Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of primary mouse hepatocytes either TCDD treated C57BL6/J and or expressing human Aryl hydrocarbon Receptor

ABSTRACT: The human and mouse aryl hydrocarbon receptor (hAHR and mAHRb) share limited (58%) transactivation domain sequence identity. Compared to the mAHRb allele, the hAHR displays 10-fold lower relative affinity for prototypical ligands such as 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD). However, in previous studies we have demonstrated that the hAHR can display a higher relative ligand binding affinity than the mAHRb for specific AHR ligands such as indirubin. Each receptor has also been shown to differentially recruit LXXLL co-activator-motif proteins and to utilize different TAD subdomains in gene transactivation. Using hepatocytes isolated from C57BL6/J mice (Ahrb/b) and AHRTtr transgenic mice which express hAHR protein specifically in hepatocytes, we investigated whether the hAHR and mAHRb differentially regulate genes. Microarray and quantitative-PCR analysis of Ahrb/b and AHRTtr primary-mouse hepatocytes treated with 10 nM TCDD revealed that a number of established AHR target genes such as Cyp1a1 and Cyp1b1 are significantly induced by both receptors. Remarkably, of the 1752 genes induced by mAHRb and 1186 genes induced by hAHR, only 265 genes (<10%) were significantly activated by both receptors in response to TCDD. Conversely of the 1100 and 779 genes significantly repressed in mAHRb and hAHR hepatocytes respectively, only 462 (<25%) genes were significantly repressed by both receptors in response to TCDD treatment. Genes identified as differentially expressed are known to be involved in a number of biological pathways, including cell proliferation and inflammatory response which suggests that compared to the mAHRb, the hAHR may play contrasting roles in TCDD-induced toxicity and endogenous AHR-mediated gene regulation. Experiment Overall Design: Isolated mouse hepatocytes from wild type and treated with 10nM TCDD for 6h were analyzed. 1 array per mouse (3 mice per treatment group) was used.GeneChipÂ® Operating Software (Affymetrix) was utilized to preprocess CAB/CEL files generated from the 12 scanned microarrays which represented hepatocytes isolated from one mouse each. Data quality was initially assessed by checking the array image, B2 oligo performance, average background to noise ratios, poly-A controls, hybridization controls and the 3' to 5' probe-set ratios for control genes (e.g. Ã-actin or GAPDH). Microarray data was normalized using Probe Logarithmic Intensity Error Approximation PLIER-MM algorithm (Affymetrix Expression Consoleâ¢ Software 1.1). Normalized microarray data outputs from TCDD and control treated Ahrb/band AHRTtr hepatocytes were compared for differential expression using Significance Analysis of Microarrays (SAM, version 2.23A (Pan 2002; Tusher et al. 2001)) with 100 permutations, KNN-10.

ORGANISM(S): Mus musculus

SUBMITTER: Gary Perdew

PROVIDER: E-GEOD-17925 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The dioxin congener 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes a wide range of toxic effects in rodent species, all of which are mediated by a ligand-dependent transcription-factor, the aryl hydrocarbon receptor (AHR). The Han/Wistar (Kuopio) (H/W) strain shows exceptional resistance to many TCDD-induced toxicities; the LD50 of >9600 µg/kg for H/W rats is higher than for any other wild-type mammal known. We have previously shown that this resistance primarily results from H/W rats expressing a variant AHR isoform that has a substantial portion of the AHR transactivation domain deleted. Despite this large deletion, H/W rats are not entirely refractory to the effects of TCDD; the variant AHR in these animals remains fully competent to up-regulate well-known dioxin-inducible genes. TCDD-sensitive (Long-Evans, L-E) and resistant (H/W) rats were treated with either corn-oil (with or without feed-restriction) or 100 µg/kg TCDD for either four or ten days. Hepatic transcriptional profiling was done using microarrays, and was validated by RT-PCR analysis of 41 genes. . A core set of genes was altered in both strains at all time points tested, including CYP1A1, CYP1A2, CYP1B1, Nqo1, Aldh3a1, Tiparp, Exoc3, and Inmt. Outside this core, the strains differed significantly in the breadth of response: three-fold more genes were altered in L-E than H/W rats. At ten days almost all expressed genes were dysregulated in L-E rats, likely reflecting emerging toxic responses. Far fewer genes were affected by feed-restriction, suggesting that only a minority of the TCDD-induced changes are secondary to the wasting syndrome. Rats from sensitive (Long-Evans, LE) and resistant (Han/Wistar, HW) strains were treated with 100 ug/kg TCDD or corn oil vehicle and sacrificed either 4 or 10 days after treatment. LE control rats were either fed normally or feed-restricted to control for the wasting effects of TCDD treatment. Each treatment group contains four or five animals (biological replicates), each of which was assayed on an individual microarray.

Project description:Wild-type and AHR-KO rats harboring a 29-bp deletion mutation in exon 2 of the AHR gene were used in this study. Wild-type and AHR-KO rats were generated from heterozygous breeding stocks generated on a Sprague-Dawley outbred background and maintained at the Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina Persistent activation of the aryl hydrocarbon receptor (AHR) is believed to play a key role in the mode-of-action for TCDD induced rat liver tumorigenesis. It has been hypothesized that the cellular responses of hepatocytes to AHR activation may differ across regions of the liver lobule and that zone-specific effects of AHR agonists may play a role in the pathogenesis of rat liver tumorigenesis. Dose-dependent changes in gene expression were observed in both populations of hepatocytes collected from WT rats which were consistent with activation of AHR signaling. No significant or dose-dependent changes in gene expression were observed in samples from AHR-KO rats. In addition, evidence of inflammatory signaling pathway activation was observed only in centrilobular hepatocytes. Evidence of cell adhesion pathway enrichment was observed only in periportial hepatocytes. Benchmark dose analysis demonstrated that dose-dependent changes in gene expression occurred at lower doses in centrilobular as compared to periportal hepatocytes. These results indicate zone-specific differences in the sensitivity and response of hepatocytes to persistent AHR activation. AHR-KO and WT rats (n = 10 rats/dose/genotype) were dosed by oral gavage (4-5 doses / week, 19 total doses) with varying concentrations of TCDD in corn oil (0, 3, 22, 100, 300, 1000 ng/kg/day). Cryopreserved liver slices collected at necropsy were thawed, cryosectioned (12 Âµm thickness), fixed with ethanol and âquick-stainedâ with hematoxylin. Centrilobular and periportal hepatocytes were collected using laser capture microdissection. Total RNA was extracted, amplified and labeled using low-yield techniques and global transcriptomic profiles were obtained using Affymetrix Gene-Titan peg arrays.

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Transcription profiling of primary mouse hepatocytes either TCDD treated C57BL6/J and or expressing human Aryl hydrocarbon Receptor

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