An Adverse Outcome Pathway Analysis Employing DNA Methylation Effects in Arsenic-Exposed Zebrafish Embryos Supports a Role of Epigenetic Events in Arsenic-Induced Chronic Disease

There is a need to identify processes underlying development of chemically induced chronic disease in humans, preferably describing key events and their relationships in an adverse outcome pathway (AOP) analysis. Epigenetic modifications, and particularly DNA methylation effects, have been implicated as a major event in susceptibility to develop chronic disease. Arsenic is a known toxicant to which large human populations around the world are exposed through drinking water and industrial activities, and the role of epigenetic events in arsenic-induced chronic disease has been suggested in multiple cases. In this article, arsenic was used as a case to design an epigenetics-based AOP framework and provide further support through detecting effects on DNA methylation in zebrafish embryos, which have the advantage of an alternative whole organism model, including the complete array of potential target tissues and their interactions. Four targets derived from literature, that is, HOXB5, HOXB9, TP53, and PAPP2c, followed by a genome-wide methylation analysis method, Digital Restriction Enzyme Analysis of Methylation (DREAM), and subsequent pyrosequencing verification. The four literature targets all showed very low baseline methylation, and notably, HOXB5 and PAPP2c showed arsenic-induced DNA methylation effects in extracts of 72 hours postfertilization arsenite-exposed whole embryos. DREAM identified hypermethylation in four additional specific DNA sites, that is, cbll1, cwc27, mvb12bb, and ybx1, which could all be related to specific cellular functions with relevance to carcinogenesis. Altogether, through pathway analysis complemented with DNA methylation analysis in zebrafish embryos, the observations in this study add to weight of evidence for the relationship between arsenic-induced epigenetic effects and late-onset disease, specifically cancer.