Project description:Mechanistic understanding of transient exposures that lead to adverse health outcomes will enhance our ability to recognize biological signatures of disease. Here, we measured the transcriptomic and epigenomic alterations due to exposure to the metabolic reprogramming agent, dichloroacetic acid (DCA). Previously, we showed that exposure to DCA increased liver cancer in B6C3F1 mice after continuous or early life exposures similarly over background level. Measures from these studies did not support direct cytotoxic, mitogenic, or genotoxic modes-of-action of tumorigenesis. Using archived formalin-fixed liver samples, we utilized modern methodologies to measure gene expression and DNA methylation levels to link to previously generated phenotypic measures. Gene expression was measured by targeted RNA sequencing (TempO-seq 1500+ toxicity panel: 2754 total genes) in liver samples collected from 10-, 32-, 57-, and 78-week old mice exposed to deionized water (controls), DCA continuously at 3.5g/L in drinking water (“Direct” group), or DCA at 3.5g/L for 10-, 32-, or 57-weeks followed by control water (“Stop” groups). Genome-scaled alterations in DNA methylation were measured by Reduced Representation Bisulfite Sequencing (RRBS) at 78-weeks. Transcriptomic changes were most robust with concurrent or adjacent timepoints after exposure stoppage. DNA methylation alterations followed a similar pattern, measuring 2720 and 567 differentially methylated regions (DMRs) in 78-week Direct and 10-week “Stop” DCA exposure groups, respectively. Gene pathway analysis indicated cellular effects linked to increased oxidative metabolism, a primary mechanism of action for DCA, closer to exposure windows especially early in life. Conversely, many gene signatures and pathways reversed patterns later in life and reflected more pro-tumorigenic patterns for both current and prior DCA exposures. DNA methylation patterns linked to early gene pathway perturbations, suggesting persistence in the epigenome and possible regulatory effects. In total, results suggested that liver metabolic reprogramming effects of DCA interact with normal age mechanisms to increase tumor burden with both continuous and prior DCA exposure in the B6C3F1 rodent model.

Project description:1,4-Dioxane (1,4-DX) is an environmental contaminant found in drinking water throughout the United States (US). While it is a suspected liver carcinogen, there is no federal or state maximum contaminant level for 1,4-DX in drinking water. Very little is known about the mechanisms by which this chemical elicits liver carcinogenicity. In the present study, we performed chronic and short-term dosing studies in female BDF-1 mice to explore the toxic effects of 1,4-DX. Histopathology studies and a multi-omics approach (transcriptomics and metabolomics) were performed to investigate potential mechanisms of toxicity. Mice were exposed to various concentrations of 1,4-DX (0, 50, 500 and 5,000 mg/L) in their drinking water for one or four weeks. Immunohistochemical analysis of the liver revealed an increase in the number of H2AXγ-positive hepatocytes (a marker of DNA double strand breaks) in mice exposed to 5,000 mg/L 1,4-DX for one and four weeks. In addition, an expansion of precholangiocytes was observed after four weeks of 5,000 mg/L 1,4-DX exposure, as reflected by CK-7 immunostaining. An increase in these markers reflect both DNA damage and repair mechanisms. Liver transcriptomics profiling showed that exposure to 5,000 mg/L 1,4-DX for four weeks resulted in the differential expression of 65 genes compared to controls. Pathway analysis of the transcriptomic data revealed 1,4-DX-induced perturbations in multiple signaling pathways in the liver, including those involved in xenobiotic metabolism, nicotine degradation and glutathione-mediated detoxification. Changes to these pathways as a result of 1,4-DX exposure reflect would be predicted to impact the oxidative stress response, detoxification, and DNA damage. Liver, kidney, stool and urine metabolomics profiling revealed no effect of 5,000 mg/L 1,4-DX exposure for one or four weeks on metabolites. We speculate that this may be reflective of DNA damage being counterbalanced by the repair response, with the net result being a null overall effect on the systemic biochemistry of the exposed mice. Our results show a novel approach for the investigation of environmental chemicals that do not elicit cell death, but have activated the repair systems in response to 1,4-DX exposure

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Time course effects of a latent carcinogen in mice

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