Global DNA methylation in the mouse liver is affected by methyl deficiency and arsenic in a sex-dependent manner

Abstract

Arsenic, a carcinogen, is assumed to induce global DNA hypomethylation by consuming the universal methyl donor S-adenosylmethionine (SAM) in the body. A previous study reported that a methyl-deficient diet (MDD) with arsenic intake greatly reduced global DNA methylation (the content of 5-methylcytosine) in the liver of male C57BL/6 mice. In the present study, we investigated the DNA methylation level, SAM content, and expression of DNA methyltransferases (DNMTs) in the liver of male and female C57BL/6 mice fed a methyl-sufficient diet (MSD), an MDD, or an MDD + arsenic. The DNA methylation level was accurately determined by measuring the content of genomic 5-methyldeoxycytidine (5medC) by high-performance liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) using stable-isotope-labeled 5medC and deoxycytidine (dC) as internal standards. The results of this study revealed that while the MDD and arsenic tended to reduce the genomic 5meC content in the male mice livers, the MDD + arsenic significantly increased the 5meC content in the female mice livers. Another unexpected finding was the small differences in 5meC content among the groups. The MDD and MDD + arsenic suppressed DNMT1 expression only in the male mice livers. In contrast, SAM content was reduced by the MDD and MDD + arsenic only in the livers of female mice, showing that the changes in 5meC content were not attributable to SAM content. The sex-dependent changes in 5meC content induced by methyl deficiency and arsenic may be involved in differences in male and female susceptibility to diseases via epigenetic modification of physiological functions.

Diurnal expression ofDnmt3bmRNA in mouse liver is regulated by feeding and hepatic clockwork

“…In our previous study, we measured the 5-mC level of the liver of mice fed a methyl-deficient diet, which has been reported to induce DNA hypomethylation, 51 by the precise LC/ESI-MS method, and found that 5 mo of feeding with methyl-deficient diet reduced the 5-mC level by 3.6% in the liver genome of male mice. 15 This study demonstrated that the 5-mC level is changed by up to 11.2% in a day and the deviation is thought to be substantial. On the other hand, we could not find the quantitative difference of DNMT3B protein between ZT 0 and 8 by western blotting analysis (Fig.…”

“…We previously reported that Dnmts mRNA expression in adult mouse liver can vary with diet and according to gender, in combination with chronic arsenic exposure. 15 We have further reported a dynamic change in Dnmt3b mRNA expression in mouse liver between the morning and the afternoon. 15 These findings suggest a novel regulatory mechanism of Dnmt3b linked to clockwork.…”

“…15 We have further reported a dynamic change in Dnmt3b mRNA expression in mouse liver between the morning and the afternoon. 15 These findings suggest a novel regulatory mechanism of Dnmt3b linked to clockwork.…”

“…By measuring the amounts of cytidine and 5-medC by high-performance liquid chromatography/electrospray ionizing mass spectrometry (LC/ESI-MS) using stableisotope-labeled surrogates as internal standards, 15 this study revealed that the level of 5-mC in liver genomic DNA varies with the time of day. A previous study that assessed global DNA methylation by fluorescently labeled cytosine extension assay reported diurnal changes in global DNA methylation in humal blood.…”

“…We also could not find Diurnal variation in 5-mC as a percentage of the total cytosine in liver genomic DNA and the effect of L-Bmal1 KO on the 5-mC percentage. In order to investigate whether the diurnal changes in Dnmt3b expression affects global DNA methylation, we accurately measured non-methylated cytidine and 5-methyldeoxycytidine (5-medC) levels in liver genomic DNA by a highly reliable method that we established previously 15 and calculated 5-mC as a percentage of total cytosine. Comparing the 5-mC percentages at ZTs 0, 4, 8, 12, 16 and 20 showed that the lowest level that was significantly different from the level at ZT 0 occurred at ZT 12 (Fig.…”