• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Chem Res Toxicol. Author manuscript; available in PMC Nov 1, 2008.
Published in final edited form as:
PMCID: PMC2515491

Hepatic transcriptional networks induced by exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin1


The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) serves as a prototype for a range of environmental toxicants and as a pharmacologic probe to study signal transduction by the aryl hydrocarbon receptor (AHR). Despite a detailed understanding of how TCDD exposure leads to the transcriptional up-regulation of cytochrome P450-dependent monooxygenases, we know little about how compounds like TCDD lead to a variety of AHR-dependent toxic endpoints such as liver pathology, terata, thymic involution and cancer. Using an acute exposure protocol and the toxic response of the mouse liver as a model system, we have begun a detailed microarray analysis to describe the transcriptional changes that occur after various TCDD doses and treatment times. Through correlation analysis of time- and dose-dependent toxicological endpoints, we are able to identify coordinately-responsive transcriptional events that can be defined as primary transcriptional events and downstream events that may represent mechanistically linked sequelae or that have potential as biomarkers of toxicity.


The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) has long been a focus of public concern. Formed by industrial processes such as combustion, bleaching of wood pulp, and chlorination of phenols, TCDD and structurally-related compounds are widely-distributed in the environment and have been shown to elicit a broad spectrum of biological responses in mammalian systems (1). At doses in the ng/kg range, TCDD leads to the up-regulation of genes important for the metabolism of xenobiotics and endogenous hormones (2). At doses in the μg/kg range, TCDD up-regulates the xenobiotic response while also causing a number of toxic endpoints such as hepatotoxicity, thymic involution, birth defects, cancer and lethality (1, 3-5).

It is widely accepted that TCDD acts through the aryl hydrocarbon receptor (AHR), a member of the Per-Arnt-Sim (PAS) family of environmental sensors (6-10). In its unliganded state, the AHR is maintained in the cytosol in a complex with chaperones such as HSP90 and ARA9 (also known as AIP1 or XAP2) (11-13). Upon binding TCDD, the AHR translocates to the nucleus where it dimerizes with another PAS protein known as ARNT (13). The AHR-ARNT complex binds to dioxin-responsive enhancers (DREs)2, modulating the expression of a battery of regulated genes (2). Interaction of AHR-ARNT dimers with DREs appears to explain the expression of many TCDD-responsive genes such as those encoding xenobiotic metabolizing enzymes (XMEs) (e.g., Cyp1a1, Cyp1a2, Cyp1b1, Ugt1a6 and members of the GST family) (2, 15, 16).

Despite our detailed understanding of AHR-regulated xenobiotic metabolism, the mechanistic links between DRE-driven gene expression and toxic endpoints such as hepatotoxicity are still unclear. A number of observations support the idea that DRE-regulated gene expression within the hepatocyte is a fundamental aspect of TCDD-induced liver toxicity. For example, recombinant mouse models with mutations in the DNA binding domain of the AHR, hepatocyte specific deletions of the AHR, or hypomorphic expression of ARNT are all resistant to TCDD-induced hepatotoxicity(17-19). While this genetic evidence points to the importance of AHR-ARNT-DRE interactions in TCDD-hepatotoxicity, it does not identify the causally related DRE-driven genes. In addition, we still do not know the number of steps that exist between the DRE-regulated battery and any given pathological endpoint.

Based upon the above ideas, we are left with a model of TCDD toxicity where a subset of DRE-regulated gene products within the hepatocyte leads directly to liver-specific pathology. This subset of DRE-regulated genes could lead directly to cell death, perhaps through stimulation of oxidative or apoptotic mechanisms (20). Alternatively the DRE-regulated subset could stimulate a multi-step sequence of physiological or developmental events, perhaps stimulation of an inappropriate differentiation pathway that results in tissue pathology. In an effort to provide evidence for these models of toxicity, we have begun an extensive transcriptomic effort to describe the batteries of genes that are deregulated by TCDD exposure. Our hypothesis is that sequences of TCDD-inducible events can be defined by monitoring coordinate transcriptional changes in gene sets. To this end, we characterized the transcriptional changes that occur over time in response to various dosages of TCDD. We then used functional and statistical analyses as methods to identify coordinately regulated gene batteries, as well as batteries that are coregulated with pathology. Through this global gene expression analysis over time and dose, we have generated a training set that can be used to estimate the number of steps in toxicity, as well as to identify those genes or gene batteries that can serve as mechanistically linked markers of specific pathological endpoints.


