on a given phenotype.

Ambiguous response genes

Six unique clones could not be unambiguously classified as

either Type-I or Type-II responses. These are shown in the

middle portion of Table 3, classified as ‘‘Ambiguous’’. For

glutamate dehydrogenase 1 (Glud1) and transforming

growth factor beta 1 induced transcript 4 (Tgfb1i4), a

second clone on the array for each of these genes emerged

under Type-I, potentially resolving the ambiguous classi-

fication (Table 3).

Measurement of mRNA levels by real-time quantitative

RT-PCR

To evaluate the validity of the microarray results, 20 genes

were selected for assessment of TCDD responsiveness by

an independent method, real-time RT-PCR (Fig. 4). These

genes were selected primarily based on their plausible

involvement in biological pathways related to toxic

responses to dioxins. Ten candidate genes that appeared as

TCDD-responsive on Clontech 4 k arrays (CYP1A1, Sdc1,

Mt1, Mt2l, Srd5a1, Lcat, Pc, Slc2a2, Tat, Igfbp) and ten on

the OCI cDNA arrays (All Type-I: Nfe2l2, Tgfb1i4, Bgn,

Glud1, Aldh3a2, Polr2c, H3144A09, Ces3, H3119G08,

Ftl1) were selected for validation by RT-PCR. In most

instances mRNA levels were quantitated by RT-PCR only

in the prototype sensitive (L–E) and resistant (H/W)

strains. However, for some genes, mRNA levels also were

quantitated in additional sensitive or resistant lines/strains

(Fig. 4).

CYP1A1 served as a benchmark for the effectiveness of

TCDD treatment since CYP1A1 is a well-established

Type-I response gene that is highly inducible via the AHR

pathway (Denison and Whitlock 1995; Nebert et al. 2004;

Okey et al. 2005). CYP1A1 mRNA was below detection

limits in livers from control dioxin-sensitive rats (L–E and

LnC) as well as in livers from control dioxin-resistant rats

(H/W and LnA). CYP1A1 mRNA was highly induced by

TCDD treatment in all four of these rat strains/lines as

quantitated by RT-PCR (Fig. 4a); hence CYP1A1 was

confirmed to be a Type-I gene.

In general, the RT-PCR measurements corroborated the

responses that were detected by microarray analyses.

However, for certain genes the RT-PCR analyses showed

some degree of discordance with the cDNA array results.

For biglycan (Bgn), the OCI cDNA array analyses indi-

cated significant upregulation by TCDD in all six rat

strains/lines (Table 3) but RT-PCR analyses performed in

L–E and H/W did not detect a significant change in Bgn

mRNA level in either strain (Fig. 4a); for Polr2c (classified

as Type-I by arrays and as Type-II by RT-PCR) results

were discordant for three of four strains tested (L–E, LnC,

H/W) (Table 3; Fig. 4b); for Ces3 (classified as Type-I by

arrays and Type-II by RT-PCR) results were discordant for

H/W, although only L–E and H/W were tested by RT-PCR

(Table 3; Fig. 4b). For some genes, RT-PCR resolved the

ambiguous genes: for Glud1, one clone was classified as

Type-I and the other as ambiguous by the array while

Fig. 3 Summary of the number of TCDD-responsive UniGene

clusters within the collective of dioxin-sensitive rats and the

collective of dioxin-resistant rats as detected on OCI cDNA arrays

The numbers in each sector of the Venn diagrams represent the

numbers of UniGene clusters whose expression was significantly

affected (P \ 0.05) by TCDD treatment in each rat strain or line. Data

were obtained from experiments with OCI cDNA arrays as described

in Materials and methods

816 Arch Toxicol (2008) 82:809–830

123

Page 9

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Arch Toxicol (2008) 82:809–830 817

123

Page 10

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818 Arch Toxicol (2008) 82:809–830

123

Page 11

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E-0

1 0.

34.

10E

-01

0.4

1.10

E-0

30.

33.

70E

-02

Rn.

1076

90N

A

NA

N

A

NA

28

4 0

mic

rotu

bule

-as

soci

ated

pro

tein

s 1A

/1B

ligh

t cha

in 3

H

3082

B10

4

0 1

5.70

E-0

60.

63.

10E

-02

0.9

1.40

E-0

1 0.

51.

20E

-01

0.6

3.70

E-0

30.

43.

70E

-02

Rn.

4141

2-0

.259

-0

.005

0-0

.027

Sim

ilar

to h

ypot

hetic

al

gene

sup

port

ed b

y B

C00

7071

H

3125

B06

4

0 0.

7 1.

30E

-05

0.4

7.50

E-0

2 0.

43.

00E

-03

0.3

3.70

E-0

1 0.

5 1.

10E

-03

0.4

1.20

E-0

2R

n.33

22

NA

N

A

NA

N

A

11

1 1

3 S

imila

r to

ald

ehyd

e de

hydr

ogen

ase

ALD

H3B

1(A

ldh

3b1)

H31

49A

06

4 0

0.1

5.00

E-0

11

1.50

E-0

31.

42.

30E

-05

0.7

7.50

E-0

2 0.

4 1.

40E

-02

0.8

4.80

E-0

5R

n.27

730

-0.9

99

-0.9

46-0

.27

-0.2

96

18

3 A

min

o ac

id tr

ansp

ort

syst

em A

3 H

3026

D12

4

0 -0

.5

9.40

E-0

3-0

.66.

40E

-02

-0.9

1.20

E-0

2-0

.71.

00E

+00

-0

.6

1.20

E-0

2-0

.74.

00E

-02

Rn.

1799

0-0

.249

-0

.001

0 0

Glu

tath

ione

pero

xida

se 1

H

3026

F02

4

0 -0

.4

4.70

E-0

2-0

.76.

40E

-04

-0.4

4.90

E-0

1 -0

.42.

60E

-01

-0.8

2.

10E

-04

-0.7

3.20

E-0

3R

n.11

323

NA

N

A

NA

N

A

Ser

ine

prot

ease

in

hibi

tor

H30

52F

12

4 0

-0.6

3.

30E

-05

-0.4

1.80

E-0

2-0

.41.

10E

-01

-0.3

4.10

E-0

1 -0

.4

4.20

E-0

3-0

.42.

90E

-02

Rn.

128

NA

N

A

NA

N

A

Tra

nsfo

rmin

g gr

owth

fa

ctor

bet

a 1

indu

ced

tran

scrip

t 4 (

Tg

fb1i

4)H

3057

G12

4

0 -0

.8

3.60

E-0

2-1

.45.

60E

-06

-1.3

5.80

E-0

1 -1

.54.

40E

-02

-0.9

1.50

E-0

2-0

.92.

80E

-01

Rn.

3545

N

A

NA

N

A

NA

NA

H

3101

H05

4

0 -0

.4

1.60

E-0

3-0

.75.

50E

-04

-0.3

7.00

E-0

2 -0

.33.

70E

-01

-0.6

1.

10E

-02

-0.5

3.20

E-0

2N

A

NA

N

A

NA

N

A

Sim

ilar

to E

25B

pr

otei

nH

3120

E02

4

0 -0

.3

6.60

E-0

4-0

.34.

00E

-02

-0.2

1.00

E+

00

-0.3

5.20

E-0

1 -0

.3

4.20

E-0

3-0

.41.

60E

-02

Rn.

1073

35N

A

NA

N

A

NA

13

Hea

t-re

spon

sive

pr

otei

n 12

H

3122

B07

4

0 -0

.3

2.40

E-0

2-0

.48.

50E

-04

-0.3

3.90

E-0

1 -0

.32.

40E

-01

-0.3

5.

00E

-03

-0.5

1.80

E-0

4R

n.69

87-0

.027

0 0

0

Arch Toxicol (2008) 82:809–830 819

123

Page 12

Ta

ble

3co

nti

nu

ed

Tra

nscr

ibed

seq

uenc

e w

ith m

oder

ate

sim

ilarit

y to

pro

tein

sp

:P04

732

(H.s

apie

ns)

MT

1E_h

uman

met

allo

thio

nein

-1E

(MT

-1E

) H

3013

D11

4

2 0.

5 2.

20E

-01

1.4

1.20

E-0

21.

16.

60E

-01

3 9.

00E

-04

1.3

9.30

E-0

31.

76.

90E

-03

Rn.

1155

49N

A

NA

N

A

NA

36

11

2 1

Cyt

ochr

ome

P45

0,

subf

amily

1B

, po

lype

ptid

e 1

(CY

P1B

1)H

3053

A08

4

2 3.

5 1.

50E

-06

1.9

3.40

E-0

22.

91.

80E

-04

1.1

1.00

E+

00

1.6

1.80

E-0

31.

37.

90E

-02

Rn.

1012

5*-1

-1

-0

0

9 2

1 A

ldeh

yde

dehy

drog

enas

e fa

mily

3,

sub

fam

ily A

2 (A

ldh

3a2)

H31

23B

09

4 2

0.7

5.10

E-0

30.

79.

00E

-04

0.7

2.30

E-0

30.

31.

00E

+00

0.

5 3.

10E

-02

0.4

2.70

E-0

1 R

n.91

13

-0.0

02

-0.0

020

-0.0

01

embr

yo-r

elat

ed p

rote

in H

3159

F04

4

2 0.

6 3.

40E

-02

0.6

8.50

E-0

40.

74.

50E

-03

0.2

1.00

E+

00

0.2

7.50

E-0

1 0.

53.

30E

-02

Rn.

9008

6N

A

NA

N

A

NA

17

4 2

1 G

luta

mat

ede

hydr

ogen

ase

1 (G

lud

1)

H30

03F

06

4 2

-1.1

1.

50E

-07

-15.

50E

-03

-12.

30E

-03

-0.8

6.00

E-0

3-0

.9

1.70

E-0

1 -0

.51.

