Halogenated quinones are a class of carcinogenic intermediates and are newly identified chlorination disinfection by-products in drinking water. We found recently that the highly reactive and biologically important hydroxyl radical ((•)OH) can be produced by halogenated quinones and H2O2 independent of transition metal ions. However, it is not clear whether these quinoid carcinogens and H2O2 can oxidize the nucleoside 5-methyl-2'-deoxycytidine (5mdC) to its methyl oxidation products and, if so, what the underlying molecular mechanism is. Here we show that three methyl oxidation products, 5-(hydroperoxymethyl)-, 5-(hydroxymethyl)-, and 5-formyl-2'-deoxycytidine, could be produced when 5mdC was treated with tetrachloro-1,4-benzoquinone (TCBQ) and H2O2. The formation of the oxidation products was markedly inhibited by typical (•)OH scavengers and under anaerobic conditions. Analogous effects were observed with other halogenated quinones and the classic Fenton system. Based on these data, we propose that the oxidation of 5mdC by TCBQ/H2O2 might be through the following mechanism: (•)OH produced by TCBQ/H2O2 may first abstract hydrogen from the methyl group of 5mdC, leading to the formation of 5-(2'-deoxycytidylyl)methyl radical, which may combine with O2 to form the peroxyl radical. The unstable peroxyl radical transforms into the corresponding hydroperoxide 5-(hydroperoxymethyl)-2'-deoxycytidine, which reacts with TCBQ and results in the formation of 5-(hydroxymethyl)-2'-deoxycytidine and 5-formyl-2'-deoxycytidine. This is the first report that halogenated quinoid carcinogens and H2O2 can induce potent methyl oxidation of 5mdC via a metal-independent mechanism, which may partly explain their potential carcinogenicity.

2,6-Dibromohydroquinone (2,6-DBrHQ) has been identified as an reactive metabolite of many brominated phenolic environmental pollutants such as tetrabromobisphenol-A (TBBPA), bromoxynil and 2,4,6-tribromophenol, and was also found as one of disinfection byproducts in drinking water. In this study, we found that the combination of 2,6-DBrHQ and Cu(II) together could induce synergistic DNA damage as measured by double strand breakage in plasmid DNA and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation, while either of them alone has no effect. 2,6-DBrHQ/Cu(II)-induced DNA damage could be inhibited by the Cu(I)-specific chelating agent bathocuproine disulfonate and catalase, but not by superoxide dismutase, nor by the typical hydroxyl radical (•OH) scavengers such as DMSO and mannitol. Interestingly, we found that Cu(II)/Cu(I) could be combined with DNA to form DNA-Cu(II)/Cu(I) complex by complementary application of low temperature direct ESR, circular dichroism, cyclic voltammetry and oxygen consumption methods; and the highly reactive •OH were produced synergistically by DNA-bound-Cu(I) with H2O2 produced by the redox reactions between 2,6-DBrHQ and Cu(II), which then immediately attack DNA in a site-specific manner as demonstrated by both fluorescent method and by ESR spin-trapping studies. Further DNA sequencing investigations provided more direct evidence that 2,6-DBrHQ/Cu(II) caused preferential cleavage at guanine, thymine and cytosine residues. Based on these data, we proposed that the synergistic DNA damage induced by 2,6-DBrHQ/Cu(II) might be due to the synergistic and site-specific production of •OH near the binding site of copper and DNA. Our findings may have broad biological and environmental implications for future research on the carcinogenic polyhalogenated phenolic compounds.