Animals and Treatments

All animal treatments were reviewed and approved by the Animal Care and Use Committee at the University of Wisconsin. TCDD dissolved in corn oil or corn oil alone was administered by oral gavage. In addition to TCDD-treated animals, a corn oil cohort was harvested at each time point. At the indicated times, the liver was removed, examined and weighed, and a section placed in 10% buffered formalin for histological preparation. The remainder of the liver was placed in “RNA Later” (Qiagen, Valencia, CA) and frozen at -80°C for subsequent preparation of RNA. In some cases, blood was withdrawn by cardiac puncture and sera sent to the Clinical Pathology Laboratory at the University of Wisconsin, School of Veterinary Medicine (Madison, WI) for quantification of alanine aminotransferase (ALT). The Cyp1a1-/- and Cyp1a2-/- animals were a generous gift from Dr. Daniel Nebert (University of Cincinnati, OH).

RNA Isolation and Quantification

Total RNA was prepared using the RNA Protect system (Qiagen, Valencia, CA). Quality and quantity of RNAs were assessed on an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA) using the RNA Nano Labchip.

cDNA Microarrays

All experiments were performed using the EDGE cDNA microarray, Liver Version 5 (http://edge.oncology.wisc.edu) (21). This microarray is composed of liver-derived cDNA clones fortified with cDNAs representing toxicologically relevant genes such as those encoding drug metabolizing enzymes, inflammatory responsive genes, and genes that we previously identified as useful for classifying chemicals (22).

cDNA Microarray Hybridization

When the amount of available total RNA exceeded 20 μg, labeling and hybridizations were carried out using the Genisphere cDNA50 system (Genisphere, Hatfield, PA). When the available total RNA was less than 20 μg, the Genisphere cDNA350 system was used. Briefly, total RNA was reverse-transcribed into cDNA using specific primers containing a Cy-3 or Cy-5 “capture sequence.” The cDNAs from both a treatment group and a pool of the corresponding control were then mixed and hybridized onto a microarray for eighteen hours. The arrays were washed according to the manufacturer’s specifications. The Cy-3 or Cy-5 fluor was included in a second hybridization where DNA complementary to the capture sequences was covalently linked to a dendrimer, containing up to 350 dye molecules. All experiments were performed in a “dye flop” experimental design where replicate hybridizations of the same sample and control are performed in each dye direction. That is, Cy-3-labeled cDNA from control sample was hybridized against Cy-5-labeled cDNA from the treated sample in dye direction #1, and Cy-5-labeled control RNA was hybridized against Cy-3-labeled treated RNA in dye direction #2.

cDNA Microarray Image Acquisition and Analysis

After hybridization, slides were scanned on a DNA Microarray Scanner, Model G2565BA (Agilent, Palo Alto, CA). Images were processed and the data was extracted using Agilent Feature Extractor software version 7.1 (Agilent, Palo Alto, CA). Further data processing were then performed by custom Perl scripts and imported into MySQL databases. Normalization was performed by the LOWESS method using the Agilent Feature Extraction Software (Agilent, Palo Alto, CA). After removing the maximum and minimum spot ratios to generate a single value, all cDNA replicates were averaged, and the values from the “dye flop” were then averaged. All microarray experiments discussed adhere to the Minimum Information About a Microarray Experiment (MIAME) guidelines. All microarray data are publicly available through the EDGE database (http://edge.oncology.wisc.edu/).

Northern Analysis

When microarray analysis identified a gene of interest, results were confirmed by Northern blot analyses. 10 μg of total RNA was electrophoresed in a 0.8% agarose gel containing 18% formaldehyde and was transferred to Hyband™-N+ membrance (Amersham Bioscience). cDNA fragments of Cyp1a2, Hectd2, Hsd17b5, Gstm1, Ugt1a6, Cyp2a5 and rGAPDH genes were randomly primed (Amersham) with 32P-dCTP and used as probes in Northern blot analysis of liver mRNA from mice treated with various doses of TCDD. Values obtained from phosphor autoradiography (STORM, Molecular Dynamics, Sunnyvale, CA) of blots probed with test and GAPDH control probes allowed for normalization of expression to eliminate mRNA loading variability.


