Biology subjects

Zacharewski, T.

Publications and source records attributed to Zacharewski, T..

6 recordsLinked to original sources

bioRxiv2024

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) elicited dose-dependent shifts in the murine urinary metabolome associated with hepatic AHR-mediated differential gene expression

Epidemiological evidence suggests an association between dioxin and dioxin-like compound (DLC) exposure and human liver disease. The prototypical DLC, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), has been shown to induce the progression of reversible hepatic steatosis to steatohepatitis with periportal fibrosis and biliary hyperplasia in mice. Although the effects of TCDD toxicity are mediated by aryl hydrocarbon receptor (AHR) activation, the underlying mechanisms of TCDD-induced hepatotoxicity are unresolved. In the present study, male C57BL/6NCrl mice were gavaged every 4 days for 28 days with 0.03 - 30 g/kg TCDD and evaluated for liver histopathology and gene expression as well as complementary 1-dimensional proton magnetic resonance (1D-1H NMR) urinary metabolic profiling. Urinary trimethylamine (TMA), trimethylamine N-oxide (TMAO), and 1-methylnicotinamide (1MN) levels were altered by TCDD at doses [≤] 3 g/kg; other urinary metabolites, like glycolate, urocanate, and 3-hydroxyisovalerate, were only altered at doses that induced moderate to severe steatohepatitis. Bulk liver RNA-seq data suggested altered urinary metabolites correlated with hepatic differential gene expression corresponding to specific metabolic pathways. In addition to evaluating whether altered urinary metabolites were liver-dependent, published single-nuclear RNA-seq (snRNA-seq), AHR ChIP-seq, and AHR knockout gene expression datasets provide further support for hepatic cell-type and AHR-regulated dependency, respectively. Overall, TCDD-induced liver effects were preceded by and occurred with changes in urinary metabolite levels due to AHR-mediated changes in hepatic gene expression.

molecular biology↗

bioRxiv2022

Generative Modeling of Single Cell Gene Expression for Dose-Dependent Chemical Perturbations

Single cell sequencing provides a new opportunity to study the heterogeneity of chemical perturbation within tissues. However, exploring the combinatorial space of all cell type-chemical combinations is experimentally and financially unfeasible. This space is significantly expanded by the dose axis of chemical perturbation. Thus, computational tools are needed to predict responses not only across tissues, but also across doses while capturing the nuances of cell type specific gene expression. Variational autoencoders simplify the single cell expression space allowing cross cell type predictions using simple vector arithmetic. However, differing sensitivities and non-linearities make cell type specific gene expression predictions following treatment at higher doses challenging. Here we introduce single cell Variational Inference of Dose-Response (scVIDR) which achieves high dose and cell type specific predictions better than other state of the art algorithms. scVIDR predicts in vivo and in vitro dose-dependent gene expression across cell types in mouse liver, peripheral blood mononuclear cells, and cancer cell lines. We use regression to interpret the outputs of scVIDR. Additionally, we use scVIDR to order individual cells based on their sensitivities to a particular chemical by assigning a pseudo-dose value to each cell. Taken together, we show that scVIDR can effectively predict the dose and cell state dependent changes associated with chemical perturbations.

bioinformatics↗

bioRxiv2022

Dose-dependent disruption of hepatic zonation by 2,3,7,8-tetrachlorodibenzo-p-dioxin in mice: integration of single-nuclei RNA sequencing and spatial transcriptomics.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) dose-dependently induces the development of hepatic fat accumulation and inflammation with fibrosis in mice initially in the portal region. Conversely, differential gene and protein expression is first detected in the central region. To further investigate cell-specific and spatially resolved dose-dependent changes in gene expression elicited by TCDD, single-nuclei RNA sequencing and spatial transcriptomics were used for livers of male mice gavaged with TCDD every 4 days for 28 days. The proportion of 11 cell (sub)types across 131,613 nuclei dose-dependently changed with 68% of all portal and central hepatocyte nuclei in control mice being overtaken by macrophages following TCDD treatment. We identified 368 (portal fibroblasts) to 1,339 (macrophages) differentially expressed genes. Spatial analyses revealed initial loss of portal identity that eventually spanned the entire liver lobule with increasing dose. Induction of R-spondin 3 (Rspo3) and pericentral Apc, suggested dysregulation of the Wnt/{beta}-catenin signaling cascade in zonally resolved steatosis. Collectively, the integrated results suggest disruption of zonation contributes to the pattern of TCDD-elicited NAFLD pathologies. SYNOPSIS O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY Single-nuclei RNA sequencing (snRNAseq) and spatial transcriptomics were integrated to investigate cell-specific and spatially resolved dose-dependent changes elicited by TCDD. We show that TCDD causes a loss of zonal characteristics that disrupts spatially defined metabolic functions. - Dose-dependent analyses show higher responsiveness of central hepatocytes despite hepatotoxicity occurring initially in the portal region. - Integration of snRNAseq and spatial transcriptomics demonstrates a loss of hepatocytes with portal characteristics. - TCDD disrupted spatially resolved expression of {beta}-catenin signaling members that are critical in maintaining liver zonation. - Spatial transcriptomics and snRNAseq shows induction of R-spondin3 from nonparenchymal cells which serve as cue for the {beta}-catenin pathway.

