J. Biochem. 115, 1141-1147 (1994)

One and Two-Electron Reduction of Quinones by Rat Liver Subcellular Fractions

Masao Nakamura and Takaaki Hayashi Department of Biophysics, Research Institute for Electronic Science, Hokkaido University, Kita-ku, Sapporo 060; and Hokkaido Institute of Public Health , N-19 W-12, Kita-ku, Sapporo 060

Received for publication, January 31, 1994

NAD(P)H-quinone (menadione, Trolox C quinone, and a-tocopherol quinone) reductase activity of rat liver subeellular fractions was observed optically at 340-400 nm, and oxygen radical generation was demonstrated using the ESR spin trap, 5,5'-dimethyl-l-pyrroline 1-oxide. NAD(P)H-menadione reductase activity of the fractions decreased in the order: cytosol > microsomes > plasma membranes. Although more than 65% of the activity of microsomes and plasma membranes was inhibited on the addition of dicoumarol, no change in the menadione-mediated formation of oxygen radicals by either fraction was observed. As judged from the intensity of ESR signals, the menadione-mediated oxygen radical formation by plasma membranes was only one-tenth as great as that by microsomes. No generation of oxygen radicals in the NAD(P)H-menadione reductase reaction by cytosol was found, and the activity was abolished in the presence of dicoumarol, an inhibitor of DT-diaphorase. It is concluded that plasma membranes reduce quinones by way of two-electron transfer and that the activity may prevent cellular quinone toxicity. NAD(P)H-a-tocopherol quinone reductase activity was confirmed in all cellular fractions [Hayashi et al. (1992) Biochem. Pharmacol. 44, 489-493] and this activity was also in hibited by dicoumarol, suggesting that it was due to DT-diaphorase.

Key words: DT-diaphorase, NADPH-cytochrome P-450 reductase, oxygen radicals, qui none toxicity, rat liver.

There is an increasing body of literature suggesting that toxicity of menadione, leading to alteration of the surface quinone-mediated formation of oxygen radicals plays a role structure of the hepatocytes (3). Metabolism of incorpo in oxidative damage to cells. A potential mechanism rated menadione in hepatocytes has also been documented through which oxygen radicals are generated in vivo is the (3, 9). one-electron reduction of quinones by NADPH-cytochrome We report reinvestigation of one and two-electron P-450 reductase (1-3). On the other hand, Ernster and reduction of menadione, and menadione-mediated forma associates (4) showed that DT-diaphorase [NAD(P)H: tion of oxygen radicals, by rat liver subcellular fractions. quinone oxidoreductase, EC 1.6.99.2], which is widely The relation of a-tocopherol quinone (a metabolite of distributed in subcellular fractions, catalyzes the two-elec a-tocopherol and an antioxidant) reductase activity and tron reduction of menadione (2-methyl-l,4-naphthoqui DT-diaphorase activity in the fractions was also examined. none), without the formation of semiquinone radicals (3, 5, 6). It has been shown that NADPH-cytochrome P-450 MATERIALSAND METHODS reductase and DT-diaphorase coexist in microsomes. Therefore, it was inferred that quinone toxicity is related to Enzymes-NADPH-cytochrome P-450 reductase and the relative activities of NADPH-cytochrome P-450 reduc NADH-cytochrome b5 reductase were donated by Dr. T. tase and DT-diaphorase (3, 6). Menadione-mediated for Iyanagi, Himeji Institute of Technology, Himeji. The mation of superoxide by flavoenzymes occurs through enzymes were prepared from rat liver microsomes, and the consecutive one-electron transfers, which results in con concentrations of NADPH-cytochrome P-450 reductase comitant oxygen consumption through the reaction of and NADH-cytochrome b5 reductase were calculated on the semiquinone with 0, (7, 8). basis of extinction coefficient values of 10.7 mM-1-cm 1 at Incubation of hepatocytes with menadione causes oxida 455 nm (10) and 10.2 mM-1 cm-1 at 460 nm (11), respec tive stress in a dose-dependent manner and pretreatment tively. Bovine erythrocyte superoxide dismutase (SOD) of the hepatocytes with dicoumarol exacerbates the cyto and bovine liver catalase were obtained from Sigma (St. Louis). Abbreviations: DMPO, 5,5'-dimethyl-l-pyrroline-1-oxide; DMPO Preparation of Rat Liver Subcellular Fractions-Male OOH, 2,2'-dimethyl-5-hydroperoxyl-l-pyrrolidinyloxyl; DMPO-OH, Sprague-Dawley rats (200-250 g) were obtained from Clea 2,2'-dimethyl-5-hydroxyl-l-pyrrolidinyloxyl; DTPA, diethylenetri Japan (Tokyo). Hepatocytes were isolated from their livers amine penta-acetic acid; Trolox C quinone, 2-hydroxy-2-carboxy-4 by the collagenase perfusion method (12). The cell suspen (3,5,6-trimethylbenzoquinone-2-yl) butane; TQ, a-tocopherol sion was diluted to a final concentration of 3-5 X 10' cells/ quinone.