Sections of mouse liver from equivalent regions were fixed in 10% buffered formalin, embedded in paraffin, and 5 μm sections were cut for Hematoxylin-eosin (H&E) staining. Two experienced personnel, who were blinded to specimen genotype and treatment, independently performed interpretation of histology. Hydropic degeneration, inflammatory cell infiltration, and steatosis were scored in a scale of 0 - 4 with 0 representing no change and 4 representing the most severe change observed. The values presented represent the average of the two interpreters.

Correlation Analysis

Probes that changed with a greater than two-fold response in at least one dose/time-point were selected for further correlation analyses. These included 373 probes from both the long-term and short-term time courses. Pearson correlation coefficients were generated, comparing the values of the time series for each probe to that of every pathological endpoint. Significance of Pearson values were assessed by generating 10,000 false time series for each probe, by randomly sampling from that probe’s true values with replacement. The actual “true” value was then ranked among the false values, creating a probability of that Pearson score arising randomly from the values generated.


Over the last few years, experiments with mutant alleles of the Ahr and Arnt loci have provided support for the idea that binding of the AHR-ARNT complex to the DRE is required for many aspects of TCDD toxicity (17-19, 23, 24). Moreover, related studies have shown that TCDD-induced toxicity is often cell-autonomous, e.g., hepatotoxicity is dependent upon AHR signal transduction within the hepatocyte (17-19, 23, 24). Despite this insight into TCDD toxicity, we still have little understanding of how signaling progresses from AHR-ARNT-DRE binding to any specific toxic endpoint. It seems clear that we have not identified all of the toxicologically-relevant DRE-regulated genes and that the genes most important in TCDD toxicity are still undefined.

In our effort to define TCDD-responsive gene batteries that might be related to hepatotoxicity, we set out to accomplish three bioinformatic tasks. The first task was to expand the number of liver transcripts that are dysregulated in response to TCDD. We hypothesize that the genes up-regulated with temporal and dose-response patterns similar to known DRE-regulated genes will have a high probability of being DRE regulated genes themselves. The second task was to identify batteries of genes that respond secondarily to the primary DRE-mediated transcriptional response. We hypothesize that these secondary or downstream genes will respond TCDD exposure with patterns that are temporally delayed from the primary battery. Our third task was to identify genes or batteries of genes that can act as surrogate markers of various pathological states. Identification of these gene batteries may prove valuable in defining biomarkers of TCDD-induced pathology.

Range finding and method validation

In an effort to define an exposure range for these studies, microarray analyses were first performed using mouse liver RNA after a 48 hour treatment with 0.1, 1.0, 10, 25, or 64 μg/kg TCDD. Data analysis revealed that 71 target genes displayed a significant change as compared to the untreated control (Figure 1). To validate the microarray approach, select transcriptional responses were assayed via northern blot analysis (Figure 2). This experiment led us to conclude that these microarray data provided a robust description of dioxin-induced gene expression, and that even small (1.5-fold) transcriptional changes can readily be detected by this approach. In all cases examined, data from the northern blots agreed with both the direction and the magnitude of the changes observed in the microarray experiments (Figure 2 and data not shown). Moreover, known DRE-driven gene products including Cyp1a1, Cyp1a2 and Ugt1a were shown to follow a classical dose-response relationship over the range employed (2).

Figure 1
Dose-response results: The heat map displays differential expression at 48 hours after five different (0.1, 1, 10, 25, and 64 μg/kg) doses of TCDD. At each dose, liver RNA was obtained from five treated mice, pooled, labeled and hybridized on ...
Figure 2
Northern blot validation: Confirmation of targets identified by microarray analyses by Northern blot. Targets identified as dose-responsive were isolated, sequence-verified and used as radiolabeled probes against a Northern blot of total RNA isolated ...

A preliminary analysis of the dose-response data revealed that a number of genes previously not thought of as “DRE-driven” were found to follow a dose-response pattern similar to Cyp1a1, Cyp1a2 and Ugt1a. These genes included Hectd2, Hsd17b2 and Cyp2a5 (NM_172637, NM_008290, NM_007812, respectively). Recently, similar studies have shown evidence supporting Hectd2 and Cyp2a5 as early dioxin responsive genes (25, 26). Although a dose-response relationship was less clear, at 64 ug/kg, TCDD upregulated three Serum Amyloid A loci (i.e,, Saa1, Saa2, and Saa3). These loci have previously been reported to be indicative of the acute phase response arm of inflammation (27) The up-regulation of these genes provides preliminary data to support the idea that some hepatic damage/inflammation is occurring at higher doses of TCDD exposure.

