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

Gerhard P Aigner,Pamela Nenning,Maja Šrut,Martina Höckner,Birgit Fiechtner

doi:10.3390/toxics10020100

Abstract

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 Lumbricus terrestris 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.

Highlights

Earthworms, as ecosystem engineers, are important organisms for soil formation because their activities have a tremendous impact on organic matter decomposition, nutrient cycles, bioturbation, aggregate formation, etc. [1]
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
Across all treatments, gene expression levels of metallothionein 2 (MT2), DNMT1 and ten–eleven translocation (TET) were significantly higher in coelomocytes compared with tissue samples

Summary

Introduction

Earthworms, as ecosystem engineers, are important organisms for soil formation because their activities have a tremendous impact on organic matter decomposition, nutrient cycles, bioturbation, aggregate formation, etc. [1]. Over the last few centuries, human activities have caused an accumulation of heavy metals in the environment [2]. The heavy metal cadmium (Cd) is one of the main soil pollutants declared as a carcinogenic substance by the WHO [3]. A Cd-polluted environment threatens soil life, including earthworms. Various ecotoxicological studies in plants, vertebrates and invertebrates have been performed explaining the toxic effects of Cd [6–10]. In the last few decades, increased research effort was put into deciphering epigenetic alterations caused by environmental stressors such as Cd. An altered epigenome can, in turn, play a role in adaption to environmental changes by influencing the activity of beneficial genes [11–13]. The environment influences epigenetic markers; these modifications can last for a long time and can even be measured across generations [15]. Epigenetic parameters were suggested as biomarkers for reflecting recent and past pollution burdens [16–18]

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