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-.     

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.     

Superoxide generation by NADPH-cytochrome P-450 reductase: the effect of iron chelators and the role of superoxide in microsomal lipid peroxidation

Superoxide generation, assessed as the rate of acetylated cytochrome c reduction inhibited by superoxide dismutase, by purified NADPH cytochrome P-450 reductase or intact rat liver microsomes was found to account for only a small fraction of their respective NADPH oxidase activities. DTPA-Fe3+ and EDTA-FE3+ greatly stimulated NADPH oxidation, acetylated cytochrome c reduction, and O(2) production by the reductase and intact microsomes. In contrast, all ferric chelates tested caused modest inhibition of acetylated cytochrome c reduction and O(2) generation by xanthine oxidase. Although both EDTA-Fe3+ and DTPA-Fe3+ were directly reduced by the reductase under anaerobic conditions, ADP-Fe3+ was not reduced by the reductase under aerobic or anaerobic conditions. Desferrioxamine-Fe3+ was unique among the chelates tested in that it was a relatively inert iron chelate in these assays, having only minor effects on NADPH oxidation and/or O(2) generation by the purified reductase, intact microsomes, or xanthine oxidase. Desferrioxamine inhibited microsomal lipid peroxidation promoted by ADP-Fe3+ in a concentration-dependent fashion, with complete inhibition occurring at a concentration equal to that of exogenously added ferric iron. The participation of O(2) generated by the reductase in NADPH-dependent lipid peroxidation was also investigated and compared with results obtained with a xanthine oxidase-dependent lipid peroxidation system. NADPH-dependent peroxidation of either phospholipid liposomes or rat liver microsomes in the presence of ADP-Fe3+ was demonstrated to be independent of O(2) generation by the reductase.     

Reconstituted microsomal lipid peroxidation: ADP-Fe3+-dependent peroxidation of phospholipid vesicles containing NADPH-cytochrome P450 reductase and cytochrome P450

A reconstituted lipid peroxidation system consisting of rat liver microsomal NADPH-cytochrome P450 reductase and cytochrome P450 incorporated into phospholipid vesicles was developed and characterized. Peroxidation of the vesicles required NADPH and ADP-Fe3+, just as in the NADPH-dependent peroxidation of microsomes. The peroxidation of the vesicles was inhibited 30-50% by superoxide dismutase, depending upon their cytochrome P450 content: those with higher cytochrome P450 contents exhibited greater rates of malondialdehyde formation which were less sensitive to inhibition by superoxide dismutase. When cytochrome P450 was incorporated into vesicles, EDTA-Fe3+ was not required for lipid peroxidation, distinguishing this system from the one previously described by Pederson and Aust [Biochem. Biophys. Res. Comm. 48, 789; 1972]. Since at least 50% of the malondialdehyde formation in the vesicular system was not inhibited by superoxide dismutase, alternative means of iron reduction (O2-.-independent) were examined. It was found that rat liver microsomes or a reconstituted mixed function oxidase system consisting of NADPH-cytochrome P450 reductase and cytochrome P450 in dilauroylphosphatidylcholine micelles reduced ADP-Fe3+ under anaerobic conditions.     

Xenobiotic-mediated production of superoxide by primary cultures of rat cerebral endothelial cells, astrocytes, and neurones

Previous works of our group demonstrated that xenobiotic metabolism by brain microsomes or cultured cerebral cells may promote the formation of reactive oxygen species. In order to characterise the risk of oxidative stress to both the central nervous system and the blood-brain barrier, we measured in the present work the release of superoxide in the culture medium of rat cerebrovascular endothelial cells during the metabolism of menadione, anthraquinone, diquat or nitrofurazone. Assays were run in the same experimental conditions on primary cultures of rat neurones and astrocytes. Quinone metabolism efficiently produced superoxide, but the production of radicals during the metabolism of diquat or nitrofurazone was very low, as a probable result of their reduced transport inside the cells. In all cell types assayed, superoxide production was time- and concentration-dependent, and cultured astrocytes always produced the highest amounts of radicals. Superoxide formation by microsomes prepared from the cultured cells was decreased by immunoinhibition of NADPH-cytochrome P450 reductase or by its irreversible inhibition by diphenyliodonium chloride, suggesting the involvement of this flavoprotein in radical production. Cerebrovascular endothelial cells cultured on collagen-coated filters produced equivalent amounts of superoxide both at their luminal side and through the artificial basement membrane, suggesting that in vivo, endothelial superoxide production may endanger adjacent astrocytes and neurones.     