Long term study

In an effort to describe the relationships between dose, time, transcriptional response, and toxic response, we next performed a time-course study at three doses of TCDD over a period of sixty-four days. Based on the dose-response study, three doses were chosen: a) sub-maximal induction of transcriptional targets (1 μg/kg or “low dose”), b) a dose that gave rise to maximal induction of known target genes (10 μg/kg or “medium dose”) and c) a dose that exceeded the maximal induction dose and that was predicted to lead to pathological endpoints such as hepatomegaly, hydropic degeneration, inflammatory cell infiltration of the liver and thymic involution (64 μg/kg or “high dose”). At 1, 4, 8, 16, 32 and 64 days post-treatment, three animals from each dose were sacrificed, and the RNA and tissues were prepared from each group. For normalization of microarray data, each pooled group was compared against a control pool comprised of eight animals that had undergone treatment with vehicle alone for the same time period.

The known DRE-driven genes, Cyp1a1 and Cyp1a2, displayed marked induction at all doses and times examined (Figure 3). The response profiles of these genes differed as a function of both dose and time. For example, induction in the high dose group remained high throughout the course while induction following the low dose returned to near-baseline levels at 64 days, most probably due to redistribution and excretion. The greatest number of transcriptional changes appeared in the high dose cohort at eight and sixteen days post treatment.

Figure 3
Long-term TCDD toxicity study: microarray results. Heat map displays differential expression of genes across 64 days in response to TCDD. Sixty-three transcripts showed either 2-fold up-regulation or the same fold down-regulation in any one of the treatments. ...

We then examined histological changes in the livers of animals from the long-term TCDD exposure study (Figure 4). We quantified levels of hydropic degeneration in the hepatocytes, fatty changes (steatosis) in the hepatocytes, evidence of hepatocellular necrosis and apoptosis, as well as the level of inflammatory cells infiltrating the liver (“inflammation”). Hydropic degeneration was the earliest pathology noted in all groups and its severity was dependent on dose and time. Significant pathology began to appear four days after treatment in all dose groups. In correlation with the substantial transcriptional changes observed at eight and sixteen days, histological examination of animals treated with the high dose of TCDD revealed severe hydropic changes, massive inflammatory cell infiltration and some evidence of apoptosis and necrosis (Figures (Figures44 and and5).5). At sixteen days, steatosis was related to the presence of both microvesicular and macrovesicular fat deposits in the high dose animals (data not shown). Steatosis was only observed in the high dose group at 16, 32 and 64 days.

Figure 4
Long-term TCDD toxicity study: Histology scoring showing dose-dependent increase in toxicity. The graphs show quantitative results of histological examination conducted using the Hematoxylin and Eosin (H&E) stained slides from respective doses ...
Figure 5
Long-term TCDD toxicity study: Dose dependent induction of hepatomegaly by TCDD. Representative H&E stained sections displaying varying grades of pathology. Histological photographs were taken from H&E stained slides at 400x magnification. ...

Short Term Study

The observation from the long-term study that histological changes were observed as early as four days led us to perform a more detailed analysis of earlier time points in a short-term study. Mice were again treated with the 1, 10, and 64 μg/kg doses of TCDD and pathology and liver gene expression were examined after 6, 12, 24, 36, 48, 60, 72 and 96 hours. In order to assess biological variance of hepatic response to TCDD, we assayed individual animals at each time-dose point. In most cases, the data were generated from three animals per individual time-dose point.