Vol. 115, No. 6, 1994 1141 1142 M. Nakamura and T. Hayashi

ml with Krebs-Ringer buffer, pH 7.4. Cell viability ranged Varian E-109B spectrometer. from 85-95%, as determined by Trypan blue exclusion . All reactions were carried out in 0.1 M potassium phos. The isolated hepatocytes were homogenized with a Potter phate buffer (pH 7.4) at 25'C, unless otherwise noted. Elvehjem homogenizer. The homogenates were centrifuged at 2,000 x g for 15 min. Plasma membranes were prepared RESULTS from the precipitate by the method of Solyom et al. (13). The 2,000 x g supernatant was centrifuged at 9,000 X g for Menadione reductase activity of NADPH-cytochrome P 10 min. The supernatant was further centrifuged at 450 reductase, NADH-cytochrome b5 reductase, and DT. 105,000 x g for 1 h to sediment the microsomes, which diaphorase has been reported (4, 5, 7, 8). These enzymes were then washed twice with 0.15 M KCl and suspended in also catalyze the reduction of Trolox C quinone and a 0.1 M phosphate, pH 7.4. To obtain the cytosol fraction , rat tocopherol quinone (Fig. 1). Reactions were performed liver was homogenized in 2 volumes of 0.25 M sucrose. The using 30 ,u M electron acceptor due to the low solubility of homogenates were centrifuged at 105 ,000 x g for 1 h and a -tocopherol quinone. As judged from the initial velocity of the supernatant was used as the cytosol. Protein concentra NAD(P)H oxidation, NAD(P)H-quinone reductase activity tions were determined by the method of Lowry et al. (14) decreased as a function of the substrate in the order: with bovine serum albumin as a standard. The isolated menadione>Trolox C quinone> a-tocopherol quinone subcellular fractions were characterized by marker en (Fig. 1). Oxygen consumption was observed during the zymes as reported previously (15). NAD(P)H-quinone reductase reactions of NADPH-cyto Chemicals-Menadione (2-methyl-l,4-naphthoquinone) chrome P-450 reductase and NADH-cytochrome b5 reduc was purchased from Nakarai (Kyoto); Trolox C from tase (data not shown), the rates being identical to those Aldrich; and DL -a -tocopherol from Sigma. Tocopherol was estimated from the NAD(P)H oxidations. The results further purified by HPLC to yield a colorless oil. Trolox C indicated that reducing equivalents from NAD(P)H were quinone and a-tocopherol quinone were obtained through transferred to oxygen through quinones. the reactions of Trolox C and a -tocopherol with FeC13 (16). Using the ESR spin trap, DMPO, oxygen radical forma The oxidation products were purified by HPLC and identi tion was confirmed in the NADPH-quinone reductase fied as the corresponding quinones by NMR, and by mass reaction of NADPH-cytochrome P-450 reductase (Fig. 2). and infrared spectrometry. NADH, NADPH, dicoumarol, From the intensities of spectra due to DMPO-OOH, the diethylenetriamine penta-acetic acid (DTPA), and the spin velocities of superoxide formation were estimated to be in trapping agent, 5,5'-dimethyl-l-pyrroline-l-oxide (DMPO), the same range as those of NADPH oxidation and oxygen were purchased from Sigma (St. Louis). uptake in the NADPH-menadione, -Trolox C quinone, and Biochemical Assays-NADH and NADPH-quinone re -a-tocopherol quinone reductase reactions catalyzed by ductase reactions of the subcellular fractions were mea NADPH-cytochrome P-450 reductase. A typical DMPO sured at 340-400 nm with a Shimadzu UV300 dual-beam spectrophotometer. The formation of oxygen radicals was measured using the spin-trapping agent, DMPO, with a