00E

+00

R

n.55

106

-1

-1

-0.1

6-0

.002

Ambiguous Tra

nsfo

rmin

g gr

owth

fa

ctor

bet

a 1

indu

ced

tran

scrip

t 4 (

Tg

fb1i

4)

H31

12G

06

4 2

-0.6

2.

80E

-02

-1.2

6.40

E-0

4-1

2.00

E-0

2-1

.26.

00E

-03

-0.7

2.

20E

-01

-0.8

5.20

E-0

2 R

n.35

45

NA

N

A

NA

N

A

NA

H

3011

C09

2

2 0

9.70

E-0

10.

25.

70E

-01

0.2

1.00

E+

00

0.1

1.00

E+

00

0.3

1.40

E-0

30.

33.

00E

-02

NA

N

A

NA

N

A

NA

Mal

ic e

nzym

e 1

H31

07D

02

2 2

1.1

2.10

E-0

11.

21.

80E

-01

1.1

1.00

E+

00

0.9

1.00

E+

00

1.3

3.30

E-0

21.

67.

60E

-03

Rn.

3519

N

A

NA

N

A

NA

S

imila

r to

RIK

EN

cD

NA

D43

0028

G21

H

3125

G07

2

2 0.

2 6.

00E

-01

0.2

1.00

E+

00

1.3

6.90

E-0

1 1

1.00

E+

00

1.1

1.60

E-0

21.

51.

70E

-02

Rn.

3499

6N

A

NA

N

A

NA

NA

H

3108

D04

2

2 0.

4 1.

60E

-02

0.4

2.20

E-0

20.

11.

00E

+00

0.

21.

00E

+00

0.

2 2.

20E

-01

0.3

2.30

E-0

1 N

A

NA

N

A

NA

N

A

Ubi

quiti

n C

H

3124

H09

2

2 0.

4 7.

40E

-04

0.4

1.60

E-0

20.

11.

00E

+00

0

1.00

E+

00

-0.1

1.

00E

+00

0.

11.

00E

+00

R

n.37

61

18(1

.000

)1(

0.00

1)0

1(0.

016)

Pro

tein

pho

spha

tase

1,

reg

ulat

ory

(inhi

bito

r)

subu

nit 1

B

H30

97C

11

2 2

0.8

1.30

E-0

21.

36.

80E

-04

0.6

1.00

E+

00

0.8

1.00

E+

00

0.7

8.00

E-0

2 0.

61.

00E

+00

R

n.70

366

NA

N

A

NA

N

A

8 1

1 S

-ad

enos

ylho

moc

yste

ine

hydr

olas

e (A

hcy

)H

3014

E01

2

2 0.

6 2.

60E

-03

0.5

8.10

E-0

30.

49.

90E

-01

0.2

1.00

E+

00

0.3

8.90

E-0

2 0.

21.

00E

+00

R

n.58

78

-1

-1

0-0

.003

Type II TR

AP

-com

plex

ga

mm

a su

buni

t H

3113

A11

2

2 0.

3 5.

70E

-03

0.3

2.60

E-0

20.

28.

40E

-01

0.3

6.80

E-0

1 0.

2 2.

00E

-01

0.2

1.00

E+

00

Rn.

3264

N

A

NA

N

A

NA

820 Arch Toxicol (2008) 82:809–830

123

Page 13

Ta

ble

3co

nti

nu

ed

cath

epsi

n L

H30

28F

03

2 2

0.3

2.40

E-0

20.

54.

40E

-02

0.4

3.90

E-0

1 0.

41.

00E

+00

0.

2 9.

70E

-01

0.2

1.00

E+

00

Rn.

1294

6(

0.02

0)1(

0.00

0)0

1(0.

001)

8 1

1 S

-ade

nosy

l ho

moc

yste

ine

hydr

olas

e (A

hcy

) H

3031

E10

2

2 0.

6 7.

10E

-04

0.7

1.70

E-0

30.

61.

70E

-01

0.3

1.00

E+

00

0.5

1.10

E-0

1 0.

41.

50E

-01

Rn.

5878

-1

-1

0

-0.0

03

8 1

1 S

-ade

nosy

l ho

moc

yste

ine

hydr

olas

e (A

hcy

)H

3144

A12

2

2 0.

5 4.

10E

-04

0.6

2.90

E-0

30.

57.

00E

-02

0.2

1.00

E+

00

0.3

5.90

E-0

2 0.

45.

60E

-02

Rn.

5878

-1

-1

0

-0.0

03

Sim

ilar

to 6

2 kD

a su

buni

t of T

FIIH

H

3108

G05

2

2 0.

6 4.

10E

-02

0.5

3.00

E-0

1 0.

81.

10E

-02

0.6

1.00

E+

00

0.3

1.80

E-0

1 0.

31.

00E

+00

R

n.24

733

NA

N

A

NA

N

A

23

7 1

1 F

errit

in li

ght c

hain

1

(Ftl

1)

H31

15H

06

2 2

0.3

4.80

E-0

30.

11.

00E

+00

0.

43.

10E

-03

0.3

4.60

E-0

1 0.

1 6.

80E

-01

0.2

8.60

E-0

1 R

n.19

05

-0.9

85

-0.9

58-0

.92

-0.0

07

23

7 1

1 F

errit

in li

ght c

hain

1

(Ftl

1)H

3020

B08

2

2 0.

3 6.

60E

-03

0 1.

00E

+00

0.

41.

10E

-03

0.2

1.00

E+

00

0.1

4.10

E-0

1 0.

21.

00E

+00

R

n.19

05

-0.9

85

-0.9

58-0

.92

-0.0

07

23

7 1

1 F

errit

in li

ght c

hain

1

(Ftl

1)H

3023

H11

2

2 0.

5 1.

90E

-03

0.2

6.00

E-0

1 0.

64.

00E

-04

0.4

2.60

E-0

1 0.

3 8.

50E

-02

0.3

2.60

E-0

1 R

n.19

05

-0.9

85

-0.9

58-0

.92

-0.0

07

Sim

ilar

to c

ompl

emen

t co

mpo

nent

1, r

su

bcom

pone

ntH

3136

D05

2

2 0.

2 3.

70E

-01

0.6

4.20

E-0

20.

63.

80E

-02

0.3

1.00

E+

00

0.2

1.00

E+

00

0.3

8.50

E-0

1 R

n.70

397

NA

N

A

NA

N

A

Sim

ilar

to c

hrom

atin

re

mod

elin

g fa

ctor

W

CR

F18

0H

3043

E03

2

2 -0

.3

2.20

E-0

1-0

.33.

70E

-01

-0.3

1.00

E+

00

-0.6

6.00

E-0

3-0

.42.

40E

-02

-0.3

5.80

E-0

1 R

n.18

64

NA

N

A

NA

N

A

NA

H

3034

A07

2

2 -0

.2

9.10

E-0

1-0

.52.

60E

-01

-0.9

1.00

E+

00

-1.1

1.70

E-0

2-0

.65.

00E

-03

-0.3

1.00

E+

00

NA

N

A

NA

N

A

NA

alph

a-2

antip

lasm

in

H31

38B

11

2 2

-0.3

1.

10E

-01

-0.4

9.50

E-0

2 -0

.21.

00E

+00

-0

.31.

00E

+00

-0

.5

1.10

E-0

2-0

.62.

40E

-02

Rn.

1699

3N

A

NA

N

A

NA

S

imila

r to

hyp

othe

tical

pr

otei

n C

LON

E24

945

H31

15D

06

2 2

-0.5

4.

20E

-01

-0.3

4.90

E-0

1 -0

.31.

00E

+00

-0

.61.

00E

+00

-0

.5

2.40

E-0

2-0

.66.

20E

-03

Rn.

4302

N

A

NA

N

A

NA

R

etin

oic

acid

rec

epto

r re

spon

der

(taz

arot

ene

indu

ced)

2

H31

37E

09

2 2

-0.1

3.

90E

-01

-0.2

2.90

E-0

1 -0

.41.

00E

-01

-0.3

2.80

E-0

1 -0

.5

1.70

E-0

5-0

.43.

70E

-02

Rn.

2853

2N

A

NA

N

A

NA

In

terf

eron

indu

ced

tran

smem

bran

e pr

otei

n 3

H

3107

D05

2

2 -0

.6

7.40

E-0

4-0

.54.

60E

-02

-0.6

7.00

E-0

2 -0

.31.

00E

+00

-0

.4

1.20

E-0

1 -0

.55.

60E

-02

Rn.

1071

66N

A

NA

N

A

NA

Fea

ture

sd

isp

lay

ing

Ty

pe-

Io

rT

yp

e-II

pat

tern

so

fre

spo

nse

toT

CD

Dw

ere

iden

tifi

edu

sin

gth

eO

CI

cDN

Aar

ray

s.E

ach

feat

ure

was

ann

ota

ted

toa

Un

iGen

ecl

ust

eran

dE

ntr

ezG

ene

IDu

sin

ga

BL

AS

Tst

rate

gy

(Un

iGen

eB

uil

dR

n.1

24

).In

som

eca

ses,

feat

ure

sco

uld

no

tb

em

app

edto

aU

niG

ene

clu

ster

and

are

des

ign

ated

as‘‘

NA

’’fo

rn

ot

avai

lab

le.

Fo

rea

chst

rain

,th

efo

ld-c

han

ge

of

dif

fere

nti

alex

pre

ssio

nb

etw

een

TC

DD

-tre

ated

and

con

tro

lsa

mp

les

issh

ow

n,

alo

ng

wit

hth

eP

val

ue

ind

icat

ing

the

stat

isti

cal

sig

nifi

can

ceo

fo

bse

rved

chan

ge.

Fo

ld-c

han

ges

that

had

a

sig

nifi

can

cev

alu

eo

fP

adju

sted\

0.0

5o

rb

ette

rar

esh

ow

nin

bo

ldfo

nt.

