Epigenetic Dysregulation of H19/IGF2 in Hepatic Cells Exposed to Toxic Metal Mixtures In Vitro [Bisulfite-seq]

Exposure to mixtures of toxic metals is known to cause adverse health effects through epigenetic alterations. Here we aimed to examine the unexplored area of aberrant DNA methylation in the H19/IGF2 domain following combined toxic metal exposure. An in vitro epigenotoxicity assay using the human normal liver epithelial cell line THLE-3 was conducted. When THLE-3 cells were exposed to specific concentrations of either organic arsenic or MeHgCl, an increase in the H19 lncRNA levels and a marked reduction in the IGF2 mRNA levels were observed. In contrast, combined exposures coupled with CdCl2 resulted in the transcriptional repression of H19 and transcriptional activation of IGF2. It should be noted that the correlation between the dysregulated expression of H19/IGF2 and the hypermethylated CpG sites within the H19 differentially methylated region (DMR) was statistically significant. Furthermore, we performed transcriptomic analysis of the hepatocytes exposed to toxic metal combinations indicating enrichment of pro-inflammatory and anti-proliferative pathways compared to the unexposed cells. Our results suggest that hazardous metal mixtures may trigger epigenetic aberrations at the H19/IGF2 locus. We propose that altered CpG methylation in the H19 DMR could be a candidate biomarker for hepatic epigenotoxicity, in part, due to environmental exposure. To investigate the effects of toxic metal exposure on DNA methylation, we performed bisulfite amplicon sequencing (BSAS) to assess the methylation status at the H19/IGF2 locus in control and metal-exposed cells. BSAS samples were collected under various experimental conditions, including control (no exposure), organoarsenic (oAs, 10 μM), cadmium chloride (CdCl2, 1 μM), and methylmercury (MeHgCl, 10 nM), either individually or in combination. The CpG sites within the H19 differentially methylated region (DMR) and imprinting control region (ICR) were analyzed to quantify methylation changes across these exposure conditions.