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Dose-response hepatic single nuclei RNA sequencing of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated mice

ABSTRACT: Dose-response is the cornerstone of safety assessments to determine risk. Innovations in transcriptomic technologies has recently led to the ability to profile gene expression at the single-cell level, enabling comprehensive assessment of adaptive and adverse responses at unprecedented resolution. To demonstrate its application of dose dependent study designs, we performed single nuclei sequencing of livers from male C57BL/6 mice gavaged with the persistent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at doses of 0.01, 0.03, 0.1, 0.3, 1, 3, 10, or 30 µg/kg or sesame oil vehicle. Following quality control, a total of 131,613 nuclei were sequenced representing 11 distinct cell (sub)types. Our findings reveal dose-dependent changes in relative levels of distinct cell (sub)types such as hepatocytes and macrophages, including the emergence of NASH-associated macrophages (NAMs) characterized by elevated Gpnmb expression. Zonally resolved hepatocytes could also be characterized demonstrating initial induction of aryl hydrocarbon receptor (AhR) target genes such as Cyp1a1 in the central region of the liver lobule while central and portal induction is observed at higher doses. Moreover, we show that zero-inflation inherent to single-cell technologies pose challenges for dose-dependent differential expression analysis and present a novel single-cell Bayesian test method that outperforms common two-group test methods. Collectively, our results demonstrate that single-cell technology can be used to further elucidate mechanisms associated within specific-cell (sub)types and interactions between different cell (sub)types to support the risk assessment paradigm.

ORGANISM(S): Mus musculus

PROVIDER: GSE184506 | GEO | 2022/05/10

REPOSITORIES: GEO

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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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Dose-response hepatic single nuclei RNA sequencing of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated mice

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