Fo

ld-c

han

ges

are

giv

enin

log

2sp

ace;

thu

sa

val

ue

of

-2

ind

icat

esth

atth

em

RN

Ale

vel

isfo

urf

old

low

erin

TC

DD

-tre

ated

anim

als

than

inco

ntr

ol

anim

als,

wh

erea

sa

val

ue

of

+3

ind

icat

esei

gh

tfo

ldg

reat

erex

pre

ssio

nin

TC

DD

-tre

ated

than

inco

ntr

ol

anim

als.

Th

eT

yp

e-I

and

Ty

pe-

IIS

core

sw

ere

gen

erat

edas

des

crib

edin

the

‘‘M

ater

ials

and

met

ho

ds’

’.T

yp

e-I

sco

res

rep

rese

nt

the

exte

nt

tow

hic

ha

gen

eis

com

mo

nly

resp

on

siv

eac

ross

all

stra

ins.

Ty

pe-

IIsc

ore

sre

pre

sen

tg

enes

wh

ose

resp

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Arch Toxicol (2008) 82:809–830 821

123

Page 14

RT-PCR corroborated the Type-I classification (Table 3;

Fig. 4a); for Aldh3a2 (classified as ambiguous by array and

as Type-II by RT-PCR) results were discordant for only one

of the four strains tested (LnC) (Table 3; Fig. 4b); for

Tgfb1i4 (classified as ambiguous by arrays and as Type-I by

RT-PCR) results were discordant for two of the four strains

tested (LnA and LnC) (Table 3; Fig. 4a). Some of the dis-

cordance between the analytical approaches may be

explained, in part, by non-specific or poor hybridization of rat

samples to mouse arrays. Of the ten genes selected from the

Clontech 4 k array, five were found to be Type-I (CYP1A1,

Sdc1, Mt1, Mt2l, Slc2a2) and five to be Type-II (Srd5a1,

Lcat, Pc, Tat, Igfbp) by RT-PCR. Of the ten genes selected

from the OCI cDNA array, seven were classified as Type-I

(Nfe2l2, Tgfb1i4, Bgn, Glud1, H3144A09, H3119G08, Ftl)

and three to be Type-II (Aldh3a2, Polr2c, Ces3).

Dose–response and time course of response for selected

genes

The 100 lg/kg TCDD dose given to rats in the cDNA array

experiments exceeds the LD50 for the three dioxin-sensi-

tive strains/lines (L–E, SD and LnC) but is far below the

LD50 for the resistant strains/lines (H/W, F1, LnA). In

order to gain some understanding of potential quantitative

differences in response to TCDD between sensitive and

resistant rats we conducted dose–response experiments

over a wide range of TCDD exposure and measured mRNA

levels by real-time RT-PCR for the benchmark gene

CYP1A1, as well as one up-regulated gene (Nfe2l2) and

one down-regulated gene (Tgfb1i4). As shown in Fig. 5

No

rmal

ized

mR

NA

exp

ress

ion

( p

erce

nt

of

max

imal

res

po

nse

)

Type-I responses

TCDDControl

Glud1

L-E H/W0

60

100

******

Slc2a2

L-E H/W

******

Nfe2l2(Nrf2)

L-E LnC H/W LnA

***

******

Ftl

L-E H/W0

60

100 ** ***Sdc1

L-E H/W

******

Tgfb1i4

L-E LnC H/W LnA0

60

100

* *** **

*

Bgn

L-E H/W

H3144A09

L-E H/W0

60

100 ******

H3119G08

L-E H/W

CYP1A1

L-E LnC H/W LnA0

60

100***

*** *** ***

Mt1a

L-E SD H/W0

60

100 ***

*****

Mt2l

L-E SD H/W

*

***

Srd5a1

L-E H/W0

60

100

*

Ces3

L-E H/W0

60

100

***

Polr2c

L-E LnC H/W LnA0

60

100 ***

Lcat

L-E H/W0

60

100 *

No

rmal

ized

mR

NA

exp

ress

ion

( p

erce

nt

of

max

imal

res

po

nse

) Igfbp

L-E H/W

*

Pc

L-E H/W

**

Tat

L-E H/W

*

Aldh3a2

L-E LnC H/W LnA

***

Type-II responses

Control TCDD

a

b

Fig. 4 Measurement of mRNA levels by real-time RT-PCR. Real-

time QRT-PCR was used to measure mRNA levels for several genes

that had been identified as TCDD-responsive in array experiments.

Genes are classified as Type-I responders (a) or Type-II responders

(b). Animals, treatments and sample preparation are the same as for

the OCI cDNA arrays described in ‘‘Materials and methods’’. For

each gene, the mRNA level that was highest for any strain or

treatment was set at 100% and all other mRNA levels for that gene

are shown as a percentage of the maximal level. All results plotted

represent the mean of four rats ±SD. Asterisks indicate groups where

the mRNA level in TCDD-treated animals differs significantly from

that of the control: *P \ 0.05; **P \ 0.01; ***P \ 0.001. Note that

for Cyp1a1 (a) the mRNA level in control animals is below the

detection limit of the assay; therefore no bars are presented for control

animals in this plot. Abbreviations for gene names: CYP1A1,

cytochrome P4501A1; Nfe2l2, nuclear factor erythroid derived 2;

Mt1a and Mt2l, methallothionein 1a and 2l; Glud1, glutamate

dehydrogenase; Slc2a2, solute carrier family 2, member 2; Ftl1,

ferritin light chain 1; Sdc1, syndecan 1; Tgfb1i4, transforming growth

factor beta 1-induced transcript 4; Bgn, biglycan; H3144A09,

unidentified clone; H3119G08, clone similar to glutathione S-

transferase 8 alpha. (b) Srd5a1, steroid 5a reductase 1; Igfbp,

insulin-like growth factor binding protein; Ces3, carboyxylesterase-3;

Pc, pyruvate carboxylase; Lcat, lecithin cholesterol acyltransferase;

Tat, tyrosine aminotransferase; Polr2c, Polymerase RNA II (DNA-

directed) polypeptide C; Aldh3a2, aldehyde dehydrogenase 3a2

b

822 Arch Toxicol (2008) 82:809–830

123

Page 15

(left panel), dose–response curves for CYP1A1 are super-

imposable for L–E and H/W rats and the curves for the

other two genes are very similar despite the substantial

deletion in the transactivation domain of the AHR in the H/

W rat and despite the fact that these two strains differ

dramatically in their susceptibility to lethality from TCDD.

The estimated ED50 for the response of CYP1A1 and

Tgfb1i4 is about 1 lg/kg, a dose which is several fold

lower than the LD50 dose in male L–E rats and more than

1,000-fold lower than the LD50 dose in H/W rats (Po-

hjanvirta and Tuomisto 1994).

To begin to differentiate candidate genes likely to be

primary ‘‘triggering’’ events in the TCDD response path-

ways from secondary effects of TCDD we used RT-PCR to

examine the time-course of alteration of mRNA expression

in response to TCDD exposure for these three genes at

time-points between 0 and 19 h. Genes that show an

alteration of mRNA expression at early time points fol-

lowing TCDD exposure are leading candidates for further

study. Genes whose expression is altered only at later time

points (days to weeks) potentially represent secondary

effects, perhaps attendant to the onset of toxicity. We did

not assess mRNA expression at time points beyond 19 h in

this study. All three genes appear to be primary events

based on the rapidity of their responses (i.e., changes in

expression were discernible within 3 h post treatment)

(Fig. 5, right panel).

Responses in Ahr-/- mice

We tested the AHR-dependence of the observed expression

changes of CYP1A1, Nfe2l2, Tgfb1i4 and Polr2c in vivo

by comparing mRNA levels in livers from Ahr-/- mice

with those in mice that have wildtype AHR (Ahr+/+) using

real-time QRT-PCR. As shown in Fig. 6, the classic AHR-

regulated gene, CYP1A1, was strongly up-regulated by

TCDD in mice with wildtype AHR but did not respond to

TCDD in Ahr-/- mice. Up-regulation of Nfe2l2 (Nrf2)

also was dependent on the AHR as was down-regulation of

Tgfb1i4 (Fig. 6). Polr2c was significantly up-regulated by

TCDD in dioxin-resistant H/W and LnA rats (Fig. 4a) but

was not significantly affected by TCDD in either wild-type

mice or in Ahr-/- mice (Fig. 6). Several other genes whose

expression was significantly affected by TCDD in our

Cyp1a1

Wild-type Ahr -/-0

20

40

60

80

100 ***Nfe2l2

Wild-type

*

Tgfbp1i4

Wild-type0

20

40

60

80

100

**

Polr2c

Wild-type

No

rmal

ized

mR

NA

exp

ress

ion

( p

erce

nt

of

max

imal

res

po

nse

)

Ahr -/-Ahr -/-

Ahr -/-

Control TCDD

ND ND ND

Fig. 6 Responses of selected genes to TCDD in Ahr-null mice versus

wildtype mice. Wildtype mice (Ahr+/+) or Ahr-null mice in a

C57BL6/J background (Ahr-/-) were treated with corn oil vehicle or

with 1,000 lg/kg TCDD. Liver was harvested 19 h after TCDD and

mRNA levels were quantitated by real-time RT-PCR. There were

three mice per group in the Ahr-/- groups and six mice per group in

the Ahr+/+ groups. For each gene, the mRNA level that was highest

for any strain or treatment was set at 100% and all other mRNA levels

for that gene are shown as a percentage of the maximal level.

