Sodium arsenite effects on DNA methylation in zebrafish embryos.. Danio rerio

There is a need to develop biomarkers in alternative testing models predictive for chemically induced chronic disease in humans, preferably describing key events and their relationships in an adverse outcome pathway analysis. Epigenetic modifications, and particularly DNA methylation effects, have been implicated as a major event in susceptibility to develop chronic disease. Arsenic exposure affects large populations around the world through drinking water and industrial activities. We sought to identify epigenetic markers of arsenic exposure. We analyzed the effect of sodium arsenite on DNA methylation in Danio rerio (zebrafish) embryos using unbiased methylation profiling by high throughput sequencing. Overall design: Pools of zebrafish embryos exposed to 0.1 mM and 1 mM sodium arsenite and non-exposed controls were analyzed in quadruplicates by DNA methylation profiling using DREAM (Digital Restriction Enzyme Analysis of Methylation). In short, genomic DNA was sequentially digested by SmaI and XmaI, which both recognize the sequence CCCGGG. SmaI is methylation sensitive, where XmaI is methylation insensitive. Distinct signatures, 5’-GGG at unmethylated sites or 5’-CCGGG at methylated sites were created by enzyme digestion and ultimately high througput sequencing was used to map these sites to the genome. Methylation ratios for each individual CCCGGG site were calculated as a proportion of methylated counts to the sum of unmethylated and methylated counts, and subsequently adjusted for differences in restriction enzyme efficiency using values obtained from spiked in standards.