Mercury-associated DNA hypomethylation in polar bear brains via the LUminometric Methylation Assay: a sensitive method to study epigenetics in wildlife

Abstract

In this paper we describe a novel approach that may shed light on the genomic DNA methylation of organisms with non-resolved genomes. The LUminometric Methylation Assay (LUMA) is permissive for genomic DNA methylation studies of any genome as it relies on the use of methyl-sensitive and -insensitive restriction enzymes followed by polymerase extension via Pyrosequencing technology. Here, LUMA was used to characterize genomic DNA methylation in the lower brain stem region from 47 polar bears subsistence hunted in central East Greenland between 1999 and 2001. In these samples, average genomic DNA methylation was 57.9% +/- 6.69 (SD; range was 42.0 to 72.4%). When genomic DNA methylation was related to brain mercury (Hg) exposure levels, an inverse association was seen between these two variables for the entire study population (P for trend = 0.17). After dichotomizing animals by gender and controlling for age, a negative trend was seen amongst male animals (P for trend = 0.07) but no associations were found in female bears. Such sexually dimorphic responses have been found in other toxicological studies. Our results show that genomic DNA methylation can be quantitatively studied in a highly reproducible manner in tissue samples from a wild organism with a non-resolved genome. As such, LUMA holds great promise as a novel method to explore consequential questions across the ecological sciences that may require an epigenetic understanding.

DNA methylation, insulin resistance and second-generation antipsychotics in bipolar disorder

“…Duplicates resulting in greater than 15% were deleted before statistical analysis leading to a loss of five subjects. After exclusion of these five subjects, the average percentage coefficient of variation (CV%) for the entire population as well as each group was <4% which resembles that of others [29–31] . Demographic data was compared using student t-tests for continuous variables and chi-square tests for dichotomous variables.…”

“…Although this type of method does not give gene specific methylation information it provides a snapshot of the entire genome’s methylation status and gives evidence for further, in-depth exploration of DNA methylation. The LUminometric Methylation Assay (LUMA) is a restriction enzymebased method that detects methylation at 5′-CCGG-3′ sequences throughout the entire genome and has been described previously [27–29] . In brief, two separate reactions are carried out, one containing approximately five units of methylation-insensitive MspI (New England Biolabs, Ipswich, MA, USA) and the other containing approximately five units of methylation-sensitive HpaII ( New England Biolabs).…”

Anthropogenic flank attack on polar bears: interacting consequences of climate warming and pollutant exposure

“…Also, the ability of some POPs to induce epigenetic changes could present a mechanistic pathway of neurodevelopmental perturbations (Mill and Petronis, 2008;Bollati and Baccarelli, 2010;Crews, 2011). Associations between neurochemical and epigenetic biomarkers and mercury levels in brain tissue have been reported for polar bears (Basu et al, 2009;Pilsner et al, 2010;Dietz et al, 2013a). Another proposed mechanism of developmental neurotoxicity is through thyroid hormone disruption, as these hormones are essential during fetal and early neonatal neurodevelopment (Crofton and Zoeller, 2005;Ahmed et al, 2008).…”

“…These combined effects may also have affected the polar bears' genetic composition and effective population size (Pertoldi et al, 2009). An epigenetic study documented Hg-associated DNA hypomethylation in brain stems of male polar bears (Pilsner et al, 2010). Epigenetic responses do not represent a permanent redirection of phenotype in the same way as mutation and natural selection (Burggren and Crews, 2014), and are "turned off " in downstream generations when the environmental stressor that created them diminishes or disappears (Burggren, 2014).…”

Epigenetics and its implications for ecotoxicology

“…Following a similar approach, but in an ecotoxicological context, the same group of authors used a luminometric methylation assay (LUMA) to study global DNA methylation in brains of polar bears that were hunted in Greenland during 3 years (Pilsner et al 2010). In male bears, they observed an inverse relationship between DNA methylation and brain mercury (Hg) exposure levels, of which the significance level was p = 0.07.…”