Asterisks indicate groups where the mRNA level in TCDD-treated

animals differs significantly from that of the control of the same

mouse strain: *P \ 0.05; **P \ 0.01; ***P \ 0.001. Note that for

Cyp1a1 the mRNA level in control animals or in TCDD-treated

Ahr-/- mice or control Ahr-/- mice is below the detection limit of

the assay (indicated by ‘‘ND’’ in the Figure)

CYP1A1

Nfe2l2(Nrf2)

Tgfb1i4

0 1.5 3 6 10 190

20

40

60

80

100

Time-course of response

0 1.5 3 6 10 190

20

40

60

80

100

0 1.5 3 6 10 190

20

40

60

80

100

No

rmalized

mR

NA

expressio

n ( p

ercent o

f maxim

al respo

nse )

Time after TCDD (hours)

L-E H/W

Dose-Response at 19 hr

TCDD Dose ( Log10 of µg/kg )

0

0

25

50

75

100

-4 -3 -2 -1 0 1 2 3 4

0

25

50

75

100

0 -4 -3 -2 -1 0 1 2 3 4

0

25

50

75

100

0 -4 -3 -2 -1 0 1 2 3 4

No

rmal

ized

mR

NA

exp

ress

ion

( p

erce

nt

of

max

imal

res

po

nse

)

Fig. 5 Dose–response and time-course of changes in mRNA expres-

sion for selected genes. For the dose–response study, four rats per

group were gavaged with corn oil (vehicle control) or with doses of

0.001, 0.01, 0.1, 1, 10, 100 and 1,000 lg/kg TCDD for the dioxin-

sensitive L–E rats. For the dioxin-resistant H/W rats the range was

expanded to 3,000 lg/kg TCDD. Liver was harvested at 19 h for the

dose–response study. Dose–response curves were fitted as described

in ‘‘Materials and methods’’. For the time-course study rats were

gavaged with 100 lg/kg TCDD and livers were removed at 3, 6, 10 or

19 h after TCDD. mRNA levels were measured by real-time RT-

PCR. For each gene, the mRNA level that was highest for any strain

or treatment was set at 100% and all other mRNA levels for that gene

are shown as a percentage of the maximal level. All results represent

the mean of four rats ±SD

Arch Toxicol (2008) 82:809–830 823

123

Page 16

cDNA array studies in rat liver previously were shown to

be AHR-dependent for their response to a high TCDD dose

in mouse liver (Tijet et al. 2006). These include: insulin-

like growth factor 1 (Igf1), cytochrome b5 (Cyb5), and

Cyp1b1 and syndecan (Sdc1).

In silico identification of putative AH response

elements

Of the 32 putative Type-I responsive genes identified by

the OCI cDNA array that could be mapped to the genome,

16 were found to contain extended AHRE-I and/or AHRE-

II motifs within the 50-flanking region of genomic sequence

(Table 3). Phylogenetic (phyloHMM) analysis indicates

that the extended AHRE-I motif is highly conserved

(phyloHMM score [0.5) for five of the Type-I genes. Of

the 15 putative Type-II responsive genes identified by the

OCI cDNA array that could be mapped to the genome, two

contain extended AHRE-I or AHRE-II motifs that are

highly conserved.

Discussion

Known Type-I response genes

A major finding of our study is that numerous genes pre-

viously known to be TCDD-inducible via the AHR

mechanism remain responsive to TCDD in dioxin-resistant

rats that have the variant H/W AHR despite the large

deletion in the AHR transactivation domain in these ani-

mals. Responsiveness in resistant as well as in sensitive rats

was demonstrated both on Clontech Atlas� Rat 1.2 and 4 k

Arrays (CYP1A1, CYP1A2, CYP1B1, Gsta3) and on the

OCI cDNA array (CYP1B1, UGT1A6, TiPARP, Nfe2l2).

Induction of drug-metabolizing enzymes such as mem-

bers of the CYP1 family is not likely to be the primary

cause of dioxin lethality in rats, although alteration of these

enzymes that bioactivate or inactivate a wide variety of

therapeutic agents and environmental chemicals clearly has

its own impact on the organism’s response to xenobiotics.

Transcriptional up-regulation of metabolic enzymes prob-

ably serves as an adaptive mechanism that enhances

clearance from the animal of potentially toxic xenobiotics

(Gu et al. 2000; Nebert et al. 2004; Okey 1990). Because

the AHR is known to mediate toxicity, some classical

dioxin-responsive genes such as CYP1A1 and CYP1A2

have been compared between dioxin-resistant and dioxin-

sensitive rat strains but no clear association with suscep-

tibility to lethality or other major dioxin toxicities has

emerged (Pohjanvirta et al. 1988; Simanainen et al. 2002,

2003; Viluksela et al. 2000). In our study, induction of

CYP1A1 and CYP1B1 by TCDD was similar in sensitive

and resistant rat collectives (Table 3; Fig. 4a). Further-

more, the log–dose response curve for CYP1A1 (Fig. 5)

lies far to the left of the LD50 in rats suggesting that even

maximal CYP1A1 induction does not lead to lethality. In

mice, however, CYP1A1 shows involvement in TCDD-

induced hepatotoxicity and lethality since knockout of the

Cyp1a1 gene substantially protects male mice from these

toxic responses (Uno et al. 2004) whereas knockout of

Cyp1a1 or Cyp1b1 genes does not affect dioxin-mediated

teratogenesis in mice (Dragin et al. 2006).

Because the standard AHR-regulated genes remain

inducible by TCDD both in dioxin-resistant rats and in

dioxin-sensitive rats, they are unlikely to account for the

dioxin-resistance phenotype or to be key components in

pathways that determine susceptibility to lethality from

TCDD. Instead, alteration of expression of these Type-I

genes by TCDD, may represent common adaptive mech-

anisms or may be central to other types of toxicity such as

thymic atrophy that occur both in L–E and in H/W rats.

In addition to the widely studied AHR-regulated cyto-

chrome P450 enzymes (Nebert et al. 2004), several other

gene products identified as Type-I responses in our studies

have potential roles in toxicity or detoxification. For

example, the nuclear receptor, Nrf2 (encoded by the Nfe2l2

gene) constitutes an important mechanism for upregulating

detoxification enzymes and protecting from oxidative

stress (Kohle and Bock 2006). Nrf2 has been shown to be

up-regulated by TCDD in an AHR-dependent fashion in

Hepa-1 mouse hepatoma cells in culture (Miao et al. 2005)

and also is inducible in rat liver (Fletcher et al. 2005). Our

results demonstrate that Nrf2 is up-regulated by TCDD in

rats in vivo (Table 3; Fig. 4a) independent of the transac-

tivation-domain deletion and that the in vivo response is

AHR-dependent as assessed in Ahr-null mice (Fig. 6). As

with other Type-I genes, however, the Nrf2 induction

response occurs in dioxin-sensitive as well as in dioxin

resistant rat strains/lines (Fig. 4a) and thus cannot explain

strain differences in TCDD susceptibility.

Biglycan (Bgn) and syndecan (Sdc1) are proteoglycans

that are important in the development of dentition and in

closure of the palate. Our array studies indicated that Bgn

was up-regulated by TCDD in a Type-I fashion but this

result could not be validated by RT-PCR (Fig. 4a). In

contrast, Sdc1 was down-regulated in both L–E and H/W

rats. Cleft palate and tooth defects are very sensitive tera-

togenic responses in TCDD-treated rats and mice (Abbott

et al. 2003; Kattainen et al. 2001; Miettinen et al. 2002). At

fetotoxic doses of TCDD, L–E rat fetuses exhibit a high

prevalence of cleft palate whereas H/W rat fetuses have

other developmental disorders (Huuskonen et al. 1994). In

contrast, molar dysgenesis in offspring of rat dams gesta-

tionally exposed to TCDD is a Type-I response (Kattainen

et al. 2001). Whether or not Sdc1 and Bgn are involved in

824 Arch Toxicol (2008) 82:809–830

123

Page 17

these phenomena remains to be determined. Previously, we

demonstrated that downregulation of Sdc1 by TCDD in

mouse liver is AHR-dependent (Tijet et al. 2006).

Tgfb1i4 is a pro-apoptotic gene inducible by TGFb1.

Tgfb1i4 can be downregulated in rat liver by non-geno-

toxic carcinogens such as clofibrate, potentially promoting

hepatocarcinogenesis by inhibiting apoptotic clearance of

mutated cells (Michel et al. 2005). TCDD is a very potent

liver tumor promoter that appears to act by inhibiting

apoptosis (Bock and Kohle 2005a); thus the tumor-pro-

moting activity of TCDD in liver might be related to down-

regulation of Tgfb1i4. Others have reported that Tgfb1i4

showed TCDD-dependent downregulation in liver of

Sprague–Dawley rats (Fletcher et al. 2005). We confirm

this result and show that it occurs in the liver of all three

dioxin-sensitive strains/lines as well as in one in dioxin-

resistant strain (H/W) (Fig. 4a). The dose–response curves

for Tgfb1i4 do not differ between sensitive and resistant rat

strains (Fig. 5), suggesting that Tgfb1i4 alteration by

TCDD is not likely to account for the large phenotypic

differences that exist between L–E and H/W rats in sus-

ceptibility to dioxin lethality, hepatotoxicity (Pohjanvirta

and Tuomisto 1994), or hepatic tumor promotion (Viluks-

ela et al. 1997).

We also found that a glucose transporter, Slc2a2, was

significantly down-regulated both in L–E and H/W rats.

Previously, Fletcher et al. (2005) reported down-regulation

of Slc2a2 in livers of dioxin-sensitive SD rats. Down-reg-

ulation of carboxylesterase 3 (Ces3) was significant in the

livers of dioxin-resistant H/W rats but the decrease in liver

of dioxin-sensitive L–E rats did not reach statistical sig-

nificance (Fig. 4b). However, the OCI cDNA arrays

demonstrate that Ces3 is significantly downregulated in all

six rat strains/lines, independent of the AHR-genotype.

Carboxylesterases metabolize xenobiotic chemicals and

also endogenous lipids. TCDD previously has been shown

to down-regulate multiple carboxylesterases in SD rat liver

(Fletcher et al. 2005; Yang et al. 2001).

Novel Type-I response genes

In addition to known Type-I response genes, our study

revealed several novel Type-I response genes that have not

previously been reported and that might play roles in

common dioxin toxicities or may be involved in adaptive

mechanisms.