Fig. 1. NAD(P)H-quinone reductase reactions catalyzed by NADPH-cytochrome P-450 reductase (A), NADH-cytochrome b5 reductase (B), and DT-diaphorase (C). A: NADPH-quinone re ductase activity of NADPH-cytochrome P-450 reductase. The concen trations were: 120,u M NADPH, 0.22,u M NADPH-cytochrome P-450 reductase, and 30,uM menadione (a), Trolox C quinone (b), or Fig. 2. ESR spectra produced in NADPH-quinone reductase a-tocopherol quinone (c). B: NADH-quinone reductase activity of reactions catalyzed by NADPH-cytochrome P-450 reductase. NADH-cytochrome b5 reductase. The concentrations were: 120MM The concentrations were: 200,uM NADPH, 0.22,uM NADPH-cyto NADH, 0.21 uM NADH-cytochrome b5 reductase, and 30 MM chrome P-450 reductase, 0.5 mM DTPA, 100 mM DMPO, and 30,u M menadione (a), Trolox C quinone (b), or a -tocopherol quinone (c). C: menadione (A), Trolox C quinone (B), or a-tocopherol quinone (C). NADPH-quinone reductase activity of DT-diaphorase. The concen The instrumental conditions were: center of magnetic field, 3,387 G; trations were: 120 k M NADPH, 0.2 mg/ml cytosol protein, and 30 modulation amplitude, 1.0 G; receiver gain, 1 x 10°; time constant, yM menadione (a), Trolox C quinone (b), or a -tocopherol quinone (c). 0.125 s; microwave power, 20 mW; and scan speed , 25 G/min. The The quinones were added at the times indicated by arrows. spectra were recorded at 2 min after NADPH had been added.

J. Biochem. Reduction of Quinones by Liver Subcellular Fractions 1143

Fig. 3. ESR spectra produced in NADPH-quinone reductase reactions (A, B) and NADH-quinone reductase reactions (C, D) catalyzed by microsomes. The concentrations were: 200,uM NADPH or NADH, 2.1 mg/ ml microsomal protein, 0.5 mM DTPA, 100 mM DMPO, and 30,u M men adione (A, C) or a-tocopherol quinone (B, D). The spectra were recorded at 2 (a) and 4 (b) min after the reactions had been started. The instrumental conditions were the same as in Fig. 2.

OOH spectrum of the superoxide adduct of DMPO was obtained immediately in NADPH-menadione reductase reactions with microsomes (Fig. 3A). The concentration of superoxide trapped with DMPO was calculated to be 9.2 ,uM at 2 min (Fig. 3Aa) and slightly decreased at 4 min (Fig. 3Ab). The signal decrease was interpreted on the basis of the accumulation of menadiol through two-electron reduction and of the decomposition of DMPO-OOH. DMPO-OOH and DMPO-OH spectra appeared concomi tantly (Fig. 3Ba), and the DMPO-OH spectrum increased with time in the NADPH a -tocopherol quinone reductase reaction of microsomes (Fig. 3Bb). It has been shown that superoxide generation in the NAD(P)H-menadione reduc tase reaction of microsomes could be verified by the reduction of acetylated cytochrome c (3). Figure 3C also shows the superoxide formation during the NADH-menadi one reductase reaction catalyzed by microsomes, though the formation was only about one-third as great as that during the NADPH-menadione reductase reaction. When menadione was replaced by a-tocopherol quinone, no generation of superoxide was observed (Fig. 3D). a -Tocopherol and its analogue are oxidized to the corre sponding quinones by hemoproteins in the presence of H202 (17, 18). We have reported that NADPH-a-tocopherol quinone reductase activity was found in plasma mem branes, microsomes and cytosol fractions (19). NAD(P)H dependent plasma membrane redox systems have also Fig. 4. ESR spectra produced in the NADPH-quinone reduc been reported (20, 21). Figure 4 shows the NAD(P)H tase reaction (A) and NADH-quinone reductase reaction (B) catalyzed by plasma membranes. The concentrations were: 200 dependent oxygen redical formation by plasma membranes h M NADPH (A) or NADH (B), 2.5 mg/ml plasma membrane protein, in the absence or presence of menadione. A signal charac 0.5 mM DTPA, 100 mM DMPO, and 30 ,uM menadione. The reac teristic of a combination of that due to DMPO-OOH and tions were carried out in the absence (a) or presence (b) of 30jM that due to DMPO-OH was observed immediately after the menadione. The instrumental conditions were the same as in Fig. 2.

