Genome‐Wide DNA Methylation Changes in Transformed Foci Induced by Nongenotoxic Carcinogens

: In vitro cell transformation assays (CTA) have been proposed as a method to identify possible nongenotoxic carcinogens. However, the current protocols do not provide information on the mechanism of action of the test articles. In this study, we combined an in vitro Bhas 42 CTA and sequencing-based DNA methylation profiling analysis to elucidate the carcinogenic mechanism associated with nongenotoxic carcinogens. Three nongenotoxic carcinogens were evaluated: cadmium chloride, methyl carbamate, and lithocholic acid. Methylation profiles were generated for the two nongenotoxic carcinogens (cadmium chloride and lithocholic acid) that were positive in Bhas 42 CTA. Methyl carbamate did not exhibit any promoter activity. Approximately 9.8% of all differentially methylated regions (DMRs) identified in cadmium chloride-induced transformed foci overlapped with DMRs in lithocholic acid-induced transformed foci. Interestingly, overlapping DMRs showed more hypermethylation than individual DMRs. In addition, the DMRs in CpG island elements common to both nongenotoxic carcinogens showed considerably more bias toward hypermethylated DMRs than those unique to either cadmium chloride or lithocholic acid. Pathway enrichment analysis revealed that genes harboring hypermethylated DMRs were significantly enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways including pathways in cancer, basal cell carcinoma, and Wnt signaling. The genes harboring hypomethylated DMRs were significantly related to mRNA surveillance pathway, RNA transport, and autophagy. Taken together, our preliminary results on genome-wide methylation analysis of cell clones from nongenotoxic carcinogen-induced foci could be exploited for CTAs improvement, but further research will be required to standardize and assess the specificity and sensitivity of this combined approach. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.