Disturbed energy metabolism and subsequent wasting

are prominent features of TCDD toxicity in rats (Po-

hjanvirta and Tuomisto 1994). Two clones representing

glutamate dehydrogenase 1 (Glud1), were significantly

down-regulated by TCDD in all rat strains examined by

array (Table 3) and by real-time RT-PCR analyses in L–E

and H/W rats (Fig. 4a). Glud1 provides a major pathway

for interconversion of alpha-amino acids and alpha-keto

acids. Glud1 activity is thought to be controlled by cellular

demand for ATP as an energy source. This enzyme may

play a key role in cellular metabolism and energy

homeostasis (Hudson and Daniel 1993), especially since

both sensitive and resistant rat strains experience an initial

reduction in feeding in response to TCDD, although H/W

rats resume feeding within 1–2 weeks, whereas wasting is

irreversible in L–E rats (Pohjanvirta and Tuomisto 1987;

Unkila 1993).

Among the novel Type-I response genes that emerged in

our RT-PCR study was an oxidative-stress-related protein,

ferritin light chain (Ftl1) (Fig. 4a), whereas the OCI cDNA

array (which includes additional sensitive and resistant

strains and lines) suggests that Ftl1 could be a Type-II

responder (Table 3). Ferritin is a major iron-storage protein

that can be induced by oxidative stress through Nrf2

pathways (Thimmulappa et al. 2002) and which may aid in

protection from the oxidative stress induced by TCDD

(Smith et al. 1998). We found that Ftl1 was significantly

up-regulated both in dioxin-sensitive L–E rats and in

dioxin-resistant H/W rats as assessed with Clontech arrays

and by RT-PCR (Fig. 4a). Glutathione peroxidase 1

(GSHPx) also aids in protection from oxidative stress. The

mRNA levels for this enzyme were repressed by TCDD in

all strains/lines (Table 3) in accordance with our previous

finding that TCDD decreases GSHPx catalytic activity both

in L–E and H/W rats (Pohjanvirta et al. 1990). Metallo-

thioneins also are important in protection from oxidative

stress and DNA damage. Induction of metallothioneins by

TCDD previously has been demonstrated in liver of SD

rats and it was proposed that this induction might protect

from TCDD-generated reactive oxygen species (Nishimura

et al. 2001). Our analyses (Fig. 4a) indicated that basal

expression of two metallothionein isoforms, Mt1a and

Mt2l, is substantially higher in the dioxin-sensitive L–E

strain than in either the dioxin-sensitive SD strain or

dioxin-resistant H/W rats; thus there are no apparent simple

relationships between basal metallothionein levels and

susceptibility to dioxin lethality. Mt1a and Mt2l were

induced by TCDD in dioxin-sensitive L–E and SD rats and

also in dioxin-resistant H/W rats (Fig. 4a), again showing

no apparent relationship of Mt induction to the dioxin-

sensitivity phenotype.

Cytosolic cysteine dioxygenase 1 (Cdo1) mRNA levels

were significantly suppressed by TCDD in all six rat

strains/lines (Table 3). Cdo1 is a key enzyme in L-cysteine

catabolism and corresponding taurine synthesis in mam-

mals. Taurine metabolic actions include bile acid

conjugation, detoxification and membrane stabilization.

Cdo1 may be required for production of sulfate for Phase II

conjugation and has therefore been proposed to play a role

in xenobiotic detoxification in liver (Parsons et al. 1998). It

Arch Toxicol (2008) 82:809–830 825

123

Page 18

is important for the amino acid cysteine to be tightly reg-

ulated by Cdo1 because at high levels cysteine is toxic

(Karlsen et al. 1981). However, cysteine insufficiency

prevents formation of glutathione, a major conjugating

tripeptide in drug detoxification and a precursor in syn-

thesis of proteins.

Type-II response genes

Our main goal was to determine whether dioxin treatment

affects mRNA expression differently between dioxin-sen-

sitive rats that have wildtype AH receptor and dioxin-

resistant rats that have a deletion in the transactivation

domain. This analysis would help to identify genes whose

expression exhibits a Type-II response to TCDD and,

therefore, might be central to dioxin lethality. Most genes

that responded significantly to TCDD did so in a Type-I

fashion (Table 3). The following is a brief description of

the potential relationship to TCDD toxicity of Type-II

genes that emerged from our array studies (Table 3;

Fig. 4b).

Our experiments with the Clontech 4 k array indicated

that steroid 5a-reductase 1 (Srd5a1) was down-regulated by

TCDD in liver of L–E but not H/W rats, consistent with a

previous report that Srd5a1 is repressed in the dioxin-

sensitive SD rat strain (Fletcher et al. 2005). Our real-time

RT-PCR measurements confirmed that Srd5a1 is signifi-

cantly down-regulated in L–E rats but not in H/W rats

(Fig. 4b). Thus Srd5a1 constitutes a candidate gene whose

differential response to TCDD in dioxin-sensitive versus

dioxin-resistant rats could potentially be involved in the

resistance phenotype. Srd5a1 is involved in converting

testosterone to the more potent androgen, dihydrotestos-

terone, but its contribution in this activity and other

possible physiological functions in liver are still uncertain.

Insulin-like growth factor pathways potentially are

important in dioxin toxicity because disturbed glucose

metabolism and profound body weight loss are prominent

features of acute dioxin toxicity in rodents (Pohjanvirta and

Tuomisto 1994). Croutch et al. (2005) previously reported

that TCDD treatment decreases serum levels of insulin-like

growth factor 1 (Igf1) in the dioxin-sensitive SD rat strain

and Fletcher et al. (2005) also detected a decrease in

Igf1 mRNA levels in livers of SD rats. Our OCI cDNA

array (Table 3) revealed a decrease in Igf1 mRNA levels in

livers of all TCDD-treated rats, i.e., a Type-I response (not

analyzed by RT-PCR) which might account for the

decrease in serum Igf1. However, in our Clontech 4 k array

experiment, confirmed by real-time RT-PCR (Fig. 4b),

TCDD caused a decrease in mRNA levels for the insulin-

like growth factor binding protein 1 (Igfbp1) but only in

dioxin-sensitive L–E rats, not in dioxin-resistant H/W rats,

indicating that an impairment in the IGF system may

underlie TCDD-induced disturbances in glucose regulation

and may contribute to the differential susceptibility of these

strains to TCDD.

Pyruvate carboxylase (Pc) is important in gluconeo-

genesis; therefore, its disruption may play a role in the

weight loss exhibited during dioxin toxicity. Pc protein

levels, mRNA levels and catalytic activity previously have

been reported to be decreased in dioxin-treated rodents

(Fletcher et al. 2005; Ilian et al. 1996; Weber et al. 1992).

Our measurements by real-time RT-PCR indicate a modest

down-regulation of Pc in H/W rats but not L–E (Fig. 4b).

Tyrosine aminotransferase (Tat) also is involved in glu-

coneogenesis wherein it catalyzes conversion of L-tyrosine

and 2-oxoglutarate to 4-hydroxyphenylpyruvate and L-

glutamate. In rodent models dioxin-like chemicals have

been reported to alter Tat activity, although not always in a

consistent fashion (Weber et al. 1994). In our current study

Tat mRNA levels were increased in L–E rats alone, in

keeping with our previous finding that a fatal dose of

TCDD (50 lg/kg) rapidly (within 24 h) decreased plasma

tyrosine concentration in L–E rats but not in H/W rats

(Viluksela et al. 1999).

Aldh3a2 could not unambiguously be classified as either

Type-I or Type-II by analysis on cDNA arrays since it

showed induction in all sensitive strains/lines but also in

one resistant strain. Real-time QRT-PCR analysis indicated

significant repression of Aldh3a2 in L–E rats (Fig. 4b). In

our previous proteomics study (Pastorelli et al. 2006) a

related gene, Aldh3a1, was highly up-regulated by TCDD

at the mRNA and protein levels in both dioxin-sensitive

L–E rats and in dioxin-resistant H/W rats after 5 days of

exposure to TCDD.

The gene list presented in Table 3, although rigorously

generated, is not presented as a final comprehensive list of

all relevant dioxin-responsive genes. Our array approach,

coupled with a genetic model of dioxin resistance, was

intended to generate testable hypotheses about mechanisms

underlying susceptibility to TCDD lethality. The 7,538

unique distinct transcripts on the cDNA arrays to which we

could assign a gene identity probably represent about one-

third of the estimated total number of genes in the rat

genome (Gibbs et al. 2004). Of those genes identified to be

TCDD-responsive, only a few were selected for further

examination at this stage based primarily on their known

functions which are related to biochemical pathways and

on pathological changes that have been observed in dioxin-

treated rodents. The remaining genes are nevertheless

worthy of follow-up.

A limitation of our study was that the cDNA library used

to construct the cDNA arrays was derived from the mRNA

taken from the tissues of control mice. Therefore, the array

contained cDNA sequences only from genes that were

constitutively expressed, and did not contain genes that

826 Arch Toxicol (2008) 82:809–830

123

Page 19

required exposure to TCDD in order to be significantly

expressed (e.g., CYP1A1). Although the OCI cDNA array

that we employed was constructed from a mouse cDNA

library, we were able to assign 68.9% of the mouse clone

sequences to a rat UniGene ID. There are precedents for

successfully hybridizing rat samples onto mouse cDNA

arrays (Wang et al. 2002b), including studies on AHR-

mediated gene expression (Kondraganti et al. 2005).

Methods have also been developed for finding orthologous

cDNA species between mouse and rat clones (Wang et al.

2002a).

Notwithstanding some experimental limitations, our

study of dioxin-sensitive versus dioxin-resistant rats helps

bring forward a refined list of genes that is worth studying

further for its potential role in the mechanisms of dioxin

lethality and major related dioxin toxicities.

