ONE-ELECTRON AND 2-ELECTRON REDUCTION OF 2-METHYL-1,4-NAPHTHOQUINONE BIOREDUCTIVE ALKYLATING-AGENTS - KINETIC-STUDIES, FREE-RADICAL PRODUCTION, THIOL OXIDATION AND DNA-STRAND-BREAK FORMATION

The one- and two-electron enzymic reduction of the bioreductive alkyla ting agents 2-methylmethoxynaphthoquinone (quinone I) and 2-chlorometh ylnaphthoquinone (quinone II) was studied with purified NADPH-cytochro me P-450 reductase and DT-diaphorase respectively, and characterized i n terms of kinetic constants, oxyradical production, thiol oxidation a nd DNA-strand-break formation. The catalytic-centre activity values in dicated that DT-diaphorase catalysed the reduction of quinone I far mo re efficiently than NADPH-cytochrome P-450 reductase, although the K-m values of the two enzymes for this quinone were similar (1.2-3.0 mu M ). The one-electron-transfer flavoenzyme also catalysed the reduction of quinone II, but the behaviour of DT-diaphorase towards this quinone did not permit calculation of kinetic constants. A salient feature of the redox transitions caused by the one- and two-electron catalysis o f these quinones was the different contributions of disproportionation and autoxidation reactions respectively. In the former case, about 26 % of NADPH consumed was accounted for in terms of autoxidation (as H2 O2 formation), whereas in the latter, the autoxidation component accou nted for most (98 %) of the NADPH consumed. This difference was abroga ted by superoxide dismutase, which enhanced autoxidation during NADPH- cytochrome P-450 catalysis to a maximal value. E.s.r. analysis indicat ed the formation of superoxide radicals, the signal of which was suppr essed by superoxide dismutase and unaffected by catalase. The one- and two-electron reduction of these quinones in the presence of GSH was a ccompanied by formation of thiyl radicals. Although superoxide dismuta se suppressed the thiol radical e.s.r. signal in both instances, the e nzyme enhanced GSSG accumulation during NADPH-cytochrome P-450 catalys is of quinone I, whereas it inhibited GSSG formation during reduction of the quinone by DT-diaphorase. One- and two-electron reduction of qu inone I led to calf thymus DNA-strand-break formation, a process that (a) was substantially decreased in experiments performed with dialysed DNA and in the presence of desferal and (b) was partially sensitive t o superoxide dismutase and/or catalase. These findings are rationalize d in terms of the occurrence of metal ions ligated to DNA, protecting against the toxic effects of superoxide radicals generated during enzy mic reduction of quinones.