In vivo – in vitro toxicogenomic comparison of TCDD-elicited gene expression in Hepa1c1c7 mouse hepatoma cells and C57BL/6 hepatic tissue

BACKGROUND: In vitro systems have inherent limitations in their ability to model whole organism gene responses, which must be identified and appropriately considered when developing predictive biomarkers of in vivo toxicity. Systematic comparison of in vitro and in vivo temporal gene expression profiles were conducted to assess the ability of Hepa1c1c7 mouse hepatoma cells to model hepatic responses in C57BL/6 mice following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). RESULTS: Gene expression analysis and functional gene annotation indicate that Hepa1c1c7 cells appropriately modeled the induction of xenobiotic metabolism genes in vivo. However, responses associated with cell cycle progression and proliferation were unique to Hepa1c1c7 cells, consistent with the cell cycle arrest effects of TCDD on rapidly dividing cells. In contrast, lipid metabolism and immune responses, representative of whole organism effects in vivo, were not replicated in Hepa1c1c7 cells. CONCLUSION: These results identified inherent differences in TCDD-mediated gene expression responses between these models and highlighted the limitations of in vitro systems in modeling whole organism responses, and additionally identified potential predictive biomarkers of toxicity

Integration of Genome-Wide Computation DRE Search, AhR ChIP-chip and Gene Expression Analyses of TCDD-Elicited Responses in the Mouse Liver

<p>Abstract</p> <p>Background</p> <p>The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor (TF) that mediates responses to 2,3,7,8-tetrachlorodibenzo-<it>p</it>-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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Differences in TCDD-elicited gene expression profiles in human HepG2, mouse Hepa1c1c7 and rat H4IIE hepatoma cells

<p>Abstract</p> <p>Background</p> <p>2,3,7,8-Tetrachlorodibenzo-<it>p</it>-dioxin (TCDD) is an environmental contaminant that elicits a broad spectrum of toxic effects in a species-specific manner. Current risk assessment practices routinely extrapolate results from <it>in vivo </it>and <it>in vitro </it>rodent models to assess human risk. In order to further investigate the species-specific responses elicited by TCDD, temporal gene expression responses in human HepG2, mouse Hepa1c1c7 and rat H4IIE cells were compared.</p> <p>Results</p> <p>Microarray analysis identified a core set of conserved gene expression responses across species consistent with the role of AhR in mediating adaptive metabolic responses. However, significant species-specific as well as species-divergent responses were identified. Computational analysis of the regulatory regions of species-specific and -divergent responses suggests that dioxin response elements (DREs) are involved. These results are consistent with <it>in vivo </it>rat vs. mouse species-specific differential gene expression, and more comprehensive comparative DRE searches.</p> <p>Conclusions</p> <p>Comparative analysis of human HepG2, mouse Hepa1c1c7 and rat H4IIE TCDD-elicited gene expression responses is consistent with <it>in vivo </it>rat-mouse comparative gene expression studies, and more comprehensive comparative DRE searches, suggesting that AhR-mediated gene expression is species-specific.</p

Comparative temporal and dose-dependent morphological and transcriptional uterine effects elicited by tamoxifen and ethynylestradiol in immature, ovariectomized mice

<p>Abstract</p> <p>Background</p> <p>Uterine temporal and dose-dependent histopathologic, morphometric and gene expression responses to the selective estrogen receptor modulator tamoxifen (TAM) were comprehensively examined to further elucidate its estrogen receptor-mediated effects. These results were systematically compared to the effects elicited by the potent estrogen receptor ligand 17α-ethynylestradiol (EE) to identify pathways similarly and uniquely modified by each compound.</p> <p>Results</p> <p>Three daily doses of 100 μg/kg TAM elicited a dose-dependent increase in uterine wet weight (UWW) in immature, ovariectomized C57BL/6 mice at 72 hrs with concurrent increases in luminal epithelial cell height (LECH), luminal circumference and glandular epithelial tubule number. Significant UWW and LECH increases were detected at 24 hrs after a single dose of 100 μg/kg TAM. cDNA microarray analysis identified 2235 differentially expressed genes following a single dose of 100 μg/kg TAM at 2, 4, 8, 12, 18 and 24 hrs, and at 72 hrs after three daily doses (3 × 24 hrs). Functional annotation of differentially expressed genes was associated with cell growth and proliferation, cytoskeletal organization, extracellular matrix modification, nucleotide synthesis, DNA replication, protein synthesis and turnover, lipid metabolism, glycolysis and immunological responses as is expected from the uterotrophic response. Comparative analysis of TAM and EE treatments identified 1209 common, differentially expressed genes, the majority of which exhibited similar profiles despite a temporal delay in TAM elicited responses. However, several conserved and treatment specific responses were identified that are consistent with proliferation (Fos, Cdkn1a, Anapc1), and water imbibition (Slc30a3, Slc30a5) responses elicited by EE.</p> <p>Conclusion</p> <p>Overall, TAM and EE share similar gene expression profiles. However, TAM responses exhibit lower efficacy, while responses unique to EE are consistent with the physiological differences elicited between compounds.</p

Tamoxifen-elicited uterotrophy: cross-species and cross-ligand analysis of the gene expression program

<p>Abstract</p> <p>Background</p> <p>Tamoxifen (TAM) is a well characterized breast cancer drug and selective estrogen receptor modulator (SERM) which also has been associated with a small increase in risk for uterine cancers. TAM's partial agonist activation of estrogen receptor has been characterized for specific gene promoters but not at the genomic level <it>in vivo</it>.Furthermore, reducing uncertainties associated with cross-species extrapolations of pharmaco- and toxicogenomic data remains a formidable challenge.</p> <p>Results</p> <p>A comparative ligand and species analysis approach was conducted to systematically assess the physiological, morphological and uterine gene expression alterations elicited across time by TAM and ethynylestradiol (EE) in immature ovariectomized Sprague-Dawley rats and C57BL/6 mice. Differential gene expression was evaluated using custom cDNA microarrays, and the data was compared to identify conserved and divergent responses. 902 genes were differentially regulated in all four studies, 398 of which exhibit identical temporal expression patterns.</p> <p>Conclusion</p> <p>Comparative analysis of EE and TAM differentially expressed gene lists suggest TAM regulates no unique uterine genes that are conserved in the rat and mouse. This demonstrates that the partial agonist activities of TAM extend to molecular targets in regulating only a subset of EE-responsive genes. Ligand-conserved, species-divergent expression of carbonic anhydrase 2 was observed in the microarray data and confirmed by real time PCR. The identification of comparable temporal phenotypic responses linked to related gene expression profiles demonstrates that systematic comparative genomic assessments can elucidate important conserved and divergent mechanisms in rodent estrogen signalling during uterine proliferation.</p