TCDD-induced hepatic transcriptomic responses in transgenic AHR-variant mice
ABSTRACT: The aryl hydrocarbon receptor (AHR) mediates most of the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, TCDD toxicity phenotypes vary widely between species, strains and even between sexes within a strain. While the exact reasons for this variation remain unclear, it is thought to be related to differences in the structure of the AHR. Previous studies comparing the downstream effects of TCDD exposure between animals with different AHR isoforms have been confounded by the genetic differences between these model systems. To address this issue conclusively, we evaluated three transgenic mouse lines, each of which express a different rat AHR isoform (rWT, DEL, and INS) from two strains of rat with highly divergent TCDD-susceptibilities, within identical genetic backgrounds. Here we profile hepatic transcriptomic responses following exposure to TCDD, and use these to identify transcripts associated with toxicity. We have confirmed that the variation in toxicity is inherent to the AHR isoform. Additionally, we note the enhanced activity of the modified transactivation domain of the DEL isoform, relative to the INS isoform, and provide further evidence that the INS isoform is responsible for the high resistance to TCDD observed in H/W rats. We also uncover several candidate genes that were consistently differentially expressed in TCDD-sensitive mice and rats. Adult male transgenic mice were treated by gavage with 0, 125, 250, 500, or 1000 µg/kg TCDD dissolved in corn oil vehicle. Mice were euthanized 4 days following treatment and liver tissue was harvested for analysis. RNA was isolated and the transcriptome for each animal assayed on separate microarrays.
Project description:In many mammals, halogenated aromatic hydrocarbon (HAH) exposure causes wasting syndrome, defined as lethal weight loss as a result of severe and persistent hypophagia. The most potent HAH in causing wasting is 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD), which exerts its toxic effects through the aryl hydrocarbon receptor (AHR) – a transcription factor. Because TCDD toxicity is thought to predominantly arise from dysregulation of AHR-transcribed genes, we hypothesized that wasting syndrome is due to TCDD-induced dysregulation of genes involved in regulation of food-intake. We therefore focused on the hypothalamus, as it is the regulatory center of food-intake and energy balance in the central nervous system. We profiled mRNA abundance in hypothalamic tissue from two rat strains with widely differing sensitivities to wasting syndrome: TCDD-sensitive Long-Evans rats and TCDD-resistant Han/Wistar rats, 23 hours after exposure to TCDD (100 μg/kg) or corn oil vehicle. We found that TCDD exposure caused minimal transcriptional dysregulation effects in the hypothalamus, with only 6 genes changed in Long-Evans rats and 15 genes in Han/Wistar rats. Two of the most dysregulated genes were Cyp1a1 and Nqo1, which are induced by TCDD across a wide range of tissues and are considered sensitive markers of TCDD exposure. The minimal response of the hypothalamic transcriptome to a lethal dose of TCDD at an early time-point suggests that the hypothalamus is not the predominant site of initial events leading to hypophagia and associated wasting. TCDD may affect feeding behaviour via events upstream or downstream of the hypothalamus, and further work is required to evaluate this at the level of individual hypothalamic nuclei and subregions. Two strains, each with drug-treated vs vehicle-control
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:2,3,7,8–tetrachlorodibenzo-p-dixion (TCDD) is a dioxin congener that causes a wide range of toxic effects in rodent species. Previous studies discovered that males and females of the same species display different sensitivities to TCDD exposure. Although it is now clear that most TCDD-induced toxic outcomes are mediated by the Aryl Hydrocarbon Receptor (AHR), a transcription factor, the mechanism of sex-specific responses to TCDD remains largely unknown. To understand the differential sensitivity in male and female animals, we profiled the hepatic transcriptomic responses to single doses of TCDD (125, 250, 500, or 1000 µg/kg) in male and female C57BL6 mice. Several key findings were revealed by our study: 1) transcriptomic profiles varied largely between sexes at all doses; 2) the mRNA abundance profiles of female mice were less altered from basal level; 3) the alteration of ‘AHR-core’ genes were consistent regardless of sex; 4) a list of sex-specific TCDD-responsive genes were identified, including Fmo3 and Nr1i3 upregulated in male mice and Sult3a1 downregulated in female mice; 5) functional analysis of these candidate genes showed various biological pathway enrichments in a sex-dependent manner. Our study shows that the sex-dependent sensitivities to TCDD exposure are associated with a set of sex-specific TCDD-responsive genes that are indirectly regulated by AHR activity. The exact roles of these genes in response to TCDD exposure are not clear and require further investigation. Adult male and female C57BL/6 mice were treated by gavage with one single-dose TCDD (125, 250, 500, or 1000 μg/kg) in corn oil or corn oil vehicle alone. Animals were euthanized at 96 hours after treatment and tissues were harvested. RNA was isolated from hepatic tissue and the transcriptome for each animal assayed on an individual microarray.
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 μg/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.
