Mitoxantrone and ametantrone inhibit hydroperoxide-dependent initiation and propagation reactions in fatty acid peroxidation

The anthracenedione antineoplastic agents mitoxantrone and ametantrone are potent inhibitors of basal and drug-stimulated lipid peroxidation in a variety of subcellular systems (Kharasch, E. D., and Novak, R. F. (1983) J. Pharmacol. Exp. Ther. 226, 500-506). The mechanism by which these compounds function as antioxidants has been investigated using enzymic and chemical systems. Mitoxantrone and ametantrone inhibited NADPH-cytochrome P-450 reductase- and xanthine oxidase-catalyzed conjugated diene formation from linoleic acid in a concentration-dependent manner with half-maximal inhibition achieved at approximately 0.5 microM anthracenedione. Inhibition of linoleic acid peroxidation was not attributable to a decrease in P-450 reductase activity, hydroxyl radical scavenging, or iron chelation by the anthracenediones. Nonenzymic fatty acid peroxidation was also inhibited by the anthracenediones. Linoleic acid oxidation initiated by superoxide (ferrous iron autoxidation) or by hydroxyl radicals (Fenton's reagent) was diminished by mitoxantrone and ametantrone after a brief delay, suggesting an effect subsequent to activated oxygen-dependent initiation. In contrast, linoleic acid oxidation initiated by iron-dependent hydroperoxide decomposition was inhibited immediately. Reinitiation of linoleic acid oxidation in an anthracenedione-inhibited system was accomplished only by superoxide generation, but not by fatty acid hydroperoxide decomposition. These results suggest the anthracenediones diminished neither oxygen radical formation nor oxygen radical-dependent initiation of peroxidation. Rather, inhibition of fatty acid peroxidation by mitoxantrone and ametantrone results from the inhibition of hydroperoxide-dependent initiation and propagation reactions.     

Oxycytochrome P-450: its breakdown to superoxide for the formation of hydrogen peroxide.

Four different experimental studies are described which were designed to evaluate the role of oxycytochrome P-450 in the formation of superoxide anions and hydrogen peroxide. The use of lipophilic copper chelates with superoxide dismutase like activity revealed that the primary site of interaction of these agents is related to the inhibition of the flavoprotein. NADPH-cytochrome P-450 reductase. Measurements of the proton assisted nucleophilic displacement of superoxide from oxycytochrome P-450 by high concentrations of sodium azide indicated an increase in the rate of hydrogen peroxide formation concomitant with the inhibition of the N-demethylation of ethylmorphine. Studies on the effect of NADH on the rate of hydrogen peroxide formation during NADPH oxidation by liver microsomes failed to reveal a stimulatory or synergistic effect in a manner analogous to results obtained during the cytochrome P-450 dependent oxidation of substrates such as ethylmorphine. These results suggest that hydrogen peroxide formation may not require the reduction of oxycytochrome P-450 to peroxycytochrome P-450. Measurements of the reduction of succinylated cytochrome c using purified cytochrome P-450 and the flavoprotein, NADPH-cytochrome P-450 reductase, directly demonstrate the formation of superoxide anions. It is concluded that oxycytochrome P-450 may decompose to generate hydrogen peroxide.     

Inhibition of adriamycin-stimulated microsomal lipid peroxidation by mitoxantrone and ametantrone,two new anthracenedione antineoplastic agents

Ametantrone and mitoxantrone, two new anthracenedione antineoplastic agents, produced a concentration-dependent inhibition of hepatic microsomal lipid peroxidation. Malondialdehyde production was diminished from 10.6 nmoles/mg/60 min to 3.3 and 5.4 nmoles/mg/60 min, in the presence of 100 μM mitoxantrone and ametantrone, respectively. Under similar conditions, Adriamycin stimulated lipid peroxidation over twofold. In addition, both mitoxantrone and ametantrone inhibited Adriamycin-stimulated lipid peroxidation, with 50% inhibition occurring at concentrations of 4 and 6 μM, respectively. Microsomal superoxide production was not significantly inhibited at anthracenedione concentrations which markedly decreased lipid peroxidation, suggesting that inhibition of lipid peroxidation was not the result of inhibition of superoxide generation. These results correlate with the lack of anthracenedione cardiotoxicity and also demonstrate anthracenedione inhibition of lipid peroxidation at micromolar concentrations; an observation with potential therapeutic significance.     