Role of cytochrome P450 in the oxidation of glycerol by reconstituted systems and microsomes.

Glycerol can be oxidized by rat liver microsomes to formaldehyde in a reaction that requires the production of reactive oxygen intermediates. Studies with inhibitors, antibodies, and reconstituted systems with purified cytochrome P4502E1 were carried out to evaluate whether P450 was required for glycerol oxidation. A purified system containing phospholipid, NADPH-cytochrome P450 reductase, P4502E1, and NADPH oxidized glycerol to formaldehyde. Formaldehyde production was dependent on NADPH, reductase, and P450, but not phospholipid. Formaldehyde production was inhibited by substrates and ligands for P4502E1, as well as by anti-pyrazole P4502E1 IgG. The oxidation of glycerol by the reconstituted system was sensitive to catalase, desferrioxamine, and EDTA but not to superoxide dismutase or mannitol, indicating a role for H2O2 plus non-heme iron, but not superoxide or hydroxyl radical in the overall glycerol oxidation pathway. The requirement for reactive oxygen intermediates for glycerol oxidation is in contrast to the oxidation of typical substrates for P450. In microsomes from pyrazole-treated, but not phenobarbital-treated rats, glycerol oxidation was inhibited by anti-pyrazole P450 IgG, anti-hamster ethanol-induced P450 IgG, and monoclonal antibody to ethanol-induced P450, although to a lesser extent than inhibition of dimethylnitrosamine oxidation. Anti-rabbit P4503a IgG did not inhibit glycerol oxidation at concentrations that inhibited oxidation of dimethylnitrosamine. Inhibition of glycerol oxidation by antibodies and by aminotriazole and miconazole was closely associated with inhibition of H2O2 production. These results indicate that P450 is required for glycerol oxidation to formaldehyde; however, glycerol is not a direct substrate for oxidation to formaldehyde by P450 but is a substrate for an oxidant derived from interaction of iron with H2O2 generated by cytochrome P450.     

Liver Nuclear Nadph-Cytochrome P-450 Reductase May be Involved in Redox Cycling of Bleomycin-Fe(III), Oxy Radical Formation and DNA Damage

When NADPH-cytochrome P-450 reductase isolated from rat liver microsomes was aerobically incubated with bleomycin, FeCl₃, NADPH and DNA parallel NADPH and oxygen were consumed and malondialdehyde was formed. A similar parallelism of NADPH- and oxygen-consumption and malondialdehyde formation was observed when ceil nuclei isolated from rat liver were incubated under the same conditions. The formation of malondialdehyde which was identified by HPLC and which was most likely released from oxidative cleavage of deoxyribose of nuclear DNA required oxygen, bleomycin, FeCl₃ and NADPH. This indicates that a nuclear NADPH-enzyme, presumably NADPH-cytochrome P-450 reductase, is able to redox cycle a bleomycin-iron-complex which in the reduced form can activate oxygen to a DNA-damaging reactive species. The data suggest that the activity of this enzyme in the cell nucleus could play an important role in the cytotoxicity of bleomycin in tumor cells.     

