DNA Methylation and Detoxification in the Earthworm Exposed to Cadmium and the DNA Demethylation Agent 5-aza-2'-deoxycytidine.

Earthworms are well-established model organisms for testing the effects of heavy metal pollution. How DNA methylation affects cadmium (Cd) detoxification processes such as the expression of metallothionein 2 (MT2), however, is largely unknown. We therefore exposed to 200 mg concentrations of Cd and 5-aza-2'-deoxycytidine (Aza), a demethylating agent, and sampled tissue and coelomocytes, cells of the innate immune system, for 48 h. MT2 transcription significantly increased in the Cd- and Cd-Aza-treated groups. In tissue samples, a significant decrease in MT2 in the Aza-treated group was detected, showing that Aza treatment inhibits basal MT2 gene activity but has no effect on Cd-induced MT2 levels. Although Cd repressed the gene expression of DNA-(cytosine-5)-methyltransferase-1 (DNMT1), which is responsible for maintaining DNA methylation, DNMT activity was unchanged, meaning that methylation maintenance was not affected in coelomocytes. The treatment did not influence DNMT3, which mediates de novo methylation, TET gene expression, which orchestrates demethylation, and global levels of hydroxymethylcytosine (5hmC), a product of the demethylation process. Taken together, this study indicates that Aza inhibits basal gene activity, in contrast to Cd-induced MT2 gene expression, but does not affect global DNA methylation. We therefore conclude that Cd detoxification based on the induction of MT2 does not relate to DNA methylation changes.