Generation of superoxide anion and hydrogen peroxide during redox cycling of 5-(4-nitrophenyl)-penta-2,4-dienal by mammalian microsomes and enzymes

5-(4-Nitrophenyl)penta-2,4-dienal (NPPD) stimulated NADPH-supported oxygen consumption by rat liver microsomes in a concentration-dependent manner. The NPPD stimulation of O2 uptake was not inhibited by metyrapone and was decreased in the presence of NADP+ and p-hydroxymercuribenzoate. These observations suggest that the NPPD initial reduction step is mediated by NADPH-cytochrome P-450 reductase and not by cytochrome P-450. Spin-trapping studies using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) revealed the formation of superoxide anion upon incubation of NPPD, NADPH, DMPO and rat liver microsomes. Hydrogen peroxide generation was also detected in these incubations, thus confirming redox cycling of NPPD under aerobic conditions. NPPD stimulated oxygen consumption, superoxide anion formation and hydrogen peroxide generation by rat kidney, testes and brain microsomes. Other enzymes capable of nitroreduction (NADH dehydrogenase, xanthine oxidase, glutathione reductase, and NADP+ ferredoxin oxidoreductase) were also found to stimulate redox cycling of NPPD. The ability of NPPD to induce superoxide anion and hydrogen peroxide formation might play a role in its reported mutagenicity.     

Adriamycin affects plasma membrane redox functions

Adriamycin (Doxorubicin) stimulates NADH oxidase activity in liver plasma membrane, but does not cause NADH oxidase activity to appear where it is not initially present, as in erythrocyte membrane. NADH dehydrogenase from rat liver and erythrocyte plasma membranes shows similar adriamycin effects with other electron acceptors. Both NADH ferricyanide reductase and vanadate-stimulated NADH oxidation are inhibited by adriamycin, as is a cyanide insensitive ascorbate oxidase activity, whereas NADH cytochrome c reductase is not affected. The effects may contribute to the growth inhibitory (control) and/or deleterious effects of adriamycin. It is clear that adriamycin effects on the plasma membrane dehydrogenase involve more than a simple catalysis of superoxide formation.     

Vanadate-dependent NAD(P)H oxidation by microsomal enzymes

Vanadate-dependent NAD(P)H oxidation, catalyzed by rat liver microsomes and microsomal NADPH-cytochrome P450 reductase (P450 reductase) and NADH-cytochrome b5 reductase (b5 reductase), was investigated. These enzymes and intact microsomes catalyzed NAD(P)H oxidation in the presence of either ortho- or polyvanadate. Antibody to P450 reductase inhibited orthovanadate-dependent NADPH oxidation catalyzed by either purified P450 reductase or rat liver microsomes and had no effect on the rates of NADH oxidation catalyzed by b5 reductase. NADPH-cytochrome P450 reductase catalyzed orthovanadate-dependent NADPH oxidation five times faster than NADH-cytochrome b5 reductase catalyzed NADH oxidation. Orthovanadate-dependent oxidation of either NADPH or NADH, catalyzed by purified reductases or rat liver microsomes, occurred in an anaerobic system, which indicated that superoxide is not an obligate intermediate in this process. Superoxide dismutase (SOD) inhibited orthovanadate, but not polyvanadate-mediated, enzyme-dependent NAD(P)H oxidation. SOD also inhibited when pyridine nucleotide oxidation was conducted anaerobically, suggesting that SOD inhibits vanadate-dependent NAD(P)H oxidation by a mechanism independent of scavenging of O2-.     

Reduction of 2,4,6-trinitrobenzenesulfonate by glutathione reductase and the effect of NADP+ on the electron transfer

Glutathione reductase has been found to catalyze an NAD(P)H-dependent electron transfer to 2,4,6-trinitrobenzenesulfonate (TNBS). In the presence of oxygen TNBS is not consumed in the reaction, but is rapidly reoxidized with concomitant production of hydrogen peroxide. Cytochrome c can replace oxygen as the final electron acceptor, indicating that a one-electron transfer takes place. The rate is slightly higher in the absence than in the presence of oxygen, ruling out superoxide anion as an obligatory intermediate in cytochrome c reduction. In the absence of oxygen (or cytochrome c), TNBS limits the reaction and accepts a total of four electrons. The TNBS-dependent NADPH (or NADH) oxidation is markedly stimulated by NADP+, and to a smaller extent also by NAD+. The TNBS-dependent reactions are inhibited by excess of NADPH but not by NADH. The kinetics of these reactions are consistent with a branching reaction mechanism in which a pathway including a ternary complex between the two-electron reduced enzyme and NADP+ has the highest turnover. NADPH-dependent reductions of ferricyanide or 2,6-dichloroindophenol catalyzed by glutathione reductase are also markedly influenced by NADP+. Evidently NADP+ facilitates a shift of the catalyzed reaction from the normal two-electron reduction of glutathione disulfide to a more unspecific one-electron reduction of other acceptors. Spectral as well as kinetic data suggest that the rate of radical formation limits the reactions with the artificial electron acceptors and that NADP+ promotes this rate-limiting step.     