pharmacology and toxicology↗

bioRxiv2021

Aryl hydrocarbon receptor (AhR) activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) dose-dependently shifts the gut microbiome consistent with the progression of steatosis to steatohepatitis with fibrosis

Gut dysbiosis with disrupted enterohepatic bile acid metabolism is commonly associated with non-alcoholic fatty liver disease (NAFLD) and recapitulated in a NAFLD-phenotype elicited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice. TCDD induces hepatic fat accumulation and increases levels of secondary bile acids including taurolithocholic acid and deoxycholic acid, microbial modified bile acids involved in host bile acid regulation signaling pathways. To investigate the effects of TCDD on the gut microbiota, cecum contents of male C57BL/6 mice orally gavaged with sesame oil vehicle or 0.3, 3, or 30 {micro}g/kg TCDD were examined using shotgun metagenomic sequencing. Taxonomic analysis identified dose-dependent increases in Lactobacillus species (i.e., Lactobacillus reuteri). Increased species were also associated with dose-dependent increases in bile salt hydrolase sequences, responsible for deconjugation reactions in secondary bile acid metabolism. Increased L. reuteri levels were further associated with mevalonate-dependent isopentenyl diphosphate (IPP) biosynthesis and menaquinone biosynthesis genes. Analysis of gut microbiomes from cirrhosis patients identified increased abundance of these pathways as identified in the mouse cecum metagenomic analysis. These results extend the association of lactobacilli with the AhR/intestinal axis in NAFLD progression and highlight the similarities between TCDD-elicited phenotypes in mice to human NAFLD.

microbiology↗

bioRxiv2021

2,3,7,8-Tetrachlorodibenzo-p-dioxin elicited decreases in cobalamin inhibits methylmalonyl-CoA mutase activity redirecting propionyl-CoA metabolism to the β-oxidation-like pathway resulting in hepatic accumulation of the toxic intermediate acrylyl-CoA

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant which induces diverse biological and toxic effects, including the reprograming of intermediate metabolism, mediated by the aryl hydrocarbon receptor (AHR). Targeted LC-MS analysis of hepatic extracts from mice gavaged with TCDD every 4 days for 28 days detected an increase in S-(2-carboxyethyl)-L-cysteine, a conjugate produced following the spontaneous reaction between the sulfhydryl group of cysteine and highly reactive acrylyl-CoA, an intermediate in the cobalamin (Cbl)-independent {beta}-oxidation-like metabolism of propionyl-CoA. In addition to repressing genes in both the canonical Cbl-dependent carboxylase and the alternate Cbl-independent {beta}-oxidation-like pathways at 30 g/kg TCDD, methylmalonyl-CoA mutase (MUT) activity was inhibited at lower doses. Moreover, TCDD decreased serum Cbl levels and hepatic cobalt levels while eliciting negligible effects on gene expression associated with Cbl absorption, transport, trafficking, or the derivatization to 5-deoxy-adenosylcobalamin (AdoCbl), the required MUT cofactor. In addition to inducing Acod1 that encodes for aconitate decarboxylase 1, the enzyme responsible for the decarboxylation cis-aconitate to itaconate, TCDD also dose-dependently increased itaconate levels in hepatic extracts. MUT inhibition is consistent with itaconate activation to itaconyl-CoA, a MUT suicide inactivator that adducts AdoCbl, that in turn, inhibits MUT activity and reduces Cbl levels. Collectively, these results suggest the decrease in MUT activity is due to Cbl depletion following TCDD treatment that redirected propionyl-CoA metabolism to the alternate Cbl-independent {beta}-oxidation-like pathway. The resulting hepatic accumulation of acrylyl-CoA likely contributes to TCDD-elicited hepatotoxicity and the multi-hit progression of steatosis to steatohepatitis with fibrosis.

pharmacology and toxicology↗

bioRxiv2020

Application of singe nuclei RNA sequencing to assess the hepatic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin

Cell-specific transcriptional responses are lost in the averages of bulk RNA sequencing. We performed single nuclei RNA sequencing (snSeq) on frozen liver samples from male C57BL/6 mice in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Approximately 19,907 hepatic genes were detected across 16,015 sequenced nuclei from control and treated samples. Eleven cell-(sub)types were identified including distinct hepatocyte sub-populations, consistent with the cell diversity of the liver. TCDD increased macrophages from 0.5% to 24.7%, while neutrophils were only present in treated samples. The number of differentially expressed genes correlated with the basal expression level of Ahr. In addition to expected functional enrichments within each cell-(sub)type, RAS signaling was enriched in nonparenchymal cells. snSeq also identified a Kupffer cell subtype highly expressing Gpnmb, consistent with a dietary NASH model. Overall, snSeq distinguished cell-specific transcriptional changes and population shifts consistent with the hepatotoxicity of TCDD.

molecular biology↗