Development of a screening system for the detection of chemically induced DNA methylation alterations in a zebrafish liver cell line.

Early molecular events with correlation to disease, such as aberrant DNA methylation, emphasize the importance of DNA methylation as a potential environmental biomarker. Currently, little is known regarding how various environmental contaminants and mixtures alter DNA methylation in aquatic organisms, and testing is both time- and labor-consuming. Therefore, the potential of an in vitro screening method was evaluated by exposing zebrafish liver cells (ZF-L) for 96 h to the nonmutagenic model substance 5'-azacytidine (AZA), as well as a selection of environmental pollutants such as sodium arsenite (NAS), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 17α-ethinylestradiol (EE2), and diethylstilbestrol (DES). Six single genes with reported and anticipated importance in cancer were selected for analysis. Methylation of gene promoter areas was monitored by bisulfite conversion and high-resolution melt (HRM) analysis after exposure to sublethal concentrations of the test compounds. Subsequently, results were validated with direct bisulfite sequencing. Exposure of ZF-L cells to 0.5 μM AZA for 96 h led to hypomethylation of genes with both low and high basal methylation indicating similarity to mechanism of action in mammals. Further, NAS, EE2, and DES were shown to induce significant alterations in methylation, whereas TCDD did not. It was concluded that cell line exposure in combination with HRM may provide an initial contaminant screening assay by quantifying DNA methylation alterations with high throughput capacity. In addition, the rapid determination of effects following contaminant exposure with this in vitro system points to the possibility for new in vivo applications to be useful for environmental monitoring.