Vol. 115, No. 6, 1994 1144 M . Nakamura and T. Hayashi

reaction was started (Fig . 4Ab). When NADPH was re activity of plasma membranes, which is stimulated by placed by NADH, a weak signal was obtained in the several hormones, has been reported (20). Although spin NADH-menadione reductase reaction of plasma mem trapping was performed in the NADH and NADPH branes (Fig. 4Bb). It should be noted here that NADH-cyto oxidase reactions of plasma membranes, a very weak signal chrome c reductase activity insensitive to SOD was 10-fold insensitive to SOD and catalase was observed (Fig. 4, Aa higher than NADPH-cytochrome c reductase activity of and Ba). plasma membranes (data not shown). NADH-oxidase The NAD(P)H-menadione reductase activities of subcel

Fig. 5. Effect of dicoumarol upon the NAD (P)H-quinone reductase reactions catalyzed by subcellular fractions. NADPH-menadione re ductase reactions catalyzed by cytosol (A), micro somes (B), and plasma membranes (C). The concen trations were: 80 uM NADPH, 20 uM menadione, 0.31 mg/ml cytosol, 0.56 mg/ml microsomal, or 0.76 mg/ml plasma membrane protein. The quinone was added to a stirred solution at the times indicated. NADH-menadione reductase reactions catalyzed by cytosol (D), microsomes (E), and plasma membranes (F). The concentrations were: 80 uM NADH, 20,uM menadione, and 0.31 mg/ml cytosol, 0.56 mg/mi microsomal or 0.76 mg/ml plasma membrane pro tein. The reactions were carried out in the absence (a) or presence (b) of 10 uM dicoumarol.

Fig. 6. Effects of catalase and SOD upon the ESR spectra the reactions had been started. B: NADPH-menadione reductase produced in the NADPH-quinone reductase reactions catalyzed reaction catalyzed by plasma membranes. The concentrations were: by microsomes and plasma membranes. A: NADPH-menadione 200uM NADPH, 30,uM menadione, 2,5mg/ml plasma membrane reductase reaction catalyzed by microsomes. The concentrations were: protein, 0.5 mM DTPA, and 100 mM DMPO. The reaction was carried 200,u M NADPH, 30p M menadione, 2.1 mg/ml mierosomal protein, out in the absence (a) or presence of 40 nM catalase (b), 0.1 uM SOD ( 0.5 mM DTPA, and 100 mM DMPO. The reaction was carried out in c), or both catalase and SOD (d) . The instrumental conditions were the absence (a) or presence of 40 nM catalase (b), 0.1 uM SOD (c), or the same as in Fig. 2. both catalase and SOD (d). The spectra were recorded at 2 min after

J. Biochem. Reduction of Quinones by Liver Subcellular Fractions 1145

TABLE I. Effect of dicoumarol upon the NAD(P)H-menadione TABLE 11. Marker enzyme activities in subcellular fractions. reductase reactions catalyzed by subcellular fractions .

'Average of 5 different samples; 'average of 7 different samples .

'Activities were estimated from the differences i n the absorbance SOD and catalase (Fig. 6Bd), the initial spectrum, which changes in the absence and presence of menadione, as shown in Fig . 5. 'NADH -oxygen reductase activity of plasma membranes was esti was a combination those of DMPO-OOH and DMPO-OH, mated to be 5.6 nmol/ma protein-min. did not change in the presence of catalase (Fig. 6Bb).