Relationship between the reduction of oxygen, artificial acceptors and cytochrome P-450 by NADPH--cytochrome c reductase.

The interaction of NADPH--cytochrome c reductase with oxygen, artificial acceptors and cytochrome P-450 was studied. The generation of superoxide anion radicals (O2-.) from the oxidation of adrenaline to adrenochrome catalysed by NADPH--cytochrome c reductase proceeds independently of the interaction of the enzyme with the artificial anaerobic acceptors cytochrome c or 2,6-dichlorophenol-indophenol. Propyl 3,4,5-trihydroxybenzoate inhibited competitively the adrenaline oxidation by isolated NADPH--cytochrome c reductase (Ki 3.2--4.7 micrometer) and inhibited non-competitively the cytochrome c reduction (Ki 92--109 micrometer). In contrast with the process of electron transfer to cytochrome c, the rate of reduction of cytochrome P-450 and the rate of oxidation of adrenaline in liver microsomal fraction are correlated. Hexobarbital increases the Vmax. of adrenaline oxidation without affecting the Km value, whereas metyrapone, a metabolic inhibitor decreases Vmax. without affecting the Km. From the results obtained, some conclusions about NADPH--cytochrome c reductase function were made.     

External alternative NADH dehydrogenase of Saccharomyces cerevisiae: a potential source of superoxide

Three rotenone-insensitive NADH dehydrogenases are present in the mitochondria of yeast Saccharomyces cerevisiae, which lack complex I. To elucidate the functions of these enzymes, superoxide production was determined in yeast mitochondria. The low levels of hydrogen peroxide (0.10 to 0.18 nmol/min/mg) produced in mitochondria incubated with succinate, malate, or NADH were stimulated 9-fold by antimycin A. Myxothiazol and stigmatellin blocked completely hydrogen peroxide formation with succinate or malate, indicating that the cytochrome bc(1) complex is the source of superoxide; however, these inhibitors only inhibited 46% hydrogen peroxide formation with NADH as substrate. Diphenyliodonium inhibited hydrogen peroxide formation (with NADH as substrate) by 64%. Superoxide formation, determined by EPR and acetylated cytochrome c reduction in mitochondria was stimulated by antimycin A, and partially inhibited by myxothiazol and stigmatellin. Proteinase K digestion of mitoplasts reduced 95% NADH dehydrogenase activity with a similar inhibition of superoxide production. Mild detergent treatment of the proteinase-treated mitoplasts resulted in an increase in NADH dehydrogenase activity due to the oxidation of exogenous NADH by the internal NADH dehydrogenase; however, little increase in superoxide production was observed. These results suggest that the external NADH dehydrogenase is a potential source of superoxide in S. cerevisiae mitochondria.     

The interaction of divalent copper and the microsomal electron transport system. A re-examination of the effects of copper chelates on the function of cytochrome P-450.

Lipophilic chelates of divalent copper, possessing superoxide dismutase-like activity, have been proposed to enhance the decay of oxycytochrome P-450 to explain their inhibitory effect on microsomal mixed-function oxidation reactions (Richter, C., Azzi, A., Weser, U., and Wendel, A. (1977) J. Biol. Chem. 252, 5061-5066). The present investigation, however, failed to provide evidence in favor of this hypothesis. In particular, it was found that the reported inhibition of cytochrome P-450-catalyzed hydroxylation reactions by copper-tyrosine is associated with an inhibition rather than a stimulation of the formation of hydrogen peroxide, the product of the dismutation of the superoxide radicals generated as a result of the decay of oxycytochrome P-450. The attenuation of both these reactions was shown to be the consequence of an impaired function of the NADPH-cytochrome P-450 reductase. Additional sites of interaction of copper chelates and the microsomal electron transport system appear to exist since divalent copper was found to undergo reduction reactions with NADPH and NADH as electron donors. These reduction reactions do not involve superoxide radicals and, therefore, are unrelated to the ability of copper chelates to function in a superoxide dismutase-like manner.     

