Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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

ABSTRACT: 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.

ORGANISM(S): Rattus norvegicus

SUBMITTER: Paul Boutros

PROVIDER: E-GEOD-13513 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Hepatic transcriptomic responses to TCDD in dioxin-sensitive and dioxin-resistant rats during the onset of toxicity.

Boutros Paul C PC Yao Cindy Q CQ Watson John D JD Wu Alexander H AH Moffat Ivy D ID Prokopec Stephenie D SD Smith Ashley B AB Okey Allan B AB Pohjanvirta Raimo R

Toxicology and applied pharmacology 20110105 2

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 previously showed that this resistance primarily results from H/W rats expressing ...[more]

PMID: 21215274

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Project description:Dioxin-like chemicals are well-known for their ability to upregulate expression of numerous genes via the AH receptor (AHR). However, recent transcriptomic analyses in several laboratories indicate that dioxin-like chemicals or AHR genotype itself also can downregulate levels of mRNAs encoded by numerous genes. The mechanism responsible for such downregulation is unknown. We hypothesized that microRNAs (miRNAs), which have emerged as powerful negative regulators of mRNA levels in several systems, might be responsible for mRNA downregulation in dioxin/AHR pathways. We used the Exiqon miRNA array platform as well as quantitative RT-PCR to measure miRNA levels in dioxin-sensitive Long-Evans (Turku/AB; L-E) rats vs. dioxin-resistant Han/Wistar(Kuopio; H/W) rats. Treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vivo caused few changes in miRNA levels in rat livers and those changes that were statistically significant were of modest magnitude. AHR genotype had little effect on hepatic miRNA levels, either in constitutive expression or in response to TCDD – only a few miRNAs differed in expression between between L-E rats (that have wildtype AHR) compared to H/W rats (whose AHR has a large deletion in the transactivation domain). It is unlikely that mRNA downregulation by dioxins is mediated by miRNAs, nor are miRNAs likely to play a significant role in dioxin toxicity in adult rodent liver. We conducted a thorough investigation to address the following questions: (1) does AHR genotype itself affect constitutive expression of microRNAs? (2) does TCDD affect microRNA levels and, if so, is this response dependent on the AHR? (3) does TCDD affect microRNA levels differently in animals that are sensitive to dioxin toxicity versus those that are dioxin-resistant? We assessed the in vivo effect of TCDD on microRNA levels in liver at multiple time points after TCDD treatment using microRNA arrays along with quantitative RT-PCR. The cumulative results of our experiments indicate that downregulation of mRNA levels by dioxins in adult rodent livers is very unlikely to involve microRNAs. Manuscript Submitted: Moffat ID, Boutros PC, Celius T, Pohjanvirta R & Okey AB. Micro-RNAs in rodent liver are refractory to dioxin treatment. Toxicological Sciences May, 2007. Keywords: miRNA expression, Time course, response to xenobiotics, genetic modification, comparative genome hybridization We examined strain differences in miRNA expression independent of TCDD: 19-h vehicle-treated dioxin-resistant H/W AHRH/W/H/W rats (HC19) vs. vehicle- control dioxin-sensitive L-E AHRWT/WT rats (LC19). In the dioxin-sensitive rats we compared miRNA expression levels 3 h (LT3) or 19 h (LT19) post-TCDD treatment vs. LC19. In the dioxin-resistant rats we compared miRNA levels 3 h (HT3), 19 h (HT19), or 96 h (HT96) post-TCDD treatment vs. HC19.

Project description:2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant that produces myriad toxicities across various experimental models. In rodents alone, there is huge divergence in the toxicological response across species, between strains within a species and even between sexes within a strain. This difference in sensitivities between sexes has been characterized in multiple rodent models, however with contrasting results: the severity of toxicity is greater in female rats than in males while male mice and guinea pigs are more sensitive to the effects of TCDD than females. While the presence of estrogens or androgens has a known impact on response, the specific transcriptional events that cause this difference remain unclear. We sought to characterize the transcriptional environment of male and female C57BL/6 mice treated with 500 M-NM-<g/kg TCDD and followed across a time-course. This dose was chosen as it is greater than the LD50 for male mice yet produces minimal effects in females. Additionally, evaluation along a time-course allows for the detection of specific changes that occur throughout the development of phenotypic toxicities. The transcriptional profile across the time-course was highly divergent between the sexes. Female TCDD-treated mice demonstrated a large number of altered transcripts as early as 6 hours following treatment, suggesting a large primary response, possibly indicative of the activation of numerous defense mechanisms. Conversely, male animals showed the greatest TCDD-mediated response 144 hours following exposure, potentially implicating significant secondary responses for the increased appearance of TCDD toxicities. Nr1i3 is statistically significantly altered at all time-points in the sensitive male animals. This mRNA encodes a transcription factor (constitutive androstane receptor; CAR) involved in the regulation of xenobiotic metabolism, as well as lipid metabolism, cell cycle and apoptosis. In particular, increased levels of CAR may result in induction of the anti-apoptotic gene Gadd45b and carboxylesterase Ces1d, both of which we identify as transcriptionally altered and may be responsible for phenotypic hepatic abnormalities, such as steatohepatitis and tumor formation. Further evaluation across studies of mice and rats into the role of Nr1i3 and associated genes are crucial to enhance our understanding of various TCDD-induced toxicities. Adult male and female C57BL/6 mice were treated by gavage with either 500 ug/kg TCDD in corn oil or corn oil vehicle alone. Animals were euthanized at either 6, 24, 72 or 144 hours after treatment and tissues were harvested. RNA was isolated from hepatic tissue and the transcriptome for each animal assayed on an individual microarray. Please note that 7 samples (out of total 65 samples) were identified as outliers and therefore the data were processed without the outliers as well. The normalized data without outliers were provided in the 'normalized_data_without_outliers.txt' file.

Dataset Information

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

Publications

Hepatic transcriptomic responses to TCDD in dioxin-sensitive and dioxin-resistant rats during the onset of toxicity.

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