DISCUSSION lular fractions and the effect of dicoumarol are shown in Fig . 5. The Km values of DT-diaphorase and NADPH-cyto Flavoenzymes are electron donor-specific but utilize many chrome P-450 reductase for menadione are 3.3 and 35 ,uM, kinds of electron acceptors, such as quinones, oxygen, and respectively (3). The NAD(P)H-menadione reductase ferricyanide (23). Since the redox properties of p-benzo activities of subcellular fractions were saturated at 30 ,uM quinone and menadione have been documented, corre menadione, and the activities of microsomes and plasma sponding semiquinone formation in the NAD(P)H-quinone membranes were reduced to 30-35% in the presence of 10 reductase reaction catalyzed by the enzymes has been ,uM dicoumarol. NAD(P)H-menadione reductase activity verified through ESR measurements (5, 8). Judging from of cytosol was essentially abolished with the same concen the primary reduction product semiquinone or hydroqui tration of dicoumarol. The results are summarized in Table none, the reduction of quinones by flavoenzymes can be I, which indicates that the NAD(P)H-menadione reductase grouped into one-, two-, and mixed-electron transfer types reactions of subcellular fractions were mainly catalyzed by (24). The redox activities of NADPH-cytochrome P-450 DT-diaphorase, without the formation of semiquinone reductase and NADH-cytochrome b5 reductase have been radicals (3, 5, 6). Therefore we have checked the effect of documented in detail (11, 25, 26), and these enzymes dicoumarol upon superoxide formation in the NADPH catalyze one-electron reduction of quinones, whereas DT menadione reductase reactions of microsomes and plasma diaphorase exclusively catalyzes two-electron reduction (5, membranes (data not shown). The oxygen radical forma 6). It has been shown that the quinone reduction mecha tion decreased by only 10% with 10,uM dicoumarol, sug nism of lipoamide dehydrogenase changed from the two to gesting that the formation was not catalyzed by DT the one-electron transfer type on modification of -SH diaphorase. The formation of superoxide in the NADPH groups of the enzyme (27). menadione reductase reaction of microsomes is ascribed to Generation of oxygen radicals through semiquinone NADPH-cytochrome P-450 reductase and NADH-cyto formation is restricted to the redox properties of semiqui chrome b5 reductase activities. The rate of superoxide nones (28-31). Menadione-mediated one-electron reduc formation in the NADPH-menadione reductase reaction of tion of oxygen by the NADPH-cytochrome P-450 reductase plasma membranes was only one-tenth as great as that in system was confirmed by the reduction of acetylated the case of microsomes. Table II shows the levels of marker cytochrome c, as a detector of superoxide (3), and by enzymes in the present preparations, it being suggested adduct formation with the ESR spin trap, DMPO. When that the plasma membranes contained about 15% of the quinones are reduced by DT-diaphorase, rather stable cytochrome P-450 found in microsomes. However, Jarasch hydroquinones are formed. The hydroquinone form of et al. (22) have pointed out that plasma membranes menadione reacts with oxygen to form superoxide, but the themselves contain relatively high amounts of cytochrome rate is pH-dependent and slow at neutral pH (5, 28). P-450, NADPH-cytochrome P-450 reductase, and NADH Ernster et al. (4) reported that DT-diaphorase is distribut cytochrome b5 reductase. Since cytochrome P-450 and ed in various rat liver subcellular fractions, including the NADPH-cytochrome P-450 reductase found in plasma cytosol, microsomes, and mitochondrial membranes. Pre membranes have not been confirmed to be identical with treatment of rats with phenobarbital or 3-methylcholan those in microsomes, we can not conclude that plasma threne induces hepatic NADPH-cytochrome P-450 reduc membranes catalyze one-electron reduction of menadione tase and DT-diaphorase (3, 6). It has been shown that these in the presence of NADPH. two enzymes coexist in microsomes. Superoxide generation The mechanism of oxygen radical formation was inves in the NAD(P)H-menadione reductase reaction of micro tigated by examining the effects of catalase and SOD. No somes was confirmed by the formation of DMPO-OOH (Fig. effect of catalase upon the formation of DMPO-OOH was 3A). The effects of dicoumarol and SOD were in good found (Fig. 6A, a and b), but a change of DMPO-OOH to agreement with the results obtained by Thor et al. (3). A DMPO-OH in the presence of SOD (Fig. 6Ac) was observed typical DMPO-OOH spectrum was obtained for the in the NADPH-menadione reductase reaction of micro NADPH-menadione reductase reaction of NADPH-cyto somes (Fig. 6A). Whereas DMPO-OH formation in the chrome P-450 reductase and microsomes, respectively, NADPH-menadione reductase reaction of plasma mem indicating the formation of superoxide (Figs. 2A and 3A). A branes was depressed very slightly in the presence of both relatively weak signal characteristic of a combination of