Liver Nuclear Nadph-Cytochrome P-450 Reductase May be Involved in Redox Cycling of Bleomycin-Fe(III), Oxy Radical Formation and DNA Damage

When NADPH-cytochrome P-450 reductase isolated from rat liver microsomes was aerobically incubated with bleomycin, FeCl₃, NADPH and DNA parallel NADPH and oxygen were consumed and malondialdehyde was formed. A similar parallelism of NADPH- and oxygen-consumption and malondialdehyde formation was observed when ceil nuclei isolated from rat liver were incubated under the same conditions. The formation of malondialdehyde which was identified by HPLC and which was most likely released from oxidative cleavage of deoxyribose of nuclear DNA required oxygen, bleomycin, FeCl₃ and NADPH. This indicates that a nuclear NADPH-enzyme, presumably NADPH-cytochrome P-450 reductase, is able to redox cycle a bleomycin-iron-complex which in the reduced form can activate oxygen to a DNA-damaging reactive species. The data suggest that the activity of this enzyme in the cell nucleus could play an important role in the cytotoxicity of bleomycin in tumor cells.     

Role of the prosthetic groups of NADPH-cytochrome P450 reductase in 3-hydroxyanthranilamide-catalyzed,NADPH-dependent superoxide anion production

Summary

The role of the prosthetic groups (FAD and FMN) of NADPH-cytochrome P450 reductase (P450 reductase)in 3-hydroxyanthranilamide (3-OH An.Amide)-catalyzed, NADPH-dependent superoxide anion (O₂-) production via the reductase was examined using the native and FMN-depleted preparations of P450 reductase which was partially purified from rat liver microsomes. NADPH-dependent O₂-production by the FMN-depleted preparation was about 10% of that by the native preparation. 3-OH An. Amide-catalyzed, NADPH-dependent O₂-production by the FMN-depleted preparation was less than 10% of that by the native preparation. FMN supplementation returned O₂-production to near normal. We observed the same results for NADPH oxidation and hydrogen peroxide formation. O₂-production, NADPH oxidation, and hydrogen peroxide formation were inhibited by native superoxide dismutase (SOD), but not by boiled, denatured SOD. These results indicate that the prosthetic groups, especially FMN, of P450 reductase play a critical role in 3-OH An.Amide-catalyzed, NADPH-dependent O₂-production via the reductase.     

Anaerobic dehalogenation of halothane by reconstituted liver microsomal cytochrome P-450 enzyme system

Cytochrome P-450 from liver microsomes of phenobarbital-treated rabbits catalyzed anaerobic dehalogenation of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) when combined with NADPH and NADPH-cytochrome P-450 reductase. Cytochromes P-450B₁ and P-448 from liver microsomes of untreated rabbits were less active. Triton X-100 accelerated the reaction. Unlike anaerobic dehalogenation of halothane in microsomes, the major product was 2-chloro-1,1,1-trifluoroethane and 2-chloro-1,1-difluoroethylene was negligible. These products were not detected under aerobic conditions, and dehalogenation activity was inhibited by carbon monoxide, phenyl isocyanide and metyrapone.     

Nitrofuran enhancement of microsomal electron transport, superoxide anion production and lipid peroxidation

Addition of nifurtimox (a nitrofuran derivative used for the treatment of Chagas' disease) to rat liver microsomes produced an increase of (a) electron flow from NADPH to molecular oxygen, (b) generation of both superoxide anion radical (O₂^?) and hydrogen peroxide, and (c) lipid peroxidation. The nifurtimox-stimulated NADPH oxidation was greatly inhibited by NADP⁺ and p-chloromercuribenzoate, and to a lesser extent by SKF-525-A and metyrapone. These inhibitions reveal the function of both the NADPH-cytochrome P-450 (c) reductase and cytochrome P-450 in nifurtimox reduction. Superoxide dismutase, catalase (in the presence of superoxide dismutase), and hydroxyl radical scavengers (mannitol, 5,5-dimethyl-1-pyrroline-1-oxide) inhibited the nifurtimox-stimulated NADPH oxidation, in accordance with the additional operation of a reaction chain including the hydroxyl radical. Further evidence supporting the role of superoxide anion and hydroxyl radicals in the nifurtimox-induced NADPH oxidation resulted from the effect of specific inhibitors on NADPH oxidation by O₂^? (generated by the xanthine oxidase reaction) and by OH. (generated by an iron chelate or the Fenton reaction). Production of O₂^? by rat kidney, testes and brain microsomes was significantly stimulated by nifurtimox in the presence of NADPH. It is postulated that enhanced formation of free radicals is the basis for nifurtimox toxicity in mammals, in good agreement with the postulated mechanism of the trypanocide effect of nifurtimox on Trypanosoma cruzi.     

