An Unexpected New Molecular Mechanism for the Formation of Nitroxide Radical from Hydroxamic Acids and Polyhalogenated Quinoid Carcinogens

43 An Unexpected New Molecular Mechanism for the Formation of Nitroxide Radical from Hydroxamic Acids and Polyhalogenated Quinoid Carcinogens Ben-Zhan Zhu1, Dan Xu1, and Chun-Hua Huang1

immediately attack DNA in a site-specific manner as demonstrated by both fluorescent and ESR spin-trapping methods. We propose 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 Cu and DNA.

doi: 10.1016/j.freeradbiomed.2016.10.085

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Research Centre for Eco-environmental Sciences, The Chinese Academy of Sciences, Beijing, People's Republic of China We have found previously that a nitroxide radical could be produced from desferrioxamine (DFO) with tetrachlorohydroquinone (TCHQ, the major genotoxic metabolite of the widely used wood preservative pentachlorophenol). However, the underlying molecualr machanism remains unclear. In this study, N-methyl acetohydroxamic acid (N-MeAHA) was used as a simple model compound for DFO for further mechanistic study. As expected, the N-MeAHA-nitroxide radical (N-MeAHA-O●) was found to be produced during the reaction between N-MeAHA and TCHQ by direct ESR. Interestingly and unexpectedly, when TCHQ was substituted by tetrachloro-1,4-benzoquinone (TCBQ), NMeAHA-O● was also observed, but no tetrachlorosemiquinone anion radical (TCSQ●–) or TCHQ was detected, suggesting the formation of N-MeAHA-O● is not a direct redox reaction between N-MeAHA and TCBQ (or TCSQ●–). To detect whether other radical intermediates were formed during the reaction of N-MeAHA and TCBQ, ESR spin-trapping method was used with 5,5-dimethyl-1pyrroline N-oxide (DMPO) as trapping agent. We found, surprisingly, that a new nitrogen-centered radical was produced, which was identified as Ac-(CH3)N● by HPLC-MS and the high resolution FTICR-MS. Taken together, we propose the production of nitroxide radical from N-MeAHA and TCHQ is via an unexpected new pathway via H-abstraction from the N-OH group by the novel nitrogen-centered radical intermediate.

doi: 10.1016/j.freeradbiomed.2016.10.084 44 Mechanism of Synergistic DNA Damage Induced by the Hydroquinone Metabolite of Brominated Phenolic Environmental Pollutants and Cu(II): Formation of DNA-Cu Complex and Site-Specific Production of Hydroxyl Radicals Ben-Zhan Zhu1 and Bo Shao1 1 Research Centre for Eco-environmental Sciences, The Chinese Academy of Sciences, Beijing, People's Republic of China 2,6-dibromohydroquinone (2,6-DBrHQ) has been identified as an reactive metabolite of many brominated phenolic environmental pollutants and was also found as one of disinfection byproducts in drinking water. Here we found that the combination of 2,6-DBrHQ and Cu(II) together could induce synergistic DNA damage as measured by double strand breakage and 8-oxodG formation. 2,6DBrHQ/Cu(II)-induced DNA damage could be inhibited by the Cu(I)-specific chelating agent and catalase, but not by SOD, nor by the typical hydroxyl radical (●OH) scavengers such as DMSO. 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, CD, cyclic voltammetry and O2 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

45 Molecular Mechanism for the Production of the More Mutagenic DIz from DNA by Halogenated Quinoid Carcinogens and Hydroperoxides: The Critical Role of Quinone-Enoxy Radicals Ben-Zhan Zhu1 and Jie Shao1 1 Research Centre for Eco-environmental Sciences, The Chinese Academy of Sciences, Beijing, People's Republic of China Halogenated quinones (XQ) are a class of carcinogenic intermediates and newly identified chlorination disinfection byproducts in drinking water. We found recently that XQ could enhance hydroperoxide decomposition and formation of alkoxyl and quinone enoxy/ketoxy radicals metal-independently. However, it is not clear whether XQ/hydroperoxides can oxidize dG to its oxidation products, and if so, what is the underlying molecular mechanism. Here we show that the major oxidation product was dIz when dG was treated with tetrachloro-1,4-benzoquinone (TCBQ) and t-BuOOH, which was markedly inhibited by typical spin-trapping agents. Further ESR and HPLC/MS results showed that the quinone enoxy radicals played a critical role in dIz formation, and O2 was also involved in dIz formation by using oxygen-18-labelled O2. We proposed that the oxidation of dG by TCBQ/t-BuOOH might be through the following mechanism: the quinone enoxy radical may first abstract H from dG, forming dG(H)● radical, which may combine with O2 to form its corresponding hydroperoxide, resulting in a cascade of decomposition reactions leading to dIz formation. This is the first report that XQ/hydroperoxides can induce potent oxidation of dG to dIz via a quinone-enoxy radical-mediated mechanism, which may partly explain their potential carcinogenicity.

doi: 10.1016/j.freeradbiomed.2016.10.086 46 Redirecting Ru(II)-Complex Into Live-Cell Nucleus for Enantioselective DNA-Imaging and Photosensitizing Via Simple Yin-Yang Ion-Pairing Ben-Zhan Zhu1 and Xi-Juan Chao1 1 Research Centre for Eco-environmental Sciences, The Chinese Academy of Sciences, Beijing, People's Republic of China Targeted delivery of diagnostic-probes and therapeutics into specific compartments inside a cell is of utmost importance in the improvement of disease detection and treatment. The molecular DNA “light-switch” Ru(II)-polypyridyl complex [Ru(DIP)2(dppz)]2+ has been shown to be accumulated only in the cytoplasm and membrane, but excluded from its intended nuclear DNA target. Here we show that chlorophenolate and flufenamate drugs can redirect [Ru(DIP)2(dppz)]2+ into live-cell nucleus while maintaining its original DNA recognition characteristics, where it acts as an

SfRBM / SFRRI 2016

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