Variability in gene expression across rat strains and

lines

Our study of six different rat strains/lines allowed, for the

first time, an assessment of the extent of variation in the

effects of dioxins on mRNA expression amongst com-

monly studied laboratory rat strains and lines. Strikingly,

the results show a high degree of divergence among strains/

lines in the effects of TCDD on mRNA expression. Of the

844 unique cDNA clones that showed significant

(Padjusted \ 0.05) changes in mRNA levels in response to

TCDD exposure, fully 75% (635/844) were altered in only

one of the six rat strains or lines; an additional 15% were

altered in only two of the six groups. These data indicate

that over 90% of changes in mRNA levels in response to

dioxin potentially are strain-specific. There even are sub-

stantial differences in the number of TCDD-responsive

UniGene clusters when comparing one sensitive strain, L–

E, with another sensitive strain, SD: 297 clusters responded

in L–E only and 23 clusters responded in SD only while 46

clusters responded both in L–E and SD (Fig. 3). Within the

resistant collective, there was less variation in the number

of clusters whose response is unique to one strain. This

may reflect the fact that there is more genetic similarity

among H/W, LnA and F1 rats than there is between L–E

and SD rats or that L–E is the most sensitive of these

strains.

The total number of UniGene clusters that responded to

dioxins was greater in the collective of sensitive rats than in

the collective of resistant rats. Numerous classic AHR-

regulated genes remain fully responsive to TCDD in

dioxin-resistant strains. However, the decrease in the total

number of responsive genes in the resistant collective may

indicate that the region deleted from the AHR transacti-

vation domain in resistant rats is required for altered

expression of many novel genes.

Our finding of large variation across rat strain/lines in

the response to TCDD is particularly intriguing given the

close genetic relationships among five of the six strains/

lines that we studied (L–E, H/W, F1, Line A, and Line C).

These results accord closely with the large differences in

mRNA expression recently observed between rats and mice

in response to TCDD by Boverhof et al. (2006). It seems

likely that TCDD-mediated alterations in mRNA levels are

modulated by genetic variations at a large number of loci,

in addition to the AHR itself.

Acknowledgments Supported by grant MOP-57903 from the

Canadian Institutes of Health Research to A.B.O. and by grants

200980 and 211120 from the Academy of Finland to R.P.; M.A.F. and

I.D.M. were supported by fellowships from the Natural Sciences and

Engineering Research Council of Canada. We thank Virpi Tiihonen

and Arja Tamminen for excellent technical assistance. Experiments

reported in this paper comply with the current laws of Canada and of

Finland.

References

Abbott BD, Buckalew AR, DeVito MJ, Ross D, Bryant PL, Schmid

JE (2003) EGF and TGF-alpha expression influence the devel-

opmental toxicity of TCDD: dose response and AhR phenotype

in EGF, TGF-alpha, and EGF + TGF-alpha knockout mice.

Toxicol Sci 71:84–95

Bock KW, Kohle C (2005a) Ah receptor- and TCDD-mediated liver

tumor promotion: clonal selection and expansion of cells

evading growth arrest and apoptosis. Biochem Pharmacol

69:1403–1408

Bock KW, Kohle C (2005b) UDP-glucuronosyltransferase 1A6:

structural, functional, and regulatory aspects. Methods Enzymol

400:57–75

Bolstad BM, Irizarry RA, Astrand M, Speed TP (2003) A comparison

of normalization methods for high density oligonucleotide array

data based on variance and bias. Bioinformatics 19:185–193

Boutros PC, Moffat ID, Franc MA, Tijet N, Tuomisto J, Pohjanvirta

R, Okey AB (2004) Dioxin-responsive AHRE-II gene battery:

identification by phylogenetic footprinting. Biochem Biophys

Res Commun 321:707–715

Boverhof DR, Tam E, Harney AS, Crawford RB, Kaminski NE,

Zacharewski TR (2004) 2,3,7,8-Tetrachlorodibenzo-p-dioxin

induces suppressor of cytokine signaling 2 in murine B cells.

Mol Pharmacol 66:1662–1670

Boverhof DR, Burgoon LD, Tashiro C, Chittim B, Harkema JR, Jump

DB, Zacharewski TR (2005) Temporal and dose-dependent

hepatic gene expression patterns in mice provide new insights

into TCDD-mediated hepatotoxicity. Toxicol Sci 85:1048–1063

Boverhof DR, Burgoon LD, Tashiro C, Sharratt B, Chittim B, Harkema

JR, Mendrick DL, Zacharewski TR (2006) Comparative toxicog-

enomic analysis of the hepatotoxic effects of TCDD in Sprague

Dawley rats and C57BL/6 mice. Toxicol Sci 94:398–416

Bunger MK, Moran SM, Glover E, Thomae TL, Lahvis GP, Lin BC,

Bradfield CA (2003) Resistance to 2,3,7,8-tetrachlorodibenzo-p-

dioxin toxicity and abnormal liver development in mice carrying

a mutation in the nuclear localization sequence of the aryl

hydrocarbon receptor. J Biol Chem 278:17767–17774

Croutch CR, Lebofsky M, Schramm KW, Terranova PF, Rozman KK

(2005) 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and

1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD) alter body

Arch Toxicol (2008) 82:809–830 827

123

Page 20

weight by decreasing insulin-like growth factor I (IGF-I)

signaling. Toxicol Sci 85:560–571

Denison MS, Whitlock JP Jr (1995) Xenobiotic-inducible transcrip-

tion of cytochrome P450 genes. J Biol Chem 270:18175–18178

Dragin N, Dalton TP, Miller ML, Shertzer HG, Nebert DW (2006)

For dioxin-induced birth defects, mouse or human CYP1A2 in

maternal liver protects whereas mouse CYP1A1 and CYP1B1

are inconsequential. J Biol Chem 281:18591–18600

Fernandez-Salguero P, Pineau T, Hilbert DM, McPhail T, Lee SS,

Kimura S, Nebert DW, Rudikoff S, Ward JM, Gonzalez FJ

(1995) Immune system impairment and hepatic fibrosis in mice

lacking the dioxin-binding Ah receptor (see comments). Science

268:722–726

Fernandez-Salguero PM, Hilbert DM, Rudikoff S, Ward JM, Gonz-

alez FJ (1996) Aryl-hydrocarbon receptor-deficient mice are

resistant to 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced toxicity.

Toxicol Appl Pharmacol 140:173–179

Fletcher N, Wahlstrom D, Lundberg R, Nilsson CB, Nilsson KC,

Stockling K, Hellmold H, Hakansson H (2005) 2,3,7,8-Tetra-

chlorodibenzo-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.

Toxicol Appl Pharmacol 207:1–24

Frericks M, Temchura VV, Majora M, Stutte S, Esser C (2006)

Transcriptional signatures of immune cells in aryl hydrocarbon

receptor (AHR)-proficient and AHR-deficient mice. Biol Chem

387:1219–1226

Fried KW, Bazzi R, Levy Lopez W, Corsten C, Schramm KW, Bell

DR, Rozman KK (2007) Relationship between aryl hydrocarbon

receptor-affinity and the induction of EROD activity by 2,3,7,8-

tetrachlorinated phenothiazine and derivatives. Toxicol Appl

Pharmacol 224:147–155

Frueh FW, Hayashibara KC, Brown PO, Whitlock JP (2001) Use of

cDNA microarrays to analyze dioxin-induced changes in human

liver gene expression. Toxicol Lett 122:189–203

Gibbs RA, Weinstock GM, Metzker ML, Muzny DM, Sodergren EJ,

Scherer S, Scott G, Steffen D, Worley KC, Burch PE, Okwuonu

G, Hines S, Lewis L, DeRamo C, Delgado O, Dugan-Rocha S,

Miner G, Morgan M, Hawes A, Gill R, Celera, Holt RA, Adams

MD, Amanatides PG, Baden-Tillson H, Barnstead M, Chin S,

Evans CA, Ferriera S, Fosler C, Glodek A, Gu Z, Jennings D,

Kraft CL, Nguyen T, Pfannkoch CM, Sitter C, Sutton GG, Venter

JC, Woodage T, Smith D, Lee HM, Gustafson E, Cahill P, Kana

A, Doucette-Stamm L, Weinstock K, Fechtel K, Weiss RB, Dunn

DM, Green ED, Blakesley RW, Bouffard GG, De Jong PJ,

Osoegawa K, Zhu B, Marra M, Schein J, Bosdet I, Fjell C, Jones

S, Krzywinski M, Mathewson C, Siddiqui A, Wye N, McPherson

J, Zhao S, Fraser CM, Shetty J, Shatsman S, Geer K, Chen Y,

Abramzon S, Nierman WC, Havlak PH, Chen R, Durbin KJ, Egan

A, Ren Y, Song XZ, Li B, Liu Y, Qin X, Cawley S, Cooney AJ,

D’Souza LM, Martin K, Wu JQ, Gonzalez-Garay ML, Jackson

AR, Kalafus KJ, McLeod MP, Milosavljevic A, Virk D, Volkov

A, Wheeler DA, Zhang Z, Bailey JA, Eichler EE, Tuzun E,

Birney E, Mongin E, Ureta-Vidal A, Woodwark C, Zdobnov E,

Bork P, Suyama M, Torrents D, Alexandersson M, Trask BJ,

Young JM, Huang H, Wang H, Xing H, Daniels S, Gietzen D,

Schmidt J, Stevens K, Vitt U, Wingrove J, Camara F, Mar Alba

M, Abril JF, Guigo R, Smit A, Dubchak I, Rubin EM, Couronne

O, Poliakov A, Hubner N, Ganten D, Goesele C, Hummel O,

Kreitler T, Lee YA, Monti J, Schulz H, Zimdahl H, Himmelbauer

H, Lehrach H, Jacob HJ, Bromberg S, Gullings-Handley J,

Jensen-Seaman MI, Kwitek AE, Lazar J, Pasko D, Tonellato PJ,

Twigger S, Ponting CP, Duarte JM, Rice S, Goodstadt L, Beatson

SA, Emes RD, Winter EE, Webber C, Brandt P, Nyakatura G,

Adetobi M, Chiaromonte F, Elnitski L, Eswara P, Hardison RC,

Hou M, Kolbe D, Makova K, Miller W, Nekrutenko A, Riemer C,

Schwartz S, Taylor J, Yang S, Zhang Y, Lindpaintner K, Andrews

TD, Caccamo M, Clamp M, Clarke L, Curwen V, Durbin R,

Eyras E, Searle SM, Cooper GM, Batzoglou S, Brudno M, Sidow

A, Stone EA, Payseur BA, Bourque G, Lopez-Otin C, Puente XS,

Chakrabarti K, Chatterji S, Dewey C, Pachter L, Bray N, Yap VB,

Caspi A, Tesler G, Pevzner PA, Haussler D, Roskin KM,

Baertsch R, Clawson H, Furey TS, Hinrichs AS, Karolchik D,

Kent WJ, Rosenbloom KR, Trumbower H, Weirauch M, Cooper

DN, Stenson PD, Ma B, Brent M, Arumugam M, Shteynberg D,

Copley RR, Taylor MS, Riethman H, Mudunuri U, Peterson J,

Guyer M, Felsenfeld A, Old S, Mockrin S, Collins F (2004)