Cytochrome P-450-mediated oxidation of 2-hydroxyestrogens to reactive intermediates

2-Hydroxyestradiol, 2-hydroxyestrone and 2-hydroxy-17α-ethynylestradiol, oxidation products of naturally occurring estrogens and synthetic estrogens in some oral contraceptives were found to be converted by rat liver microsomes to reactive metabolites that become irreversibly bound to microsomal protein. The irreversible binding required microsomes, oxygen and NADPH. The NADPH could be replaced by a xanthine-xanthine oxidase system which is known to generate superoxide anions. The irreversible binding was substantially inhibited by superoxide dismutase, 30% in those incubations containing NADPH and 98% in those incubations containing the xanthine-xanthine oxidase system. Further studies with 2-hydroxyestradiol showed that microsomal cytochrome P-450 was rate limiting in the NADPH-dependent irreversible binding, because the binding was inhibited 62% by an antibody against NADPH-cytochrome $\text{c}$ reductase and 70% in an atmosphere of CO:O₂ (9:1) when compared to an atmosphere of N₂:O₂ (9:1). Phenobarbital, a known inducer of cytochrome P-450, had no effect on the irreversible binding of 2-hydroxyestradiol, whereas another inducer of P-450, pregnenolone-16α-carbonitrile, markedly increased the irreversible binding. In contrast, cobaltous chloride, an inhibitor of the synthesis of cytochrome P-450, decreased both P-450 and the irreversible binding. These results are consistent with a mechanism for irreversible binding of estrogens and 2-hydroxyestrogens to microsomes that requires oxidation of the catechol nucleus by cytochrome P-450-generated superoxide anion.     

Effect of interferon inducers on superoxide anion generation from rat liver microsomes detected by lucigenin chemiluminescence

Microsomal superoxide anion (O2-) production was detected using the chemiluminigenic probe, bis-N-Methylacridinium nitrate (lucigenin). Superoxide dismutase (SOD) inhibited 55% of the light emission but in the presence of a detergent (Triton X100) SOD inhibited the light emission by 94%. Lucigenin chemiluminescence from rat liver microsomes supplemented with NADPH was found to be selective and sensitive in detecting the O2- production. Treatment of rats with poly IC and LPS resulted in a decrease of the hepatic microsomal cytochrome P450 content by 44% and 37% respectively. The decrease in the cytochrome P450 contents was accompanied by a decrease in LgCl from the hepatic microsomal fractions by 61% for the poly IC and by 51% for the LPS treated rats. This is the first report to demonstrate that decreased P450 in the presence of normal amounts of cytochrome P450(c) reductase produce correspondingly less O2- from the microsomes.     

The role of iron chelates in hydroxyl radical production by rat liver microsomes,NADPH-cytochrome P-450 reductase and xanthine oxidase