Project description:Rodents exposed to the environmental contaminant, TCDD, suffer from a number of acute and chronic toxicities, including lethality and a wasting syndrome. Hypothesizing that the wasting syndrome may be caused by changes in neural control of energy flux and metabolism, we profiled the transcriptional response of rat hypothalamus to TCDD. We employed two separate rat strains: the Long-Evans strain is sensitive to TCDD toxicities while the Han/Wistar strain is over four orders of magnitude more resistant. Surprisingly, few transcriptional changes were induced by TCDD in either strain. Only four genes were altered in Long-Evans rats, including three classic TCDD-responsive genes: Cyp1a1, Cyp1b1, and Nqo1. These three genes were also altered in Han/Wistar rats, along with 133 additional genes. However, the magnitudes of alteration of these additional genes was very modest, with most changes well below two-fold in magnitude. We therefore concluded that rat hypothalamus is mostly refractory to TCDD exposure, at least at the doses and time-points surveyed here. Two strains, each with drug-treated vs. vehicle-control
Project description:Rodents exposed to the environmental contaminant, TCDD, suffer from a number of acute and chronic toxicities, including lethality and a wasting syndrome. Hypothesizing that the wasting syndrome may be caused by changes in adipose tissue -- either in its hormonal regulation or in homeostatic effects -- we profiled the transcriptional response of rat white adipose to TCDD. We employed two separate rat strains: the Long-Evans strain is sensitive to TCDD toxicities while the Han/Wistar strain is over four orders of magnitude more resistant. One day after TCDD exposure few genes were altered in either strain, but after four days a modest number of transcriptional alterations were observed. Strikingly, TCDD had far fewer effects than did a feed-restriction protocol intended to mimic the wasting syndrome itself. Notably several classic TCDD-responsive genes were modulated at all time-points, including Cyp1a1, Cyp1b1, and Nqo1. We therefore concluded that rat adipose tissue is unlikely to be the primary driver of the wasting syndrome, and that another tissue is likely involved. Two strains, each with drug-treated vs. vehicle-control
Project description:Objective - The TRIB1 locus has been linked to hepatic triglyceride metabolism in mice and to plasma triglycerides and coronary artery disease (CAD) in humans. The lipid associated SNPs identified by genome-wide association studies (GWAS) are located ~ 30 kb downstream from TRIB1 suggesting complex regulatory effects on genes or pathways relevant to hepatic triglyceride metabolism. The goal of this study was to investigate the functional relationship between common SNPs at the TRIB1 locus and plasma lipid traits. Methods & Results - Characterization of the risk locus reveals that it encompasses a gene, TRIB1 associated locus (TRIBAL) comprised of a well conserved promoter region and an alternatively spliced transcript. Bioinformatic analysis and re-sequencing identified a single nucleotide polymorphism (SNP), rs2001844, within the promoter region that associates with increased plasma triglycerides, reduced HDL-C and CAD risk. Furthermore, we show that rs2001844 is an expression trait locus (eQTL) for TRIB1 expression in blood and alters TRIBAL promoter activity in a reporter assay model. The TRIBAL transcript has features typical of long noncoding RNAs (lncRNA), including poor sequence conservation. Modulation of TRIBAL expression had limited impact on either TRIB1 or lipid regulatory genes mRNA levels in human hepatocyte models. In contrast, TRIB1 knockdown markedly increased TRIBAL expression in HepG2 cells and primary human hepatocytes. Conclusions - These studies demonstrate an interplay between a novel locus,TRIBAL, and TRIB1. TRIBAL is located in the GWAS identified risk locus, responds to altered expression of TRIB1, harbors a risk SNP that is an eQTL for TRIB1 expression and associates with plasma triglyceride concentrations. HepG2 hepatoma cells were stably infected with TRIBAL1 or no insert carrying lentiviruses
Project description:Shigella is an intracellular bacterial pathogen known to activate numerous innate immunity and inflammatory signaling pathways. Many of these pathways are also involved in mTOR signaling. We used HEK293T cell cultures and sought to understand how Shigella infection and rapamycin treatment affect the transcriptome and in particular whether there were commonly affected pathways in the two experimental conditions. 8 samples were used - 2 control that were uninfected/untreated -- 1 sample lost due to RNA degradation, 3 infected with Shigella for 4 hours, and 3 treated with Rapamycin (50 ug/mL) for 4 hours. All samples were from independent experiments. We used the program AltAnalyze to perform pairwise comparisons between Uninfected with Infected, and Uninfected with Rapamycin using default parameters.
Project description:Behaviour is derived from complex molecular interactions within the brain, in response to specific environmental stimuli. In some rare cases, the direct causes of behaviour have been attributed to the interactions of a single or small group of gene transcripts and proteins. We conducted two experiments with the hope of defining some of the molecular interactors for four separate behaviours: sugar feeding, locomotor activity in a novel field, and acoustic startle reflex, and prepulse inhibition of the acoustic startle reflex, which have been linked to prefrontal cortex dopaminergic function or as predictors of sensorimotor gating in diseases such as schizophrenia. Rats with high and low response phenotypes were selected to determine the differences between these two extremes of behaviour. From our analyses, transcripts of Homer1, a neuronal scaffolding protein which interacts with group1 metabotropic glutamate receptors, were found to be significantly correlated with array data in both experiments, and with behaviour data across three separate tests in the second experiment, indicating that this gene's transcripts and probably downstream protein interactions have a significant correlation with behaviour phenotype in the inbred Lewis rat. Future areas of pursuit for this data should involve modification of the expression of Homer1 in an isolated fashion to determine a pharmacological threshold for differences in behaviour. This SuperSeries is composed of the following subset Series:; GSE14902: The relationship between brain mRNA levels and behaviour among inbred Lewis rats: Experiment 1; GSE14903: The relationship between brain mRNA levels and behaviour among inbred Lewis rats: Experiment 2 Experiment Overall Design: Refer to individual Series