Microarray analyses were performed to describe the difference in global gene expression in response to the three doses of TCDD. Induction of Cyp1a1 and Cyp1a2 occurred within six hours, with average peak induction occurring twenty-four hours after treatment (Figure 6). During the time course, 103 targets, or 6.4% of those genes assayed were either up-regulated or down-regulated at least 2-fold. Assessment of pathology from the short-term time course experiment revealed distinct differences in the dose-response. As with the long-term study, histological examination of animals from all three doses displayed hydropic degeneration, with severity being dose-responsive (Figure 7). At 96 hours, we observed inflammatory cell infiltration in the high dose cohort. In this experimental series, we added a direct measure of hepatocyte damage, with the quantification of serum levels of alanine aminotransferase (ALT). The ALT levels did not significantly vary from the control values at any time point except for 72 and 96 hours in the high dose. This rise corresponded in time to the infiltration of inflammatory cells (Figure 7).

Figure 6
Short-term TCDD toxicity study: microarray results. Heat map displays differential expression of genes from three doses of TCDD across eight time points. Animals were dosed with the indicated dose and sacrificed and the specified time. RNA from individual ...
Figure 7
Short-term TCDD toxicity study: Histology scores showing dose-dependent increase in toxicity. The graphs show quantitative results of histological examination conducted using the Hematoxylin and Eosin (H&E) stained slides from respective doses ...

Looking for batteries of coregulated genes

Our approach to identify batteries of coregulated genes is to identify transcripts that respond to TCDD with similar dose-and time dependent profiles. In our approach, we incorporated all of the available expression data from the “dose response”, “short-term” and “long-term” experiments in a correlation analysis with specific model transcripts or with the presentation of various pathological endpoints (Table 1 and Figure 8). After running a 100,000 bootstrap-permutation test, we selected those response pairs where a statistically significant correlation was observed (p < 0.001). These correlations, which ranged from 1 to - 0.885, were then organized using a hierarchical cluster analysis (Figure 8)(28). This analysis revealed four major groups that were then annotated by using the DAVID analysis tool from the National Cancer Institute (29). We postulate that genes with high positive correlations values over various treatments and times are candidates for coregulation and that transcripts with negative correlations represent regulation of transcripts that are moving in the opposite direction to the model transcript. Negatively correlated transcripts may represent a linked, yet opposing mechanism of gene regulation and they may be as valuable as positively correlated transcripts as biomarkers of pathology or for insight into mechnism.

Figure 8
Results of correlation-permutation test. All transcripts that changed significantly at least one time point across both short-term and long-term experiments were individually correlated with pathological endpoints and prototype marker genes. Positive ...
Table 1
Novel target transcripts consistent with DRE-mediation. Transcripts that showed a positive Pearson correlation coefficient with known DRE-driven genes were subjected to permutation significance analysis. All genes included are considered hypothetical ...

Primary targets and potential DRE-driven genes

Our approach to identify primary targets of TCDD was to identify transcripts that respond similarly to the known DRE-driven genes Cyp1a1, Cyp1a2, GSTm1 and Ugt1a (Table 1). Using this approach, we identified twenty-seven genes as being co-regulated with known DRE-driven genes (Fig 8). That is, over the dose-response, long-term and short-term experiments, these 27 genes responded similarly (i.e., with a positive correlation) to Cyp1a1, Cyp1a2, Gstm1 and/or Ugt1a (Table 1). Given that our model predicts that the initial induction of DRE-driven genes lies at the heart of dioxin hepatotoxicity; this list may include those DRE-driven transcripts that underlie some of the toxic effects of dioxin. Given that these microarrays are only sampling about one quarter of the mouse transcriptome it is also possible that a number of primary response genes will only be detected by a similar study using whole genome arrays.

Secondary response genes

As a first attempt to identify downstream (e.g., secondary and tertiary) transcriptional responses to TCDD, the correlation analysis was interrogated to identify transcripts that correlated with two prototype transcripts that were selected because they provided robust transcriptional changes that were temporally distinct from known DRE-driven gene products. The Car3 gene was selected because its TCDD response was down-regulated at high doses and temporally distinct from classical DRE-driven responses (Figure 3). The Saa1 gene was selected because its TCDD response was upregulated at high doses and temporally distinct from classical DRE-driven responses (Figure 3). Correlation analysis with these transcripts revealed the potential for the existence of two large TCDD-responsive gene batteries that can be described by the prototypes, Car3 and Saa1 (Figure 8). The gene batteries that correlate with these model transcripts appear to represent downstream responses to TCDD as their expression is markedly delayed in comparison to classical DRE regulated genes and occurs only at higher doses of TCDD exposure where toxicity is significant.