Uninduced rat liver microsomes and NADPH-Cytochrome P-450 reductase, purified from phenobarbital-treated rats, catalyzed an NADPH-dependent oxidation of hydroxyl radical scavenging agents. This oxidation was not stimulated by the addition of ferric ammonium sulfate, ferric citrate, or ferric-adenine nucleotide (AMP, ADP, ATP) chelates. Striking stimulation was observed when ferric-EDTA or ferric-diethylenetriamine pentaacetic acid (DTPA) was added. The iron-EDTA and iron-DTPA chelates, but not unchelated iron, iron-citrate or iron-nucleotide chelates, stimulated the oxidation of NADPH by the reductase in the absence as well as in the presence of phenobarbital-inducible cytochrome P-450. Thus, the iron chelates which promoted NADPH oxidation by the reductase were the only chelates which stimulated oxidation of hydroxyl radical scavengers by reductase and microsomes. The oxidation of aminopyrine, a typical drug substrate, was slightly stimulated by the addition of iron-EDTA or iron-DTPA to the microsomes. Catalase inhibited potently the oxidation of scavengers under all conditions, suggesting that H₂O₂ was the precursor of the hydroxyl radical in these systems. Very high amounts of superoxide dismutase had little effect on the iron-EDTA-stimulated rate of scavenger oxidation, whereas the iron-DTPA-stimulated rate was inhibited by 30 or 50% in microsomes or reductase, respectively. This suggests that the iron-EDTA and iron-DTPA chelates can be reduced directly by the reductase to the ferrous chelates, which subsequently interact with H₂O₂ in a Fenton-type reaction. Results with the reductase and microsomal systems should be contrasted with results found when the oxidation of hypoxanthine by xanthine oxidase was utilized to catalyze the production of hydroxyl radicals. In the xanthine oxidase system, ferric-ATP and -DTPA stimulated oxidation of scavengers by six- to eightfold, while ferric-EDTA stimulated 25-fold. Ferric-desferrioxamine consistently was inhibitory. Superoxide dismutase produced 79 to 86% inhibition in the absence or presence of iron, indicating an iron-catalyzed Haber-Weiss-type of reaction was responsible for oxidation of scavengers by the xanthine oxidase system. These results indicate that the ability of iron to promote hydroxyl radical production and the role that superoxide plays as a reductant of iron depends on the nature of the system as well as the chelating agent employed.     

Increased microsomal interaction with iron and oxygen radical generation after chronic acetone treatment

In vivo administration of acetone influences a variety of reactions catalyzed by rat liver microsomes. The effect of chronic treatment with acetone (1% acetone in the water for 10-12 days) on interaction with iron and subsequent oxygen radical generation by liver microsomes was evaluated. Microsomes from the acetone-treated rats displayed elevated rates of H2O2 generation, an increase in iron-dependent lipid peroxidation, and enhanced chemiluminescence upon the addition of t-butylhydroperoxide. The ferric EDTA-catalyzed production of formaldehyde from DMSO or of ethylene from 2-keto-4-thiomethylbutyrate was increased 2-fold after acetone treatment. This increase in hydroxyl radical generation was accompanied by a corresponding increase in NADPH utilization and was sensitive to inhibition by catalase and a competitive scavenger, ethanol, but not to superoxide dismutase. In vitro addition of acetone to microsomes had no effect on oxygen radical generation. Associated with the chronic acetone treatment was a 2-fold increase in the microsomal content of cytochrome P-450 and in the activity of NADPH-cytochrome-P-450 reductase. It appears that increased oxygen radical generation by microsomes after chronic acetone treatment reflects the increase in the major enzyme components which comprise the mixed-function oxidase system.     

Reductase and oxidase activity of rat liver cytochrome P450 with 2,3,5,6-tetramethylbenzoquinone as substrate.