Genome sequence of the Brown Norway rat yields insights into

mammalian evolution. Nature 428:493–521

Gu YZ, Hogenesch JB, Bradfield CA (2000) The PAS superfamily:

sensors of environmental and developmental signals. Annu Rev

Pharmacol Toxicol 40:519–561

Hayes KR, Vollrath AL, Zastrow GM, McMillan BJ, Craven MW,

Jovanovich SB, Walisser JA, Rank DR, Penn SG, Reddy JK,

Thomas RS, Bradfield CA (2005) EDGE: a centralized resource

for the comparison, analysis and distribution of toxicogenomic

information. Mol Pharmacol 67:1360–1368

Hayes KR, Zastrow GM, Nukaya M, Pande K, Glover E, Maufort JP,

Liss AL, Liu Y, Moran SM, Vollrath AL, Bradfield CA (2007)

Hepatic transcriptional networks induced by exposure to 2,3,7,8-

tetrachlorodibenzo-p-dioxin. Chem Res Toxicol

Hudson RC, Daniel RM (1993) L-glutamate dehydrogenases: distri-

bution, properties and mechanism. Comp Biochem Physiol B

106:767–792

Ilian MA, Sparrow BR, Ryu BW, Selivonchick DP, Schaup HW

(1996) Expression of hepatic pyruvate carboxylase mRNA in

C57BL/6J Ah(b/b) and congenic Ah((d/d)) mice exposed to

2,3,7,8-tetrachlorodibenzo-p-dioxin. J Biochem Toxicol 11:51–

56

Karlsen RL, Grofova I, Malthe-Sorenssen D, Fonnum F (1981)

Morphological changes in rat brain induced by L-cysteine

injection in newborn animals. Brain Res 208:167–180

Karyala S, Guo J, Sartor M, Medvedovic M, Kann S, Puga A, Ryan P,

Tomlinson CR (2004) Different global gene expression profiles

in benzo[a]pyrene- and dioxin-treated vascular smooth muscle

cells of AHR-knockout and wild-type mice. Cardiovasc Toxicol

4:47–74

Kattainen H, Tuukkanen J, Simanainen U, Tuomisto JT, Kovero O,

Lukinmaa PL, Alaluusua S, Tuomisto J, Viluksela M (2001) In

utero/lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure

impairs molar tooth development in rats. Toxicol Appl Pharma-

col 174:216–224

Kohle C, Bock KW (2006) Activation of coupled Ah receptor and

Nrf2 gene batteries by dietary phytochemicals in relation to

chemoprevention. Biochem Pharmacol 72:795–805

Kolluri SK, Weiss C, Koff A, Gottlicher M (1999) p27(Kip1)

induction and inhibition of proliferation by the intracellular Ah

receptor in developing thymus and hepatoma cells. Genes Dev

13:1742–1753

Kondraganti SR, Muthiah K, Jiang W, Barrios R, Moorthy B (2005)

Effects of 3-methylcholanthrene on gene expression profiling in

the rat using cDNA microarray analyses. Chem Res Toxicol

18:1634–1641

Kooperberg C, Fazzio TG, Delrow JJ, Tsukiyama T (2002) Improved

background correction for spotted DNA microarrays. J Comput

Biol 9:55–66

Lee K, Burgoon LD, Lamb L, Dere E, Zacharewski TR, Hogenesch

JB, Lapres JJ (2006) Identification and characterization of genes

susceptible to transcriptional cross-talk between the hypoxia and

dioxin signaling cascades. Chem Res Toxicol 19:1284–1293

Lin TM, Ko K, Moore RW, Buchanan DL, Cooke PS, Peterson RE

(2001) Role of the aryl hydrocarbon receptor in the development

828 Arch Toxicol (2008) 82:809–830

123

Page 21

of control and 2,3,7,8-tetrachlorodibenzo-p-dioxin-exposed male

mice. J Toxicol Environ Health A 64:327–342

Martinez JM, Afshari CA, Bushel PR, Masuda A, Takahashi T,

Walker NJ (2002) Differential toxicogenomic responses to

2,3,7,8-tetrachlorodibenzo-p-dioxin in malignant and nonmalig-

nant human airway epithelial cells. Toxicol Sci 69:409–423

Miao W, Hu L, Scrivens PJ, Batist G (2005) Transcriptional

regulation of NF-E2 p45-related factor (NRF2) expression by

the aryl hydrocarbon receptor–xenobiotic response element

signaling pathway: direct cross-talk between phase I and II

drug-metabolizing enzymes. J Biol Chem 280:20340–20348

Michel C, Roberts RA, Desdouets C, Isaacs KR, Boitier E (2005)

Characterization of an acute molecular marker of nongenotoxic

rodent hepatocarcinogenesis by gene expression profiling in a

long term clofibric acid study. Chem Res Toxicol 18:611–618

Miettinen HM, Alaluusua S, Tuomisto J, Viluksela M (2002) Effect

of in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin

exposure on rat molar development: the role of exposure time.

Toxicol Appl Pharmacol 184:57–66

Mimura J, Yamashita K, Nakamura K, Morita M, Takagi TN, Nakao

K, Ema M, Sogawa K, Yasuda M, Katsuki M, Fujii-Kuriyama Y

(1997) Loss of teratogenic response to 2,3,7,8-tetrachlo-

rodibenzo-p-dioxin (TCDD) in mice lacking the Ah (dioxin)

receptor. Genes Cells 2:645–654

Moffat ID, Roblin S, Harper PA, Okey AB, Pohjanvirta R (2007) Aryl

hydrocarbon receptor splice variants in the dioxin-resistant rat:

tissue expression and transactivational activity. Mol Pharmacol

72:956–966

Nebert DW, Dalton TP, Okey AB, Gonzalez FJ (2004) Role of aryl

hydrocarbon receptor-mediated induction of the CYP1 enzymes

in environmental toxicity and cancer. J Biol Chem 279:23847–

23850

Nishimura N, Miyabara Y, Suzuki JS, Sato M, Aoki Y, Satoh M,

Yonemoto J, Tohyama C (2001) Induction of metallothionein in

the livers of female Sprague–Dawley rats treated with 2,3,7,8-

tetrachlorodibenzo-p-dioxin. Life Sci 69:1291–1303

Okey AB (1990) Enzyme induction in the cytochrome P-450 system.

Pharmacol Ther 45:241–98

Okey AB (2007) An aryl hydrocarbon receptor Odyssey to the Shores

of Toxicology: the Deichmann lecture, International Congress of

Toxicology-XI. Toxicol Sci 98:5–38

Okey AB, Franc MA, Moffat ID, Tijet N, Boutros PC, Korkalainen

M, Tuomisto J, Pohjanvirta R (2005) Toxicological implications

of polymorphisms in receptors for xenobiotic chemicals: the case

of the aryl hydrocarbon receptor. Toxicol Appl Pharmacol

207:S43–S51

Ovando BJ, Vezina CM, McGarrigle BP, Olson JR (2006) Hepatic

gene downregulation following acute and subchronic exposure to

2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol Sci 94:428–438

Pande K, Moran SM, Bradfield CA (2005) Aspects of dioxin toxicity

are mediated by interleukin 1-like cytokines. Mol Pharmacol

67:1393–1398

Parsons RB, Ramsden DB, Waring RH, Barber PC, Williams AC

(1998) Hepatic localisation of rat cysteine dioxygenase. J

Hepatol 29:595–602

Pastorelli R, Carpi D, Campagna R, Airoldi L, Pohjanvirta R,

Viluksela M, Hakansson H, Boutros PC, Moffat ID, Okey AB,

Fanelli R (2006) Differential expression profiling of the hepatic

proteome in a rat model of dioxin resistance: correlation with

genomic and transcriptomic analyses. Mol Cell Proteomics

5:882–894

Pohjanvirta R, Tuomisto J (1987) Han/Wistar rats are exceptionally

resistant to TCDD. II. Arch Toxicol Suppl 11:344–347

Pohjanvirta R, Tuomisto J (1994) Short-term toxicity of 2,3,7,8-

tetrachlorodibenzo-p-dioxin in laboratory animals: effects,

mechanisms, and animal models. Pharmacol Rev 46:483–549

Pohjanvirta R, Juvonen R, Karenlampi S, Raunio H, Tuomisto J

(1988) Hepatic Ah-receptor levels and the effect of 2,3,7,8-

tetrachlorodibenzo-p-dioxin (TCDD) on hepatic microsomal

monooxygenase activities in a TCDD-susceptible and -resistant

rat strain. Toxicol Appl Pharmacol 92:131–140

Pohjanvirta R, Tuomisto L, Tuomisto J (1989) The central nervous

system may be involved in TCDD toxicity. Toxicology 58:167–

174

Pohjanvirta R, Sankari S, Kulju T, Naukkarinen A, Ylinen M,

Tuomisto J (1990) Studies on the role of lipid peroxidation in the

acute toxicity of TCDD in rats. Pharmacol Toxicol 66:399–408

Pohjanvirta R, Wong JMY, Li W, Harper PA, Tuomisto J, Okey AB

(1998) Point mutation in intron sequence causes altered

carboxyl-terminal structure in the aryl hydrocarbon receptor of

the most 2,3,7,8-tetrachlorodibenzo-p-dioxin-resistant rat strain.

