Comparative approaches were used to identify human, mouse and rat dioxin response elements (DREs) in genomic sequences unambiguously assigned to a nucleotide RefSeq accession number. A total of 13 bona fide DREs, all including the substitution intolerant core sequence (GCGTG) and adjacent variable sequences, were used to establish a position weight matrix and a matrix similarity (MS) score threshold to rank identified DREs. DREs with MS scores above the threshold were disproportionately distributed in close proximity to the transcription start site in all three species. Gene expression assays in hepatic mouse tissue confirmed the responsiveness of 192 genes possessing a putative DRE. Previously identified functional DREs in well-characterized AhR-regulated genes including Cyp1a1 and Cyp1b1 were corroborated. Putative DREs were identified in 48 out of 2437 human–mouse–rat orthologous genes between −1500 and the transcriptional start site, of which 19 of these genes possessed positionally conserved DREs as determined by multiple sequence alignment. Seven of these nineteen genes exhibited 2,3,7,8-tetrachlorodibenzo- p -dioxin-mediated regulation, although there were significant discrepancies between in vivo and in vitro results. Interestingly, of the mouse–rat orthologous genes with a DRE between −1500 and +1500, only 37% had an equivalent human ortholog. These results suggest that AhR-mediated gene expression may not be well conserved across species, which could have significant implications in human risk assessment.

In an effort to further characterize conserved and species-specific mechanisms of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)–mediated toxicity, comparative temporal and dose-response microarray analyses were performed on hepatic tissue from immature, ovariectomized Sprague Dawley rats and C57BL/6 mice. For temporal studies, rats and mice were gavaged with 10 or 30 μg/kg of TCDD, respectively, and sacrificed after 2, 4, 8, 12, 18, 24, 72, or 168 h while dose-response studies were performed at 24h. Hepatic gene expression profiles were monitored using custom cDNA microarrays containing 8567 (rat) or 13,361 (mouse) cDNA clones. Affymetrix data from male rats treated with 40 μg/kg TCDD were also included to expand the species comparison. In total, 3087 orthologous genes were represented in the cross-species comparison. Comparative analysis identified 33 orthologous genes that were commonly regulated by TCDD as well as 185 rat-specific and 225 mouse-specific responses. Functional annotation using Gene Ontology identified conserved gene responses associated with xenobiotic/chemical stress and amino acid and lipid metabolism. Rat-specific gene expression responses were associated with cellular growth and lipid metabolism while mouse-specific responses were associated with lipid uptake/metabolism and immune responses. The common and species-specific gene expression responses were also consistent with complementary histopathology, clinical chemistry, hepatic lipid analyses, and reports in the literature. These data expand our understanding of TCDD-mediated gene expression responses and indicate that species-specific toxicity may be mediated by differences in gene expression which may help explain the wide range of species sensitivities and will have important implications in risk assessment strategies.

In an effort to further characterize the mechanisms of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated toxicity, comprehensive temporal and dose–response microarray analyses were performed on hepatic tissue from immature ovariectomized C57BL/6 mice treated with TCDD. For temporal analysis, mice were gavaged with 30 μg/kg of TCDD or vehicle and sacrificed after 2, 4, 8, 12, 18, 24, 72, or 168 h. Dose–response mice were gavaged with 0, 0.001, 0.01, 0.1, 1, 10, 100, or 300 μg/kg of TCDD and sacrificed after 24 h. Hepatic gene expression profiles were monitored using custom cDNA microarrays containing 13,362 cDNA clones. Gene expression analysis identified 443 and 315 features which exhibited a significant change at one or more doses or time points, respectively, as determined using an empirical Bayes approach. Functional gene annotation extracted from public databases associated gene expression changes with physiological processes such as oxidative stress and metabolism, differentiation, apoptosis, gluconeogenesis, and fatty acid uptake and metabolism. Complementary histopathology (H&E and Oil Red O stains), clinical chemistry (i.e., alanine aminotransferase [ALT], triglyceride [TG], free fatty acids [FFA], cholesterol) and high-resolution gas chromatography/mass spectrometry assessment of hepatic TCDD levels were also performed in order to phenotypically anchor changes in gene expression to physiological end points. Collectively, the data support a proposed mechanism for TCDD-mediated hepatotoxicity, including fatty liver, which involves mobilization of peripheral fat and inappropriate increases in hepatic uptake of fatty acids.

