Common mechanisms cannot explain time- and dose-dependent DNA methylation changes in earthworms exposed to cadmium.

DNA hypermethylation caused by environmental pollutants like cadmium (Cd) has already been demonstrated in many invertebrates, including earthworms. However, the exact epigenetic mechanisms that drive this hypermethylation are largely unknown and even basic DNA methylation and demethylation processes are hardly characterized. Therefore, we used an important bioindicator, the earthworm Lumbricus terrestris, as a model organism to determine time- and dose-dependent effects of Cd on global and gene-specific DNA methylation and its underlying mechanisms. We revealed Cd-induced adenine and cytosine hypermethylation using specific antibodies in dot blots and found that the methylation level of adenine compared to cytosine changed even to a bigger extent. However, the levels of hydroxymethylated cytosine did not differ between treatment groups. General methylation and demethylation components like methyltransferases (DNMT1 and 3), and ten-eleven translocation (TET) genes were confirmed in L. terrestris by quantitative RealTime PCR. However, neither gene expression, nor DNMT and TET enzyme activity showed significant differences in the Cd exposure groups. Using bisulfite conversion and sequencing, gene body methylation (gbm) of metallothionein 2 (MT2), one of the most important detoxification proteins, was characterized. Cd-dependent changes in MT2 gbm could, however, not be correlated to MT2 gene activity evaluated by quantitative RealTime PCR. Future directions as well as missing links are discussed in the present study hinting towards the importance of studying epigenetic marks and mechanistic insights in a broad variety of species to deepen our knowledge on the effects of changing environmental conditions.