Mechanisms of hydroxyl radical formation and ethanol oxidation by ethanol-inducible and other forms of rabbit liver microsomal cytochromes P-450

The hydroxyl radical-mediated oxidation of 5,5-dimethyl-1-pyrroline N-oxide, benzene, ketomethiolbutyric acid, deoxyribose, and ethanol, as well as superoxide anion and hydrogen peroxide formation was quantitated in reconstituted membrane vesicle systems containing purified rabbit liver microsomal NADPH-cytochrome P-450 reductase and cytochromes P-450 LM2, P-450 LMeb , or P-450 LM4, and in vesicle systems devoid of cytochrome P-450. The presence of cytochrome P-450 in the membranes resulted in 4-8-fold higher rates of O-2, H2O2, and hydroxyl radical production, indicating that the oxycytochrome P-450 complex constitutes the major source for superoxide anions liberated in the system, giving as a consequence hydrogen peroxide and also, subsequently, hydroxyl radicals formed in an iron-catalyzed Haber-Weiss reaction. Depletion of contaminating iron in the incubation systems resulted in small or negligible rates of cytochrome P-450-dependent ethanol oxidation. However, small amounts (1 microM) of chelated iron (e.g. Fe3+-EDTA) enhanced ethanol oxidation specifically when membranes containing the ethanol and benzene-inducible form of cytochrome P-450 (cytochrome P-450 LMeb ) were used. Introduction of the Fe-EDTA complex into P-450 LMeb -containing incubation systems caused a decrease in hydrogen peroxide formation and a concomitant 6-fold increase in acetaldehyde production; consequently, the rate of NADPH consumption was not affected. In iron-depleted systems containing cytochrome P-450 LM2 or cytochrome P-450 LMeb , an appropriate stoichiometry was attained between the NADPH consumed and the sum of hydrogen peroxide and acetaldehyde produced. Horseradish peroxidase and scavengers of hydroxyl radicals inhibited the cytochrome P-450 LMeb -dependent ethanol oxidation both in the presence and in the absence of Fe-EDTA. The results are not consistent with a specific mechanism for cytochrome P-450-dependent ethanol oxidation and indicate that hydroxyl radicals, formed in an iron-catalyzed Haber-Weiss reaction and in a Fenton reaction, constitute the active oxygen species. Cytochrome P-450-dependent ethanol oxidation under in vivo conditions would, according to this concept, require the presence of non-heme iron and endogenous iron chelators.     

Effect of superoxide dismutase on hydroxylase activity and hydrogen peroxide formation in anthranilamide hydroxylation by a rat liver microsomal monooxygenase system

Anthranilamide was slightly hydroxylated by a reconstituted rat liver microsomal monooxygenase system with NADPH, but a large amount of hydrogen peroxide was formed with a consumption of NADPH during the reaction. Superoxide dismutase stimulated the hydroxylation by depressing the hydrogen peroxide formation, in that there was a reverse correlation between the two effects due to the dismutase. In addition, a trace of 3-hydroxyanthranilamide, one of the products, not only stimulated NADPH-dependent hydrogen peroxide formation via NADPH-cytochrome c (P-450) reductase, but also inhibited the reduction of cytochrome P-450 by NADPH in the reconstituted system. These effects of 3-hydroxyanthranilamide were also diminished by superoxide dismutase.     

Mechanism of inactivation of rat liver microsomal cytochrome P-450c by 2-bromo-4'-nitroacetophenone