Mol Pharmacol 54:86–93

Pohjanvirta R, Viluksela M, Tuomisto JT, Unkila M, Karasinska J,

Franc M-A, Holowenko M, Giannone JV, Harper PA, Tuomisto

J, Okey AB (1999) Physicochemical differences in the AH

receptors of the most TCDD-susceptible and the most TCDD-

resistant rat strains. Toxicol Appl Pharmacol 155:82–95

Puga A, Maier A, Medvedovic M (2000) The transcriptional signature

of dioxin in human hepatoma HepG2 cells. Biochem Pharmacol

60:1129–1142

Schwanekamp JA, Sartor MA, Karyala S, Halbleib D, Medvedovic

M, Tomlinson CR (2006) Genome-wide analyses show that

nuclear and cytoplasmic RNA levels are differentially affected

by dioxin. Biochim Biophys Acta 1759:388–402

Siepel A, Haussler D (2004) Combining phylogenetic and hidden

Markov models in biosequence analysis. J Comput Biol 11:413–

428

Simanainen U, Tuomisto JT, Tuomisto J, Viluksela M (2002)

Structure–activity relationships and dose responses of polychlo-

rinated dibenzo-p-dioxins for short-term effects in 2,3,7,8-

tetrachlorodibenzo-p-dioxin-resistant and -sensitive rat strains.

Toxicol Appl Pharmacol 181:38–47

Simanainen U, Tuomisto JT, Tuomisto J, Viluksela M (2003) Dose–

response analysis of short-term effects of 2,3,7,8-tetrachlo-

rodibenzo-p-dioxin in three differentially susceptible rat lines.

Toxicol Appl Pharmacol 187:128–136

Slatter JG, Cheng O, Cornwell PD, de Souza A, Rockett J, Rushmore

T, Hartley D, Evers R, He Y, Dai X, Hu R, Caguyong M,

Roberts CJ, Castle J, Ulrich RG (2006) Microarray-based

compendium of hepatic gene expression profiles for prototypical

ADME gene-inducing compounds in rats and mice in vivo.

Xenobiotica 36:902–937

Smith AG, Clothier B, Robinson S, Scullion MJ, Carthew P, Edwards

R, Luo J, Lim CK, Toledano M (1998) Interaction between iron

metabolism and 2,3,7,8-tetrachlorodibenzo-p-dioxin in mice

with variants of the Ahr gene: a hepatic oxidative mechanism.

Mol Pharmacol 53:52–61

Smyth GK (2003) Linear models and empirical Bayes methods for

assessing differential expression in microarray experiments. Stat

Appl Genetics Mol Biol 3:1–26

Storey JD, Tibshirani R (2003) Statistical significance for genome-

wide studies. Proc Natl Acad Sci USA 100:9440–9445

Tanaka TS, Jaradat SA, Lim MK, Kargul GJ, Wang X, Grahovac MJ,

Pantano S, Sano Y, Piao Y, Nagaraja R, Doi H, Wood WH III,

Becker KG, Ko MS (2000) Genome-wide expression profiling of

mid-gestation placenta and embryo using a 15,000 mouse

developmental cDNA microarray. Proc Natl Acad Sci USA

97:9127–9132

Thimmulappa RK, Mai KH, Srisuma S, Kensler TW, Yamamoto M,

Biswal S (2002) Identification of Nrf2-regulated genes induced

by the chemopreventive agent sulforaphane by oligonucleotide

microarray. Cancer Res 62:5196–5203

Arch Toxicol (2008) 82:809–830 829

123

Page 22

Thurmond TS, Silverstone AE, Baggs RB, Quimby FW, Staples JE,

Gasiewicz TA (1999) A chimeric aryl hydrocarbon receptor

knockout mouse model indicates that aryl hydrocarbon receptor

activation in hematopoietic cells contributes to the hepatic

lesions induced by 2,3,7, 8- tetrachlorodibenzo-p-dioxin. Toxicol

Appl Pharmacol 158:33–40

Tijet N, Boutros PC, Moffat ID, Okey AB, Tuomisto J, Pohjanvirta R

(2006) The aryl hydrocarbon receptor regulates distinct dioxin-

dependent and dioxin-independent gene batteries. Mol Pharma-

col 69:140–153

Tuomisto J (2005) Does mechanistic understanding help in risk

assessment-the example of dioxins. Toxicol Appl Pharmacol

207:2–10

Tuomisto JT, Viluksela M, Pohjanvirta R, Tuomisto J (1999) The AH

receptor and a novel gene determine acute toxic responses to

TCDD: segregation of the resistant alleles to different rat lines.

Toxicol Appl Pharmacol 155:71–81

Unkila M (1993) Differential effect of TCDD on brain serotonin

metabolism in a TCDD-susceptible and a TCDD-resistant rat

strain. Chemosphere 27:401–406

Uno S, Dalton TP, Sinclair PR, Gorman N, Wang B, Smith AG,

Miller ML, Shertzer HG, Nebert DW (2004) Cyp1a1(-/-) male

mice: protection against high-dose TCDD-induced lethality and

wasting syndrome, and resistance to intrahepatocyte lipid

accumulation and uroporphyria. Toxicol Appl Pharmacol

196:410–421

Vartiainen T, Lampi P, Tuomisto JT, Tuomisto J (1995) Poly-

chlorodibenzo-p-dioxin and polychlorodibenzofuran con-

centrations in human fat samples in a village after pollution of

drinking water with chlorophenols. Chemosphere 30:1429–1438

Vezina CM, Walker NJ, Olson JR (2004) Subchronic exposure to

TCDD, PeCDF, PCB126, and PCB153: effect on hepatic gene

expression. Environ Health Perspect 112:1636–1644

Viluksela M, Bager Y, Tuomisto JT, Scheu G, Unkila M, Pohjanvirta

R, Flodstrom S, Warngard L, Tuomisto J (1997) Comparison of

liver tumor promoting activity of TCDD in a TCDD-sensitive

and a TCDD-resistant rat strain. Pharmacol Toxicol 80:152–156

Viluksela M, Unkila M, Pohjanvirta R, Tuomisto JT, Stahl BU,

Rozman KK, Tuomisto J (1999) Effects of 2,3,7,8-tetrachlo-

rodibenzo-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 73:323–

336

Viluksela M, Bager Y, Tuomisto JT, Scheu G, Unkila M, Pohjanvirta

R, Flodstrom S, Kosma VM, Maki-Paakkanen J, Vartiainen T,

Klimm C, Schramm KW, Warngard L, Tuomisto J (2000) Liver

tumor-promoting activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin

(TCDD) in TCDD-sensitive and TCDD-resistant rat strains.

Cancer Res 60:6911–6920

Walisser JA, Bunger MK, Glover E, Bradfield CA (2004) Gestational

exposure of Ahr and Arnt hypomorphs to dioxin rescues vascular

development. Proc Natl Acad Sci USA 101:16677–16682

Wang P, Ding F, Chiang H, Thompson RC, Watson SJ, Meng F

(2002a) ProbeMatchDB—a web database for finding equivalent

probes across microarray platforms and species. Bioinformatics

18:488–489

Wang W, Wyckoff JB, Frohlich VC, Oleynikov Y, Huttelmaier S,

Zavadil J, Cermak L, Bottinger EP, Singer RH, White JG, Segall

JE, Condeelis JS (2002b) Single cell behavior in metastatic

primary mammary tumors correlated with gene expression

patterns revealed by molecular profiling. Cancer Res 62:6278–

6288

Weber LW, Lebofsky M, Stahl BU, Kettrup A, Rozman K (1992)

Comparative toxicity of four chlorinated dibenzo-p-dioxins

(CDDs) and their mixture. Part II: structure–activity relation-

ships with inhibition of hepatic phosphoenolpyruvate

carboxykinase, pyruvate carboxylase, and gamma-glutamyl

transpeptidase activities. Arch Toxicol 66:478–483

Weber LW, Palmer CD, Rozman K (1994) Reduced activity of

tryptophan 2,3-dioxygenase in the liver of rats treated with

chlorinated dibenzo-p-dioxins (CDDs): dose–responses and

structure–activity relationship. Toxicology 86:63–69

Workman C, Jensen LJ, Jarmer H, Berka R, Gautier L, Nielser HB,

Saxild HH, Nielsen C, Brunak S, Knudsen S (2002) A new non-

linear normalization method for reducing variability in DNA

microarray experiments. Genome Biol 3:research0048

Yang YH, Speed T (2002) Design issues for cDNA microarray

experiments. Nat Rev Genet 3:579–588

Yang D, Li Y, Yuan X, Matoney L, Yan B (2001) Regulation of rat

carboxylesterase expression by 2,3,7,8-tetrachlorodibenzo-p-

dioxin (TCDD): a dose-dependent decrease in mRNA levels

but a biphasic change in protein levels and activity. Toxicol Sci

64:20–27

Zeytun A, McKallip R, Fisher M, Camacho I, Nagarkatti M,

Nagarkatti P (2002) Analysis of 2,3,7,8-tetrachlorodibenzo-p-

dioxin-induced gene expression profile in vivo using pathway-

specific cDNA arrays. Toxicology 178:241

Zhang L, Zheng W, Jefcoate CR (2003) Ah receptor regulation of

mouse Cyp1B1 is additionally modulated by a second novel

complex that forms at two AhR response elements. Toxicol Appl

Pharmacol 192:174–190

830 Arch Toxicol (2008) 82:809–830

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