Potential biomarkers

In our attempt to identify biomarkers of TCDD hepatotoxicity, transcriptional responses were correlated with three pathological endpoints and those pairs of transcriptional and pathological changes where a statistically significant correlation or anti-correlation was observed were selected (p < 0.001). The underlying idea of this experiment was that transcripts that correlated with pathological events that were temporally distinct and later than DRE driven events are later steps in toxic signaling and represent potential biomarkers of toxicity. Hierarchical clustering revealed that three large classes of transcripts showed a relationship with the toxic endpoints studied. Moreover, two of these groups of genes showed a relationship with the expression of our models genes Saa1 and Car3.

Our prior work profiling the liver’s transcriptional response to many hepatotoxic agents leads us to predict that many of the transcripts that are coregulated with Car3 are related to a state of general hepatic stress (http://genome.oncology.wisc.edu/edge2/edge.php)(21). In our earlier work, we found that hepatotoxicants commonly downregulated many of the transcripts in this group, including the transcripts for Mup1-5 and Temt. Both these and related genes are down-regulated by TCDD as pathological endpoints worsen (i.e., they are anticorrelated). This observation leads us to suggest that common pathological states within the liver are the cause or consequence of this battery of down-regulated genes. It follows that transcripts from this group may provide molecular biomarkers for the correlated pathological endpoints induced by TCDD or a wide spectrum of hepatotoxic agents.

In our previous arrays work, we also observed that treatment of animals with hepatotoxic chemicals often results in a transient response from inflammatory cytokines such as IL1 α and tumor necrosis factor alpha (TNFα)(21). The action of these cytokines in downstream steps of TCDD hepatotoxicity is evident in the current data set. In this regard, the third largest group of correlated genes was found to contain a number of acute-phase genes, such as Saa1. As TCDD is not known to directly stimulate the IL-1α pathway, and the battery is temporally delayed from the DRE-cluster, we conclude that the acute phase is a secondary response battery. The observation that the acute phase response is inversely related to induction of cytochrome P450s (Figure 8 and EDGE website) is probably due to the well-known activity of cytokines to repress the expression of particular P450s (30).

Testing of Downstream Batteries

Armed with an objective list of primary TCDD targets, we inquired as to how these genes might be responsible for the regulation of secondary gene batteries by using null alleles for candidates. We hypothesized that downstream transcriptional responses were secondary to a DRE-regulated transcript. To test this idea we examined two well understood direct targets, Cyp1a1 and Cyp1a2, using null animal models (Figure 9a). These results supported the existence of downstream targets of the primary DRE-battery implicating apolilpoprotein-A4 and cytochrome P450 2a5 as downstream targets of Cyp1a2 (Figure 9a and data not shown). Cytochrome P450 2a5 and Apoa4 were selected as prototype downstream targets of Cyp1a2 and confirmed in independent biological samples (Figure 9b, 9c and data not shown). The probe interrogated on the cDNA microarray shows greater than ninety percent homology to both Cyp2a4 and Cyp2a5, but since only Cyp2a5 is expressed in male animals, we conclude that the Cyp2a5 is the affected gene (31). We suggest that these data show that Cyp2a5, previously labeled as AHR-responsive, is better described as Cyp1a2 responsive (32). The mechanism by which Cyp1a2 regulates Cyp2a5 expression is unclear. Given that the Cyp1a1 and Cyp1a2 null genotypes had no effect on our defined histopathological endpoints, we suggest that Cyp1a1 and Cyp1a2, as well as their downstream targets, might play a lesser role in the causality of TCDD toxicity.

Figure 9
Examination of the effects of Cyp1A1-/- and Cyp1A2-/- loci on TCDD-mediated toxicity. A. Hierarchical cluster of 19 targets that responded with a greater than 3-fold change in response to 64μg/kg TCDD over four days. Results indicate that the ...

In this study, we have characterized both the breadth and magnitude of the response of hepatic gene expression to TCDD. Through correlation analysis with prototype DRE-driven genes, we defined a list of primary target genes that directly respond to TCDD. Secondary and downstream targets were identified by a similar approach to genes and pathologies that were temporally distinct from primary events. We have further begun to separate the list of primary response genes into those that we believe to be direct (e.g. HectD2) from those that are dependent (e.g. Cyp2a5). Moreover, we have identified a series of transcriptional surrogates for hepatotoxicity that will aid in the quantitation of toxicity in future studies. This approach also allows for prioritization of candidate genes for future mechanistic studies.