The mechanism by which 2-bromo-4'-nitroacetophenone (BrNAP) inactivates cytochrome P-450c, which involves alkylation primarily at Cys-292, is shown in the present study to involve an uncoupling of NADPH utilization and oxygen consumption from product formation. Alkylation of cytochrome P-450c with BrNAP markedly stimulated (approximately 30-fold) its rate of anaerobic reduction by NADPH-cytochrome P-450 reductase, as determined by stopped flow spectroscopy. This marked stimulation in reduction rate is highly unusual in that Cys-292 is apparently not part of the heme- or substrate-binding site, and its alkylation by BrNAP does not cause a low spin to high spin state transition in cytochrome P-450c. Under aerobic conditions the rapid oxidation of NADPH catalyzed by alkylated cytochrome P-450c was associated with rapid reduction of molecular oxygen to hydrogen peroxide via superoxide anion. The intermediacy of superoxide anion, formed by the one-electron reduction of molecular oxygen, established that alkylation of cytochrome P-450c with BrNAP uncouples the catalytic cycle prior to introduction of the second electron. The generation of superoxide anion by decomposition of the Fe2+ X O2 complex was consistent with the observations that, in contrast to native cytochrome P-450c, alkylated cytochrome P-450c failed to form a 430 nm absorbing chromophore during the metabolism of 7-ethoxycoumarin. Alkylation of cytochrome P-450c with BrNAP did not completely uncouple the catalytic cycle such that 5-20% of the catalytic activity remained for the alkylated cytochrome compared to the native protein depending on the substrate assayed. The uncoupling effect was, however, highly specific for cytochrome P-450c. Alkylation of nine other rat liver microsomal cytochrome P-450 isozymes with BrNAP caused little or no increase in hydrogen peroxide formation in the presence of NADPH-cytochrome P-450 reductase and NADPH.     

NAD(P)H oxidation elicits anion superoxide formation in radish plasmalemma vesicles

Radish plasmalemma-enriched fractions show an NAD(P)H-ferricyanide or NAD(P)H-cytochrome c oxidoreductase activity which is not influenced by pH in the 4.5-7.5 range. In addition, at pH 4.5-5.0, NAD(P)H elicits an oxygen consumption (NAD(P)H oxidation) inhibited by catalase or superoxide dismutase (SOD), added either before or after NAD(P)H addition. Ferrous ions stimulate NAD(P)H oxidation, which is again inhibited by SOD and catalase. Hydrogen peroxide does not stimulate NADH oxidation, while it does stimulate Fe2+-induced NADH oxidation. NADH oxidation is unaffected by salicylhydroxamic acid and Mn2+, is stimulated by ferulic acid, and inhibited by KCN, EDTA and ascorbic acid. Moreover, NADH induces the conversion of epinephrine to adrenochrome, indicating that anion superoxide is formed during its oxidation. These results provide evidence that radish plasma membranes contain an NAD(P)H-ferricyanide or cytochrome c oxidoreductase and an NAD(P)H oxidase, active only at pH 4.5-5.0, able to induce the formation of anion superoxide, that is then converted to hydrogen peroxide. Ferrous ions, sparking a Fenton reaction, would stimulate NAD(P)H oxidation.     

Interaction of heme nonapeptide derived from cytochrome c with microsomal reductases

The interaction of heme nonapeptide (a proteolytic product of cytochrome c) with purified NADH:cytochrome b5 (EC 1.6.2.2) and NADPH:cytochrome P-450 (EC 1.6.2.4) reductases was investigated. In the presence of heme nonapeptide, NADH or NADPH were enzymatically oxidized to NAD+ and NADP+, respectively. NAD(P)H consumption was coupled to oxygen uptake in both enzyme reactions. In the presence of carbon monoxide the spectrum of a carboxyheme complex was observed during NAD(P)H oxidation, indicating the existence of a transient ferroheme peptide. NAD(P)H oxidation could be partially inhibited by cyanide, superoxide dismutase and catalase. Superoxide and peroxide ions (generated by enzymic xanthine oxidation) only oxidized NAD(P)H in the presence of heme nonapeptide. Oxidation of NAD(P)H was more rapid with O2- than O2-2. We suggest that a ferroheme-O2 and various heme-oxy radical complexes (mainly ferroheme-O-2 complex) play a crucial role in NAD(P)H oxidation.     

Changes in electron transport pathways in endoplasmic reticulum of rapeseed in response to cold

