ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 252, No. 1, January, pp. 41-48,1987 Formation of Glutathione-Conjugated Semiquinones by the Reaction of Quinones with Glutathione: An ESR Study NAOHIRO TAKAHASHI,’ JijRG SCHREIBER, VOLKER FISCHER, AND RONALD P. MASON2 Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, P.O. BRT 122.33, Research Triangle Park, North Carolina 27709 Received June 11,1986, and in revised form August 26,1986 The nonenzymatic reaction of the cytotoxic compounds menadione (2methyl-1,4- naphthoquinone) and 1,4-naphthoquinone (a reactive metabolite of l-naphthol) with reducing agents such as NADPH and glutathione led to the formation of semiquinone- free radicals, which were detected with electron spin resonance spectroscopy. In the presence of glutathione as a reducing agent, menadione and 1,Cnaphthoquinone un- derwent net one-electron reduction and conjugation with glutathione. At higher con- centrations of glutathione, 1,4-naphthoquinone formed the semiquinones of both the monoconjugate and the diconjugate. The naphthoquinone-glutathione conjugates should redox cycle in a manner already known for the menadione conjugate. The semiquinone intermediates could be detected only under a nitrogen atmosphere and are probably the primary oxygen-reactive species responsible for the redox cycling of menadione- and naphthoquinone-glutathione conjugates. Q 1987 Academic PESS, IM. Quinones are naturally occurring pig- ments in a variety of plants and fungi, and some are clinically important antitumor drugs (1). They are substrates for flavoen- zymes and can undergo either one- or two- electron reduction, a property of impor- tance in determining the cytotoxic and antitumor effects of quinones (2-4). One- electron reduction is catalyzed by micro- somal NADPH-cytochrome P-450 reduc- tase and mitochondrial NADH-ubiquinone oxidoreductase to form semiquinone radi- cals (5, 6). Most semiquinones rapidly re- duce molecular oxygen to form superoxide anion radical (0~) with concomitant re- generation of the quinones (7). This pro- cess, known as redox cycling, leads to con- ditions of oxidative stress and toxicity due 1 Permanent address: Takarazuka Research Center, Sumitomo Chemical Co., Ltd., 4-2-l Takatsukasa, Takarazuka, Hyogo 665, Japan. *To whom correspondence should be addressed. to the formation of 05 and of more reactive oxygen-derived species. The two-electron reduction of quinones to quinols without formation of semiqui- none radicals is catalyzed by NAD(P)H- quinone reductase, known as DT-diapho- rase, which serves as a cellular protective mechanism against quinone toxicity (2). Quinols can be oxidized to semiquinones. When the oxidizing agent is the corre- sponding quinone, this reaction is called comproportionation. Although menadione (I, 2-methyl-1,4- naphthoquinone; vitamin K3) is used ther- 8 0 7/ 6 1 Q CH3 3 5 0 I apeutically, it is also cytotoxic and causes the marked decrease of intracellular thiols 41 0003-9861/8-i $3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.