Analyses of 8-Methyldeoxyadenosine and 8-Methyldeoxyguanosine as Markers of Free Radical-mediated DNA Methylation in Mouse

Abstract

To examine whether methyl radical-induced DNA damage occurs in vivo, cumene hydroperoxide (CuOOH) and methionine sulfoxide (MetO) were administered to mice as methyl radical generators, and 8-methyldeoxyadenosine (m 8 dA) and 8-methyldeoxyguanosine (m 8 dG) in the tissue DNA were analyzed by the LC/MS/MS method. After mouse skin was treated with CuOOH and Fe 2＋ , both m 8 dA and m 8 dG were detected in the epidermal DNA. m 8 dA formation was also detected in the liver DNA from MetO-treated, non-alcoholic steatohepatitis (NASH) mice. Since 5-methyldeoxycytidine, m 8 dA, and m 8 dG are formed in DNA by methyl radicals, these results support our hypothesis that de novo DNA methylation at the cytosine C-5 position is triggered by a free radical mechanism and may initiate epigenetic changes.

Free radical-mediated cytosine C-5 methylation triggers epigenetic changes during carcinogenesis

“…The higher incidence of hepatocarcinoma may be due in part to the m 5 dC formation by methyl radicals in vivo . In addition, we confirmed the formation of m 8 dA in the MetO-treated mouse liver DNA, suggesting that methyl radicals are actually produced in the mouse liver by those treatments (44) . Notably, MetO is generated in proteins by inflammation, aging, and smoking (21 -24) , which are recognized as DNA hypermethylation inducers (11,13,14) .…”

“…To determine whether a skin tumor promoter could induce radical methylation of DNA in vivo , mouse skin was treated with CuOOH in the presence of Fe 2 + , and the methylated purine nucleosides in the epidermal DNA were analyzed by an LC-MS-MS method (44) . 8-Methyldeoxyadenosine (m 8 dA) and m 8 dG were detected at levels of 2.65 ± 1.41 and 0.98 ± 0.14 per 10 7 nucleotides, respectively, suggesting that radical DNA methylations, including the formation of m 5 dC, may occur in cellular DNA.…”