C. Giulivi et E. Cadenas, 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, Biochemical journal, 301, 1994, pp. 21-30
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.