Persistent gene expression and DNA methylation alterations linked to carcinogenic effects of dichloroacetic acid [RRBS]
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. Overall design: Male B6C3F1 mice were treated with 3.5 g/L DCA in deionized water in a stop exposure study design over a period of 78 weeks. Mice were either provided only deionized water (control), continuous DCA (direct) or in a series of stop-dosage timepoints after which the mice were switched to deionized water for the duration of the experiment. The stop group treatments were as follows: 10 weeks of DCA followed by 68 weeks of water (S1), 32 weeks of DCA and 46 weeks of water (S2) and 57 weeks of DCA with 21 weeks of water (S3). The direct group were exposed to 3.5 g/L for the entirety of the 78-week study. At each timepoint (10, 32, 57 or 78 weeks), a subset of the control, direct and stop treatment block mice were humanely euthanized with CO2 following EPA approved Animal Care and Use Committee protocols.  Portions of the livers were fresh frozen at each sacrifice timepoint along with the preparation of FFPE paraffin blocks. Figure 1 outlines study collection points and treatments. FFPE-derived samples were used to measure gene expression and frozen samples were used for DNA methylation determination.