Vol. 115, No. 6, 1994 1146 M. Nakamura and T. Hayashi

DMPO-OOH and DMPO-OH was observed for the the ESR spin trap, attempts to detect oxygen radicals in NADPH-menadione reductase reaction of plasma mem hepatocytes and the menadione system were unsuccessful, branes (Fig. 4Ab). As judged on comparison of the inten suggesting the two-electron reduction of menadione with sities of the ESR spectra for the NADPH-menadione hepatocytes (data not shown). The present preparations reductase reactions of plasma membranes (Fig. 4Ab) and showed that the viability of hepatocytes ranged from 85 to microsomes (Fig. 3Aa), superoxide generation by the 95%. Therefore, we could not rule out the possibility that plasma membrane system was about 10% of that by the cytosolic SOD inhibits the detection of oxygen radicals. microsomes. The results are compatible with the ratio of NADH-oxidase activity sensitive to growth factors and NADPH-cytochrome P-450 reductase activity of plasma glucagon has been reported in plasma membranes (20). The membranes to that of microsomes (Table II). transmembrane redox system accompanied by H+ trans Because hydroxyl radicals are strong oxidants and react port has also been reported, but its architecture is not fully rapidly with biomolecules, the observation of DMPO-OH is understood. It has been reported that NADPH-ubiquinone noteworthy. If such reactions were to occur in biomem reductase found in the rat liver cytosol catalyzes the branes, deleterious effects would be expected (32). There reduction of quinones with a long isoprenoid side chain, are several lines of evidence that the characteristic ESR though such quinones are not good electron acceptors for spectrum for DMPO-OH should not be used as definitive DT-diaphorase (41). The evidence that pretreatment of proof for the spin trapping of hydroxyl radicals in biological isolated hepatocytes with both inhibitors of DT-diaphorase systems (33, 34). The DMPO-OH signal is observed in the and SOD exacerbates menadione toxicity confirms that NADPH-oxidase reaction of hamster hepatic nuclei (35). both enzymes protect hepatocytes against the toxicity (3). Incubation of endothelial cells with both menadione and Tables I and II indicate that the ratio of the relative DMPO gave a combination spectrum due to DMPO-OOH activities of DT-diaphorase to NADPH-cytochrome P-450 and DMPO-OH (36), It has been suggested that the reac reductase in plasma membranes is higher than that in tion of semiquinone radicals with H202 is associated with microsomes. Although the main outlines of the defense the formation of hydroxyl radicals (29, 30, 37). However, mechanism against quinone toxicity have been established, the reaction mechanism is controversial and the formation plasma membrane DT-diaphorase plays a key role in the of hydroxyl radicals is concluded to be catalyzed by trace mechanism at the cellular level. metals (38). A very weak signal characteristic of DMPO OH was observed in the NAD(P)H-oxidase reaction of We wish to thank Dr. Richard S. Magliozzo (Albert Einstein College of Medicine of Yoshiva University) for reading the manuscript. plasma membranes (Fig. 4, Aa and Ba). The ESR signal appeared even in the presence of DTPA, SOD, and catalase (data not shown). As indicated by a number of studies, the REFERENCES conversion of DMPO-OOH to DMPO-OH by the glutath 1. Powis, G. (1989) Free Radical Biol. Med. 6, 63-101 ione peroxidase system and by other reduct.ants has been 2. Chesis, P.L., Levin, D.E., Smith, M.T., Ernster, L., & Ames, reported (34). The observation of a DMPO-OH signal may B.N. (1984) Proc. Natl. Acad. Sci. USA 81, 1696-1700 be ascribed to the enzyme system and trace metals. 3. Thor, H., Smith, M.T., Hartzell, P., Bellomo, G., Jewell, S.A., & a-Tocopherol quinone, a metabolite of a-tocopherol, is Orrenius, S. (1982) J. Biol. 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