In vivo metabolism of tert-butyl hydroperoxide to methyl radicals. EPR spin-trapping and DNA methylation studies

Metabolic activation of peroxides and hydroperoxides to free radicals is as sociated with the tumor promoting activity of these compounds. tert-Butyl h ydroperoxide (t-BOOH) metabolism has been extensively studied as a model of peroxide biotransformation. In vivo studies are limited, and the hemoglobi n-thiyl radical was the only species thus far identified in the blood of tr eated rats. Here we further examine t-BOOH metabolism in vivo with regard t o free radical and DNA adduct production. Spin-trapping experiments with ph enyl-N-tert-butylnitrone (PBN) led to the detection of EPR signals in the b lood, bile, and organic extracts of the liver and stomach of rats treated w ith t-BOOH. Analysis of these signals demonstrated that t-BOOH metabolism i n vivo produces alkyl radicals, detected in the bile and organic extracts o f liver and stomach, in addition to the previously identified hemoglobin-th iyl radical. To characterize the produced alkyl radicals, experiments were performed with C-13-labeled t-BOOH and two spin traps, PEN and alpha-(4-pyr idyl-1-oxide)-N-tert-butylnitrone (POBN). The latter was used because the E PR signals obtained with PEN were too weak to be unambiguous. Nevertheless, the EPR signals present in the bile of animals treated with C-13-labeled t -BOOH and PEN or POBN mere consistent with adducts of C-13-labeled methyl r adical and an unidentified alkyl radical. The latter is probably derived fr om lipids oxidized by the metabolically produced primary radicals, methyl a nd its precursor, tert-butoxyl. The presence of 8-methylguanine and 7-methy lguanine in hydrolysates of DNA from liver and stomach of rats treated with t-BOOH was also examined. g-Methylguanine, a typical product of methyl rad ical attack on DNA, was detectable in both the Liver and stomach of treated rats. The results may be relevant to the understanding of the genotoxic pr operties of other peroxides, particularly of cumene hydroperoxide.