Genome-wide DNA methylation analysis in differentiated liver HepaRG cells treated with low doses of aflatoxin B1, aflatoxin B2, benzo[a]pyrene, benzo[e]pyrene, and methapyrilene.

The increasing number of man-made chemicals in the environment that may pose a carcinogenic risk highlights the need for developing reliable time- and cost-effective approaches for carcinogen detection and identification. To address this issue, we investigated the utility of high-throughput microarray gene expression and next-generation genome-wide DNA methylation sequencing for the in vitro identification of genotoxic and non-genotoxic carcinogens. Terminally differentiated and metabolically competent human liver HepaRG cells were treated with (i) the genotoxic human liver carcinogen aflatoxin B1 (AFB1) and its structural non-carcinogenic analog aflatoxin B2 (AFB2); (ii) the genotoxic human lung carcinogen benzo[a]pyrene (B[a]P) and its non-carcinogenic isomer benzo[e]pyrene (B[e]P); and (iii) the non-genotoxic liver carcinogen methapyrilene. Cells were treated at minimally cytotoxic concentrations of chemicals for 72 hours and transcriptomic and DNA methylation profiles were examined. Treatment of HepaRG cells with the liver carcinogens AFB1 and methapyrilene, but not with the non-carcinogen AFB2, generated distinct gene-expression profiles. No treatment-related transcriptomic changes were found in cells treated with the genotoxic lung carcinogen B[a]P or its non-carcinogenic isomer B[e]P. In contrast to transcriptomic alterations, treatment of HepaRG cells with the carcinogenic and non-carcinogenic chemicals resulted in profound changes in the DNA methylation footprint; however, the correlation between gene-specific DNA methylation and gene expression changes was negligible. Among the carcinogen-altered genes, the transferrin (TF) gene and corresponding TF protein emerged as sensitive markers for an initial screening of chemicals for their potential liver carcinogenicity. This may allow identifying and selecting chemicals with potential liver carcinogenic activity for the subsequent transcriptomic analyses, which could differentiate genotoxic from non-genotoxic carcinogens by their distinct gene expression profiles. This approach may substantially enhance the identification and assessment of potential liver carcinogens.