Effects of methylmercury on epigenetic markers in three model species: Mink, chicken and yellow perch

Abstract

We previously reported that methylmercury (MeHg) exposure is associated with DNA hypomethylation in the brain stem of male polar bears. Here, we conveniently use archived tissues obtained from controlled laboratory exposure studies to look for evidence that MeHg can disrupt DNA methylation across taxa. Brain (cerebrum) tissues from MeHg-exposed mink (Neovison vison), chicken (Gallus gallus) and yellow perch (Perca flavescens) were analyzed for total Hg levels and global DNA methylation. Tissues from chicken and mink, but not perch, were also analyzed for DNA methyltransferase (DNMT) activity. In mink we observed significant reductions in global DNA methylation in an environmentally-relevant dietary exposure group (1 ppm MeHg), but not in a higher group (2 ppm MeHg). DNMT activity was significantly reduced in all treatment groups. In chicken or yellow perch, no statistically significant effects of MeHg were observed. Dose-dependent trends were observed in the chicken data but the direction of the change was not consistent between the two endpoints. Our results suggest that MeHg can be epigenetically active in that it has the capacity to affect DNA methylation in mammals. The variability in results across species may suggest inter-taxa differences in epigenetic responses to MeHg, or may be related to differences among the exposure scenarios used as animals were exposed to MeHg through different routes (dietary, egg injection), for different periods of time (19–89 days) and at different life stages (embryonic, juvenile, adult).

Introduction

Methylmercury (MeHg) is a ubiquitous toxicant that biomagnifies through aquatic food webs (Scheuhammer et al., 2007). Once inside the body, MeHg is effectively absorbed from the bloodstream and can readily enter sensitive tissues such as the brain (Clarkson and Magos, 2006). Numerous studies have documented that under real-world exposure scenarios, MeHg causes adverse effects to the health of individuals and populations of various taxa, including fish (Depew et al., 2012a), birds (Depew et al., 2012b), and mammals (Basu and Head, 2010).

The toxic actions of MeHg are diverse owing to its high affinity for thiol groups (Clarkson and Magos, 2006). A myriad of effects have been described at a molecular and cellular level including changes in neurotransmission, oxidative stress, and gene expression. There is now preliminary evidence that MeHg, like other toxic metals, may cause epigenetic changes (Pilsner et al., 2010). Epigenetics refers to heritable factors affecting gene expression that occur outside of modifications to the DNA sequence itself (Robertson and Wolffe, 2000, Head et al., 2012). These include DNA methylation and histone modification. Epigenetic marks are susceptible to environmental influences such as exposure to chemicals, and these changes may be inherited as cells divide mitotically, or even between generations. Epigenetic mechanisms may even help explain the observation of temporal disconnects between exposure and effect, as early-life exposure may leave epigenetic marks that result in adverse health outcomes later in life. This is noteworthy as MeHg is known to have a long latency of effect in many organisms (Clarkson and Magos, 2006, Basu and Head, 2010).

In the biomedical sciences, MeHg-associated changes in epigenetic markers are starting to be found in several experimental model systems. Developmental exposure of mice to MeHg resulted in a range of epigenetic changes in brain-derived neurotrophic factor (BDNF) in male offspring, including increased DNA methylation and altered methylation and acetylation of histones (Onishchenko et al., 2008). These epigenetic changes were associated with depressive symptoms. In female offspring of rats exposed to MeHg throughout pregnancy, reduced hepatic expression of DNA methyltransferases (DNMT1, DNMT3a, DNMT3b) was reported (Desaulniers et al., 2009). DNA methyltransferases are a family of enzymes that establish and maintain patterns of DNA methylation in the genome. An in vitro study using mouse embryonic cells found that acute exposure to Hg^2 + impairs histone production and reduces H3-K27 methylation (Gadhia et al., 2012). Such epigenetic findings have been extended to humans as increased methylation of GSTM1/5 promoter in blood was associated with MeHg exposure in women undergoing in vitro fertilization (Hanna et al., 2012), and hypomethylation of SEPP1 was associated with hair Hg levels among a cohort of male dentists (Goodrich et al., in press). Collectively these aforementioned studies provide founding evidence that mercury compounds can cause epigenetic changes.

Of all organisms, fish-eating wildlife are among those with the greatest exposures to MeHg (Scheuhammer et al., 2007, Basu and Head, 2010). However, little is known about MeHg-associated epigenetic effects in these organisms, (Vandegehuchte and Janssen, 2011, Head et al., 2012). We previously observed reduced global DNA methylation in association with MeHg exposure in brain stem tissues of male polar bears (Pilsner et al., 2010). While the results of this polar bear study were informative, the DNA methylation results were highly variable and likely influenced by a range of known (e.g., other toxicants, health status, age, gender, tissue quality) and unknown factors that could not be well-controlled when studying tissues from wild-caught animals. Given the need to generate data from well-controlled laboratory experiments, the current study was performed to increase understanding of possible MeHg-associated epigenetic changes using a mammalian, avian, and fish model species. All three classes contain species with well-documented sensitivities to MeHg (Scheuhammer et al., 2007). For each class we chose to study a model ecotoxicological test organism, namely mink (Neovison vison), chicken (Gallus gallus domesticus), and yellow perch (Perca flavescens). As part of other research projects, animals of each species were previously exposed in the laboratory to levels of MeHg ranging from ecologically relevant to high. Here we opportunistically used tissues preserved from those studies. The brain tissue (focus: cerebrum) was investigated in each model since this organ is the principal target of MeHg (Clarkson and Magos, 2006, Basu and Head, 2010). We examined global genomic DNA methylation using the LUMA assay and the enzymatic activity of DNMT via a commercially available ELISA kit. The guiding hypothesis of this study was that brain global genomic DNA methylation and DNMT enzyme activity would be reduced in association with methylmercury exposure in each of the three model species.