1This work was supported by National Institutes of Health Grants R01-ES012752, P30-CA014520 and T32-CA009135.

2Here we define DRE-regulated (a.k.a. AHRE or XRE) genes to be any gene driven by the classical DRE (TNGCGTG), the recently reported DRE-II element (CATGNNNNNNC(T/A)TG) or any other undiscovered AHR-ARNT-target sequence (14).


(1) Whitlock JP., Jr. Mechanistic aspects of dioxin action. Chem. Res. Toxicol. 1993;6:754–763. [PubMed]
(2) Whitlock JP, Jr., Chichester CH, Bedgood RM, Okino ST, Ko HP, Ma Q, Dong L, Li H, Clarke-Katzenberg R. Induction of drug-metabolizing enzymes by dioxin. Drug Metab. Rev. 1997;29:1107–1127. [PubMed]
(3) Yang JH, Rhim JS. 2,3,7,8-Tetrachlorodibenzo-p-dioxin: molecular mechanism of carcinogenesis and its implication in human in vitro model. Critical Reviews in Oncology-Hematology. 1995;18:111–127. [PubMed]
(4) Couture LA, Abbott BD, Birnbaum LS. A critical review of the developmental toxicity and teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin: Recent advances toward understanding the mechanism. Teratology. 1990;42:619–627. [PubMed]
(5) Skene SA, Dewhurst IC, Greenberg M. Polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans: The risks to human health. A review. Human Toxicol. 1989;8:173–203. [PubMed]
(6) Safe S. Molecular biology of the Ah receptor and its role in carcinogenesis. Toxicol. Lett. 2001;120:1–7. [PubMed]
(7) Gonzalez FJ, Fernandez-Salguero P. The aryl hydrocarbon receptor: studies using the AHR-null mice. Drug Metab. Dispos. 1998;26:1194–1198. [PubMed]
(8) Poland A, Glover E. 2,3,7,8-Tetrachlorodibenzo-p-dioxin: A potent inducer of δ-aminolevulinic acid synthetase. Science. 1973;179 [PubMed]
(9) Poland A, Glover E, Kende AS. Stereospecific, high affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol. Evidence that the binding species is receptor for induction of aryl hydrocarbon hydroxylase. J. Biol. Chem. 1976;251:4936–4946. [PubMed]
(10) Hankinson O. The aryl hydrocarbon receptor complex. Annu. Rev. Pharmacol. Toxicol. 1995;35:307–340. [PubMed]
(11) Carver LA, LaPres JJ, Jain S, Dunham EE, Bradfield CA. Characterization of the Ah receptor-associated Protein, ARA9. J. Biol. Chem. 1998;273:33580–36159. [PubMed]
(12) Meyer BK, Pray-Grant MG, Vanden Heuvel JP, Perdew GH. Hepatitis B virus X-associated protein 2 is a subunit of the unliganded aryl hydrocarbon receptor core complex and exhibits transcriptional enhancer activity. Mol. Cell. Biol. 1998;18:978–988. [PMC free article] [PubMed]
(13) Gu Y-Z, Hogenesch J, Bradfield C. Annu. Rev. Pharmacol. Toxicol. Academic Press; 2000. The PAS superfamily: Sensors of environmental and developmental signals; pp. 519–561. [PubMed]
(14) Boutros PC, Moffat ID, Franc MA, Tijet N, Tuomisto J, Pohjanvirta R, Okey AB. Dioxin-responsive AHRE-II gene battery: identification by phylogenetic footprinting. Biochem. Biophy.s Res. Commun. 2004;321:707–715. [PubMed]
(15) Dong L, Ma Q, Whitlock JP., Jr. DNA binding by the heterodimeric Ah receptor. Relationship to dioxin-induced CYP1A1 transcription in vivo. J. Biol. Chem. 1996;271:7942–7948. [PubMed]
(16) Swanson HI, Chan WK, Bradfield CA. DNA binding specificities and pairing rules of the Ah receptor, ARNT, and SIM proteins. J. Biol. Chem. 1995;270:26292–26302. [PubMed]
(17) Bunger MK, Moran SM, Glover E, Thomae TL, Lahvis GP, Lin BC, Bradfield CA. 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. 2003;278:17767–17774. [PubMed]
