Two-color fluorescent cytosine extension assay for the determination of global DNA methylation.

Here, we present a DNA restriction enzyme-based, fluorescent cytosine extension assay (CEA) to improve normalization and technical variation among sample-to-sample measurements. The assay includes end-labeling of parallel methylation-sensitive and methylation-insensitive DNA restriction enzyme digests along with co-purification and subsequent co-measurement of incorporated fluorescence. This non-radioactive, two-color fluorescent CEA (TCF-CEA) was shown to be a relatively rapid and accurate, with 3-fold greater precision than the one-color CEA. In addition, TCF-CEA provided an index of global DNA methylation that was sensitive to differences >5%. TCF-CEA results were highly correlated with LUminometric Methylation Assay (LUMA) results using human liver cell lines (HepG2, HepaRG, HC-04) as well as a human liver primary cell culture. Hypomethylation was observed in cells treated with the de-methylating agent 5-aza-2'-deoxycytidine. These results demonstrate that TCF-CEA provides a simple method for measuring relative degrees of global DNA methylation that could potentially be scaled up to higher-throughput formats.

Sodium bisulfite pyrosequencing revealed that developmental exposure to environmental contaminant mixtures does not affect DNA methylation of DNA repeats in Sprague-Dawley rats.

Hypomethylation of DNA repeats has been linked to diseases and cancer predisposition. Human studies suggest that higher blood concentrations of environmental contaminants (EC) correlate with levels of hypomethylation of DNA repeats in blood. The objective of this study was to examine the effect of in utero and/or lactational exposure to EC on the methylation of DNA repeats (LINE-1 and identifier element) in Sprague-Dawley rat pups at birth, at postnatal day (PND) 21, and in adulthood (PND78-86). From gestation day 0 to PND20, dams were exposed to a mixture "M" of polychlorinated biphenyls (PCB), pesticides, and methylmercury (MeHg), at 0.5 or 1 mg/kg/d (0.5M and M). At birth, some control (C) and M litters were cross-fostered to create the following in utero/postnatal exposure groups: C/C, M/C, C/M, M/M. Additional dams received 1.8 ng/kg/d of a mixture of aryl-hydrocarbon receptor (AhR) agonists (non-ortho-PCB, PC-dibenzodioxins, and PC-dibenzofurans) without or with 0.5M (0.5MAhR). Measurements of EC residue levels confirmed differences in their accumulation across treatments, age, and tissues. Although induction of hepatic detoxification enzyme activities (cytochrome P-450) demonstrated biological effects of treatments, the assessment of methylation in DNA repeats by sodium bisulfite pyrosequencing of liver, spleen, and thymus samples revealed no marked treatment-related effects but significant tissue- and age-related methylation differences. Further studies are required to determine whether absence of significant observable treatment effects on methylation of DNA repeats in the rat relate to tissue, strain, or species differences.

Effects of mixtures of polychlorinated biphenyls, methylmercury, and organochlorine pesticides on hepatic DNA methylation in prepubertal female Sprague-Dawley rats.

DNA methylation is one of the epigenetic mechanisms that regulates gene expression, chromosome structure, and stability. Our objective was to determine whether the DNA methylation system could be a target following in utero and postnatal exposure to human blood contaminants. Pregnant rats were dosed daily from gestation day 1 until postnatal day 21 with 2 dose levels of either organochlorine pesticides (OCP; 0.019 or 1.9 mg/kg/day), methylmercury chloride (MeHg; 0.02 or 2 mg/kg/day), polychlorinated biphenyls (PCBs; 0.011 or 1.1 mg/kg/day), or a mixture (Mix; 0.05, or 5 mg/kg/day) including all 3 groups of chemicals. Livers from 1 female offspring per litter were collected at postnatal day 29. Hepatic analysis revealed that the mRNA abundance for DNA methyltransferase (DNMT)-1, -3a, and -3b were significantly reduced by the high dose of PCB, that the high dose of MeHg also reduced mRNA levels for DNMT-1, and -3b, but that OCP had no significant effects compared with control. The high dose of PCB and Mix reduced the abundance of the universal methyl donor S-adenosylmethionine, and Mix also reduced global genome DNA methylation (5-methyl-deoxycytidine/5-methyl-deoxycytidine + deoxycytidine). The latter is consistent with pyrosequencing methylation analysis, revealing that the high-dose groups (except OCP) generally decreased the methylation of CpG sites (position -63 to -29) in the promoter of the tumor suppressor gene p16(INK4a). Overall, these hepatic results suggest that the DNA methylation system can be affected by exposure to high doses of blood contaminants, and that OCP is the least potent chemical group from the investigated mixtures.