In Vivo Metabolism of tert-Butyl Hydroperoxide to Methyl Radicals. EPR Spin-Trapping and DNA Methylation Studies

Abstract

Metabolic activation of peroxides and hydroperoxides to free radicals is associated with the tumor promoting activity of these compounds. tert-Butyl hydroperoxide (t-BOOH) metabolism has been extensively studied as a model of peroxide biotransformation. In vivo studies are limited, and the hemoglobin-thiyl radical was the only species thus far identified in the blood of treated rats. Here we further examine t-BOOH metabolism in vivo with regard to free radical and DNA adduct production. Spin-trapping experiments with phenyl-N-tert-butylnitrone (PBN) led to the detection of EPR signals in the blood, bile, and organic extracts of the liver and stomach of rats treated with t-BOOH. Analysis of these signals demonstrated that t-BOOH metabolism in vivo produces alkyl radicals, detected in the bile and organic extracts of liver and stomach, in addition to the previously identified hemoglobin-thiyl radical. To characterize the produced alkyl radicals, experiments were performed with (13)C-labeled t-BOOH and two spin traps, PBN and alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN). The latter was used because the EPR signals obtained with PBN were too weak to be unambiguous. Nevertheless, the EPR signals present in the bile of animals treated with (13)C-labeled t-BOOH and PBN or POBN were consistent with adducts of (13)C-labeled methyl radical and an unidentified alkyl radical. The latter is probably derived from lipids oxidized by the metabolically produced primary radicals, methyl and its precursor, tert-butoxyl. The presence of 8-methylguanine and 7-methylguanine in hydrolysates of DNA from liver and stomach of rats treated with t-BOOH was also examined. 8-Methylguanine, a typical product of methyl radical 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 properties of other peroxides, particularly of cumene hydroperoxide.

Myeloperoxidase-Dependent Oxidation of Etoposide in Human Myeloid Progenitor CD34+ Cells

“…Few reports have been published demonstrating endogenous production of free radicals in cells or in animals. An ESR spin-trapping system was successfully implemented to detect free radicals produced in rodents after treatment with tert-butyl hydroperoxide (Hix et al, 2000). A spin-trapping technique was used as well for the detection of radicals inside macrophages (Lopes de Menezes and Augusto, 2001).…”

Ortho‐thioquinones and mediterranean diet: The sulfur connection

“…The more active compounds of this set, namely, derivatives 1c-e, were also tested to verify their ability to protect against DNA damage induced by both cumene hydroperoxide (CumOOH), a direct generating radical species [18,19], and ferric ions/glutathione (Fe 3+ /GSH), a system able to initiate the Fenton reaction that causes the formation of highly reactive ROS, including the hydroxyl radical OH…”

Nonadditivity of methyl group in single‐electron hydrogen bond of methyl radical‐water complex

“…In most studies on the single-electron hydrogen bond, methyl radical is often taken as a proton acceptor. This is because the methyl radical is a simple prototype for a wide class of organic radicals which play a key role as an intermediate in the field of chemistry and biochemistry [18,19]. In the single-electron hydrogen bond, the proton donor, such as HF [20,21] and H 2 O [22], is like that in conventional hydrogen bonds.…”