(18) Walisser JA, Bunger MK, Glover E, Harstad EB, Bradfield CA. Patent ductus venosus and dioxin resistance in mice harboring a hypomorphic Arnt allele. J Biol. Chem. 2004;279:16326–16331. [PubMed]
(19) Walisser JA, Bunger MK, Glover E, Bradfield CA. Gestational exposure of Ahr and Arnt hypomorphs to dioxin rescues vascular development. Proc. Natl. Acad. Sci. U.S.A. 2004;101:16677–16682. [PMC free article] [PubMed]
(20) Lin PH, Lin CH, Huang CC, Chuang MC, Lin P. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces oxidative stress, DNA strand breaks, and poly(ADP-ribose) polymerase-1 activation in human breast carcinoma cell lines. Toxicol. Lett. 2007;172:146–158. [PubMed]
(21) Hayes KR, Vollrath AL, Zastrow GM, McMillan BJ, Craven M, Jovanovich S, Rank DR, Penn S, Walisser JA, Reddy JK, Thomas RS, Bradfield CA. EDGE: a centralized resource for the comparison, analysis, and distribution of toxicogenomic information. Mol. Pharmacol. 2005;67:1360–1368. [PubMed]
(22) Thomas RS, Rank DR, Penn SG, Zastrow GM, Hayes KR, Pande K, Glover E, Silander T, Craven MW, Reddy JK, Jovanovich SB, Bradfield CA. Identification of toxicologically predictive gene sets using cDNA microarrays. Mol. Pharmacol. 2001;60:1189–1194. [PubMed]
(23) Yim SH, Shah Y, Tomita S, Morris HD, Gavrilova O, Lambert G, Ward JM, Gonzalez FJ. Disruption of the Arnt gene in endothelial cells causes hepatic vascular defects and partial embryonic lethality in mice. Hepatology. 2006;44:550–560. [PMC free article] [PubMed]
(24) Tomita S, Sinal CJ, Yim SH, Gonzalez FJ. Conditional disruption of the aryl hydrocarbon receptor nuclear translocator (ARNT) gene leads to loss of target gene induction by the aryl hydrocarbon receptor and hypoxia-inducible factor 1 alpha. Mol. Endocrinol. 2000;14:1674–1681. [PubMed]
(25) Tijet N, Boutros PC, Moffat ID, Okey AB, Tuomisto J, Pohjanvirta R. Aryl hydrocarbon receptor regulates distinct dioxin-dependent and dioxin-independent gene batteries. Mol. Pharmacol. 2006;69:140–153. [PubMed]
(26) Boverhof DR, Burgoon LD, Tashiro C, Chittim B, Harkema JR, Jump DB, Zacharewski TR. Temporal and dose-dependent hepatic gene expression patterns in mice provide new insights into TCDD-Mediated hepatotoxicity. Toxicol. Sci. 2005;85:1048–1063. [PubMed]
(27) Jensen LE, Whitehead AS. Regulation of serum amyloid A protein expression during the acute-phase response. Biochem. J. 1998;334(Pt 3):489–503. [PMC free article] [PubMed]
(28) Eisen MB, Spellman PT, Brown PO, Botstein D. Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. U.S.A. 1998;95:14863–14868. [PMC free article] [PubMed]
(29) Dennis G, Jr., Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, Lempicki RA. DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol. 2003;4:P3. [PMC free article] [PubMed]
(30) Morgan ET. Regulation of cytochromes P450 during inflammation and infection. Drug Metab. Rev. 1997;29:1129–1188. [PubMed]
(31) Sueyoshi T, Yokomori N, Korach KS, Negishi M. Developmental action of estrogen receptor-alpha feminizes the growth hormone-Stat5b pathway and expression of Cyp2a4 and Cyp2d9 genes in mouse liver. Mol. Pharmacol. 1999;56:473–477. [PubMed]
(32) Arpiainen S, Raffalli-Mathieu F, Lang MA, Pelkonen O, Hakkola J. Regulation of the Cyp2a5 gene involves an aryl hydrocarbon receptor-dependent pathway. Mol. Pharmacol. 2005;67:1325–1333. [PubMed]
PubReader format: click here to try


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...