Persistent gene expression and DNA methylation alterations linked to carcinogenic effects of dichloroacetic acid [RNA-seq]

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.