We studied changes induced by cold on electron transfer pathways (linked to NADH or NADPH oxidation) in endoplasmic reticulum of rapeseed hypocotyls (Brassica napus L.) from a freezing-sensitive variety (ISL) and freezing-tolerant variety (Tradition). Plantlets were grown at 22 degrees C then submitted to a cold shock of 13 or 35 days at 4 degrees C. We measured the content in NADH, NADPH, NAD and NADP of the hypocotyls and the redox power was estimated by the reduced versus oxidized nucleotide ratio. The contents in cytochromes b (5) and P-450, electron acceptors of NADH and NADPH respectively, were determined by differential spectrophotometry. Finally, activity of both NADH-cytochrome b (5) reductase (E.C.1.6.2.2) and NADPH cytochrome P-450 reductase (E.C.1.6.2.4) was determined by reduction of exogenous cytochrome c. Results show that during cold shock, along with an increase of linolenic acid content, there was a general activation of the NADPH pathway which was observed more quickly in Tradition plantlets than in ISL ones. Due to transfer of electrons that can occur between NADPH reductase and cytochrome b (5), this could favor fatty acid desaturation in Tradition, explaining why linolenic acid accumulation was more pronounced in this variety. Besides, more cytochrome P-450 accumulated in ISL that could compete for electrons needed by the FAD3 desaturase, resulting in a relative slower enrichment in 18:3 fatty acid in these plantlets.     

Role of superoxide in the N-oxidation of N-(2-methyl-1-phenyl-2-propyl)hydroxylamine by the rat liver cytochrome P-450 system

The N-oxidation of N-(2-methyl-1-phenyl-2-propyl)hydroxylamine (N-hydroxyphentermine, MPPNHOH) and the N-hydroxylation of 2-methyl-1-phenyl-2-propylamine (phentermine) by reconstituted systems that contained cytochromes P-450 purified from rat liver microsomes were demonstrated. The oxidation of MPPNHOH, but not of phentermine, could also be mediated by a superoxide and hydrogen peroxide generating system that contained xanthine and xanthine oxidase. Superoxide dismutase completely inhibited the oxidation of MPPNHOH by the xanthine/xanthine oxidase system and inhibited by 70% the oxidation mediated by a reconstituted cytochrome P-450 oxidase system. The majority of the microsomal oxidation was inhibited by an antibody raised against the major isozyme of cytochrome P-450 purified from livers of phenobarbital-pretreated rats. 2-Methyl-2-nitroso-1-phenylpropane (MPPNO) was found to be an intermediate in the overall oxidation of MPPNHOH to 2-methyl-2-nitro-1-phenylpropane (MPPNO2). Superoxide dismutase appeared to inhibit the first step, the conversion of MPPNHOH to MPPNO. These observations are accounted for by a sequence of two mechanistically distinct P-450-mediated oxidations. In the first reaction, N-hydroxylation of phentermine occurs by a normal cytochrome P-450 pathway. The formed hydroxylamine then uncouples the cytochrome P-450 system to generate superoxide and hydrogen peroxide. The superoxide oxidizes MPPNHOH to MPPNO which is then oxidized to MPPNO2, the ultimate product. This superoxide-mediated oxidation represents another pathway for N-oxidation by cytochrome P-450.     

Generation of superoxide by the mitochondrial Complex I

Superoxide production by inside-out coupled bovine heart submitochondrial particles, respiring with succinate or NADH, was measured. The succinate-supported production was inhibited by rotenone and uncouplers, showing that most part of superoxide produced during succinate oxidation is originated from univalent oxygen reduction by Complex I. The rate of the superoxide (O2*-)) production during respiration at a high concentration of NADH (1 mM) was significantly lower than that with succinate. Moreover, the succinate-supported O2*- production was significantly decreased in the presence of 1 mM NADH. The titration curves, i.e., initial rates of superoxide production versus NADH concentration, were bell-shaped with the maximal rate (at 50 microM NADH) approaching that seen with succinate. Both NAD+ and acetyl-NAD+ inhibited the succinate-supported reaction with apparent Ki's close to their Km's in the Complex I-catalyzed succinate-dependent energy-linked NAD+ reduction (reverse electron transfer) and NADH:acetyl-NAD+ transhydrogenase reaction, respectively. We conclude that: (i) under the artificial experimental conditions the major part of superoxide produced by the respiratory chain is formed by some redox component of Complex I (most likely FMN in its reduced or free radical form); (ii) two different binding sites for NADH (F-site) and NAD+ (R-site) in Complex I provide accessibility of the substrates-nucleotides to the enzyme red-ox component(s); F-site operates as an entry for NADH oxidation, whereas R-site operates in the reverse electron transfer and univalent oxygen reduction; (iii) it is unlikely that under the physiological conditions (high concentrations of NADH and NAD+) Complex I is responsible for the mitochondrial superoxide generation. We propose that the specific NAD(P)H:oxygen superoxide (hydrogen peroxide) producing oxidoreductase(s) poised in equilibrium with NAD(P)H/NAD(P)+ couple should exist in the mitochondrial matrix, if mitochondria are, indeed, participate in ROS-controlled processes under physiologically relevant conditions.     