Since its inception, there have been high expectations for the science of toxicogenomics to decrease the uncertainties associated with the risk assessment process by providing valuable insights into toxic mechanisms of action. However, the application of these data into risk assessment practices is still in the early stages of development, and proof of principle experiments have yet to emerge. The following discusses some potential applications as well as impediments that warrant a concerted investigation from all stakeholders in order to facilitate the acceptance and subsequent incorporation of toxicogenomics into regulatory decision making.

Background: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor (TF) that mediates responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Integration of TCDD-induced genome-wide AhR enrichment, differential gene expression and computational dioxin response element (DRE) analyses further elucidate the hepatic AhR regulatory network.Results: Global ChIP-chip and gene expression analyses were performed on hepatic tissue from immature ovariectomized mice orally gavaged with 30 μg/kg TCDD. ChIP-chip analysis identified 14,446 and 974 AhR enriched regions (1% false discovery rate) at 2 and 24 hrs, respectively. Enrichment density was greatest in the proximal promoter, and more specifically, within ± 1.5 kb of a transcriptional start site (TSS). AhR enrichment also occurred distal to a TSS (e.g. intergenic DNA and 3' UTR), extending the potential gene expression regulatory roles of the AhR. Although TF binding site analyses identified over-represented DRE sequences within enriched regions, approximately 50% of all AhR enriched regions lacked a DRE core (5'-GCGTG-3'). Microarray analysis identified 1,896 number of TCDD-responsive genes (|fold change| ≥ 1.5, P1(t) > 0.999). Integrating this gene expression data with our ChIP-chip and DRE analyses only identified 625 differentially expressed genes that involved an AhR interaction at a DRE. Functional annotation analysis of differentially regulated genes associated with AhR enrichment identified overrepresented processes related to fatty acid and lipid metabolism and transport, and xenobiotic metabolism, which are consistent with TCDD-elicited steatosis in the mouse liver.Conclusions: Details of the AhR regulatory network have been expanded to include AhR-DNA interactions within intragenic and intergenic genomic regions. Moreover, the AhR can interact with DNA independent of a DRE core suggesting there are alternative mechanisms of AhR-mediated gene regulation.

The aryl hydrocarbon receptor (AhR) mediates alterations in hepatic lipid composition elicited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In order to further investigate the effects of TCDD, liver, serum, and gonadal white adipose tissue (gWAT) fatty acid methyl esters (FAMEs) and lipids were examined in fasted 4-week-old female mice orally gavaged with 30 µg/kg TCDD at 24, 72, and 168h postdose. Mean hepatic FAME levels increased (236.7 µmol/g in controls compared with 392.2 µmol/g in TCDD treated) with minimal changes in gWAT and serum. In the liver, TCDD decreased saturated fatty acids (SFAs 16:0, 18:0, 20:0, and 22:0) and increased monounsaturated fatty acids (MUFAs 16:1n7, 18:1n9, and 20:1n9). Hepatic polyunsaturated fatty acids (PUFAs) 20:2n6, 20:3n6, 18:3n3, and 22:5n3 also increased, whereas 20:4n6 and 22:6n3 levels decreased. gWAT PUFAs 20:2n6 and 20:3n6 exhibited modest increases, whereas serum 18:0 decreased and 18:1n9 increased. Serum analyses also identified a ~25% decrease in total cholesterol (CHOL), low-density lipoprotein (LDL), and high-density lipoprotein following TCDD treatment. The decrease in serum CHOL was consistent with the induction of hepatic reverse CHOL transport genes Lcat (2.0-fold), Apoa1 (1.7-fold), and Ldlr (3.6-fold), and the repression of CHOL biosynthesis genes Hmgcs1 (−2.1-fold) and Hmgcr (−2.3-fold). In addition, TCDD decreased serum Apob100 (4.4-fold) and Apob48 (2.2-fold) protein levels, suggesting serum lipid clearance and decreased hepatic efflux. Collectively, the TCDD-elicited decreases in serum lipid levels are consistent with AhR-mediated enhancement of dietary fat distribution to the liver.