Dataset Information

Single-Nuclei RNA Sequencing Assessment of the Hepatic Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin.

ABSTRACT: BACKGROUND AND AIMS:Characterization of cell specific transcriptional responses to hepatotoxicants is lost in the averages of bulk RNA-sequencing (RNA-seq). Single-cell/nuclei RNA-seq technologies enable the transcriptomes of individual cell (sub)types to be assessed within the context of in vivo models. METHODS:Single-nuclei RNA-sequencing (snSeq) of frozen liver samples from male C57BL/6 mice gavaged with sesame oil vehicle or 30 ?g/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) every 4 days for 28 days was used to demonstrate the application of snSeq for the evaluation of xenobiotics. RESULTS:A total of 19,907 genes were detected across 16,015 nuclei from control and TCDD-treated livers. Eleven cell (sub)types reflected the expected cell diversity of the liver including distinct pericentral, midzonal, and periportal hepatocyte subpopulations. TCDD altered relative proportions of cell types and elicited cell-specific gene expression profiles. For example, macrophages increased from 0.5% to 24.7%, while neutrophils were only present in treated samples, consistent with histological evaluation. The number of differentially expressed genes (DEGs) in each cell type ranged from 122 (cholangiocytes) to 7625 (midcentral hepatocytes), and loosely correlated with the basal expression level of Ahr, the canonical mediator of TCDD and related compounds. In addition to the expected functions within each cell (sub)types, RAS signaling and related pathways were specifically enriched in nonparenchymal cells while metabolic process enrichment occurred primarily in hepatocytes. snSeq also identified the expansion of a Kupffer cell subtype highly expressing Gpnmb, as reported in a dietary NASH model. CONCLUSIONS:We show that snSeq of frozen liver samples can be used to assess cell-specific transcriptional changes and population shifts in models of hepatotoxicity when examining freshly isolated cells is not feasible.

SUBMITTER: Nault R

PROVIDER: S-EPMC7674514 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Single-Nuclei RNA Sequencing Assessment of the Hepatic Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin.

Nault Rance R Fader Kelly A KA Bhattacharya Sudin S Zacharewski Tim R TR

Cellular and molecular gastroenterology and hepatology 20200810 1

<h4>Background and aims</h4>Characterization of cell specific transcriptional responses to hepatotoxicants is lost in the averages of bulk RNA-sequencing (RNA-seq). Single-cell/nuclei RNA-seq technologies enable the transcriptomes of individual cell (sub)types to be assessed within the context of in vivo models.<h4>Methods</h4>Single-nuclei RNA-sequencing (snSeq) of frozen liver samples from male C57BL/6 mice gavaged with sesame oil vehicle or 30 μg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) ...[more]

PMID: 32791302

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Project description:2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant that disrupts normal hepatic function. This disruption leads to the development and progression of non-alcoholic fatty liver disease (NAFLD) pathologies, including steatosis, steatohepatitis, and fibrosis. TCDD exerts hepatotoxic effects through activation of the aryl hydrocarbon receptor (AHR) eliciting transcriptional changes. Although diverse liver cell types contribute to TCDD-induced hepatotoxicity, cell-specific transcriptional changes in response to acute TCDD exposure remain unclear. The objective of this project was to elucidate the cell-specific outcomes of AHR activation following acute TCDD exposure. We conducted liver single-nuclei RNA-sequencing from male C57BL/6 mice administered 30 μg/kg of TCDD or sesame oil and sacrificed at 2, 4, 8, 12, 18, 24, or 72 hours post-oral gavage. Leiden clustering and annotation identified 10 major liver cell types, and 2 macrophage and 8 endothelial cell (EC) subtypes. While most cell types maintained their relative abundance, neutrophils exhibited an increase at 72 hours in response to TCDD. Hepatocytes, ECs, and hepatic stellate cells (HSCs) were the only cell types with cell-specific gene expression changes with known AHR binding sites (1,550, 102, and 40, respectively). Functional enrichment analysis in response to TCDD revealed disruptions in the metabolism of steroids, primary bile acids, vitamins, retinols, and glutathione in addition to one-carbon metabolism. EC differential gene expression was associated with PI3-AKT signaling, as well as protein processing and transport pathways. HSCs were enriched in protein processing, ABC transporters, and the dysregulation of tryptophan and linoleic acid metabolism. Altogether, these results suggest that early responses to TCDD trigger cell-specific changes in gene expression in the liver, contributing to hepatotoxicity and the emergence of hepatic pathologies related to NAFLD.

Project description:Background2,3,7,8-tetrachlorodibenzo-p-dixion (TCDD) is the most potent of the dioxin congeners, capable of causing a wide range of toxic effects across numerous animal models. Previous studies have demonstrated that males and females of the same species can display divergent sensitivity phenotypes to TCDD toxicities. Although it is now clear that most TCDD-induced toxic outcomes are mediated by the aryl hydrocarbon receptor (AHR), the mechanism of differential responses to TCDD exposure between sexes remains largely unknown. To investigate the differential sensitivities in male and female mice, we profiled the hepatic transcriptomic responses 4 days following exposure to various amounts of TCDD (125, 250, 500 or 1000 μg/kg) in adult male and female C57BL/6Kuo mice.ResultsSeveral key findings were revealed by our study. 1) Hepatic transcriptomes varied significantly between the sexes at all doses examined. 2) The liver transcriptome of males was more dysregulated by TCDD than that of females. 3) The alteration of "AHR-core" genes was consistent in magnitude, regardless of sex. 4) A subset of genes demonstrated sex-dependent TCDD-induced transcriptional changes, including Fmo3 and Nr1i3, which were significantly induced in livers of male mice only. In addition, a meta-analysis was performed to contrast transcriptomic profiles of various organisms and tissues following exposure to equitoxic doses of TCDD. Minimal overlap was observed in the differences between TCDD-sensitive or TCDD-resistant models.ConclusionsSex-dependent sensitivities to TCDD exposure are associated with a set of sex-specific TCDD-responsive genes. In addition, complex interactions between the aryl hydrocarbon and sex hormone receptors may affect the observable differences in sensitivity phenotypes between the sexes. Further work is necessary to better understand the roles of those genes altered by TCDD in a sex-dependent manner, and their association with changes to sex hormones and receptors.

Dataset Information

Single-Nuclei RNA Sequencing Assessment of the Hepatic Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin.

Publications

Single-Nuclei RNA Sequencing Assessment of the Hepatic Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin.

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