Temporary decrease in renal quinone reductase activity induced by chronic administration of estradiol to male Syrian hamsters. Increased superoxide formation by redox cycling of estrogen

Cytochrome P-450-mediated redox cycling between the synthetic estrogen diethylstilbestrol (DES) and diethylstilbestrol-4',4"-quinone (DES Q) has previously been demonstrated. Cytochrome P-450 reductase catalyzes the reduction of DES Q presumably via a semiquinone formed by one-electron reduction. A reducing action of NAD(P)H quinone reductase (EC 1.6.99.2) mediating two-electron reduction of DES Q has been investigated in the present work. Quinone reductase catalyzed the conversion in the presence of NADH or NADPH of DES Q to 53-65% Z-DES, a marker product of reduction. Dicumarol (15 microM), a known specific inhibitor of quinone reductase, inhibited this reduction almost completely. Using microsomes from Syrian hamster kidney, a target organ of estrogen-induced carcinogenesis, the reduction of DES Q was only partially inhibited by dicumarol. Apparent Km values of quinone reductase and cytochrome P-450 reductase were 17.25 and 11.9 microM, respectively. These data demonstrate that in hamster kidney, quinone reductase and cytochrome P-450 reductase compete for the reduction of DES Q. Microsomal 02-. radical generation was stimulated 10-fold over base levels by the addition of 100 microM DES Q. The formation of 02-. radicals was inhibited by addition of superoxide dismutase (0.2 mg/ml) or by 2'-AMP or NADP, known inhibitors of cytochrome P-450 reductase. In contrast, dicumarol enhanced microsome-mediated 02-. formation. It is concluded that cytochrome P-450 reductase in hamster kidney microsomes mediates one-electron reduction of estrogen quinones to free radicals (semiquinones), which may subsequently enter redox cycling with molecular oxygen to form 02-.. Moreover, quinone reductase reduces DES Q directly to E- and Z-DES, and thus may prevent the formation of toxic intermediates during redox cycling of estrogens. Measurements of quinone reductase activity in liver and kidney of hamsters treated with estrogen for various lengths of time revealed a temporary decrease in activity by 80% specifically in the kidney after 1 month of chronic treatment with estradiol. Thus, a temporary decrease in quinone reductase activity, which occurred specifically in estrogen-exposed hamster kidney, may enhance the formation of free radical intermediates generated during biotransformation of estrogens.     

Hydrogen peroxide formation and iron ion oxidoreduction linked to NADH oxidation in radish plasmalemma vesicles

Previously, we showed the presence in radish (Raphanus sativus L.) plasmalemma vesicles of an NAD(P)H oxidase, active at pH 4.5-5.0, which elicits the formation of anion superoxide (Vianello and Macrí (1989) Biochim. Biophys. Acta 980, 202-208). In this work, we studied the role of hydrogen peroxide and iron ions upon this oxidase activity. NADH oxidation was stimulated by ferrous ions and, to a lesser extent, by ferric ions. Salicylate and benzoate, two known hydroxyl radical scavengers, inhibited both basal and iron-stimulated NADH oxidase activity. The iron chelators EDTA (ethylenediaminetetraacetic acid) and DFA (deferoxamine melysate) at high concentrations (2 mM) inhibited the NADH oxidation, whereas they were ineffective at lower concentrations (80 microM); the subsequent addition of ferrous ions caused a rapid and limited increase of oxygen consumption which later ceased. Hydrogen peroxide was not detected during NADH oxidation but, in the presence of salicylate, its formation was found in significant amounts. NADH oxidase activity was also associated to a Fe2+ oxidation which was only partially inhibited by salicylate. Ferrous ion oxidation was partially inhibited by catalase and prevented by superoxide dismutase, while ferric ion reduction was abolished by catalase and unaffected by superoxide dismutase. These results show that during NADH oxidation iron ions undergo oxidoreduction and that hydrogen peroxide is produced and rapidly consumed. As previously suggested, this oxidation appears linked to the univalent oxidoreduction of iron ions by a reduced flavoprotein of radish plasmalemma which is then converted to a radical form. The latter, reacting with oxygen generates the superoxide anion which dismutases to H2O2. Hydrogen peroxide, through a Fenton's reaction, may react with Fe2+ to produce hydroxyl radicals, or with Fe3+ to generate the superoxide anion.