The main objective of the present study was to investigate the proposed role of cytochrome P450 in the reductive metabolism of quinones as well as in the formation of reduced oxygen species in liver microsomes from phenobarbital (PB-microsomes) and beta-naphthoflavone (beta NF-microsomes) pretreated rats. In the present study, 2,3,5,6-tetramethylbenzoquinone (TMQ) was chosen as a model quinone. Anaerobic one-electron reduction of TMQ by PB-microsomes showed relatively strong electron spin resonance (ESR) signals of the oxygen-centered semiquinone free radical (TMSQ), whereas these signals were hardly detectable with beta NF-microsomes. Under aerobic conditions TMSQ formation was diminished and concomitant reduction of molecular oxygen occurred in PB-microsomes. Interestingly, TMQ-induced superoxide anion radicals, measured by ESR (using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide), and hydrogen peroxide generation was found to occur with beta NF-microsomes as well. Furthermore, SK&F 525-A (a type I ligand inhibitor of cytochrome P450) inhibited TMQ-induced hydrogen peroxide formation in both PB- and beta NF-microsomes. However, metyrapone and imidazole (type II ligand inhibitors of cytochrome P450) inhibited molecular oxygen reduction in beta NF-microsomes and not in PB-microsomes. The present study indicates that cytochrome P450-mediated one-electron reduction of TMQ to TMSQ and subsequent redox cycling of TMSQ with molecular oxygen constitutes the major source for superoxide anion radical and hydrogen peroxide generation in PB-microsomes (i.e. from the reductase activity of cytochrome P450). However, most of the superoxide anion radical formed upon aerobic incubation of TMQ with beta NF-microsomes originates directly from the dioxyanion-ferri-cytochrome P450 complex (i.e. from the oxidase activity of cytochrome P450). In conclusion, both the one-electron reduction of TMQ and molecular oxygen were found to be cytochrome P450 dependent. Apparently, both the reductase and oxidase activities of cytochrome P450 may be involved in the reductive cytotoxicity of chemotherapeutic agents containing the quinoid moiety.     

Disruption of rat hepatic microsomal electron transport chains by the selenium-containing anti-inflammatory agent Ebselen

The influence of Ebselen, an organoselenium anti-inflammatory agent, on the two electron transport chains present in rat liver microsomes has been studied. At low micromolar concentrations, Ebselen markedly inhibited the flow of reducing equivalents from NADPH-cytochrome P450 reductase to both its natural electron acceptor, cytochrome P450, and its artificial electron acceptor, cytochrome c. Similarly, the microsomal NADH-cytochrome c reductase system consisting of cytochrome b5 and its flavoprotein, NADH-cytochrome b5 reductase, was also significantly inhibited by Ebselen. The inhibition appears to be due to the inability of the reduced pyridine nucleotide to transfer electrons to the flavin (FAD and/or FMN) in the flavoprotein reductase. This was shown with the purified NADPH-cytochrome P450 reductase, which in the presence of Ebselen was not converted to the semiquinone form following the addition of NADPH. The addition of Ebselen to a suspension of hepatic microsomes from either untreated or phenobarbital-treated rats did not result in any spectral change characteristic of type I, type II, or reverse type I.     

P-450 HFLa, a form of cytochrome P-450 purified from human fetal livers, is the 16 alpha-hydroxylase of dehydroepiandrosterone 3-sulfate

In a reconstituted system containing NADPH, dilauroyl-L-3-phosphatidylcholine, and NADPH-cytochrome P-450 reductase purified from rat liver microsomes, cytochrome P-450 (P-450 HFLa) purified from human fetal livers catalyzed the 16 alpha-hydroxylation of dehydroepiandrosterone 3-sulfate (DHEA-sulfate). Addition of cytochrome b5 purified from rat liver microsomes to the reconstituted system resulted in a remarkable increase in the hydroxylase activity. The level of P-450 HFLa in liver homogenates from human fetuses highly correlated with the activity of DHEA-sulfate 16 alpha-hydroxylase. Antibodies to P-450 HFLa inhibited the 16 alpha-hydroxylation of DHEA-sulfate in a dose-dependent manner. The NH2-terminal amino acid sequence of P-450 HFLa was similar to that of P-450NF (Beaune, P. H., Umbenhauer, D. R., Bork, R. W., Lloyd, R. S., and Guengerich, F. P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8064-8068). We conclude that P-450 HFLa is a form of cytochrome P-450 involved in the 16 alpha-hydroxylation of DHEA-sulfate.     

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.     

Cytochrome P-450 mediated reductive dehalogenation of the perhalogenated aromatic compound hexachlorobenzene

Hexachlorobenzene (HCB) elicits concentration-dependent and saturable type 1 binding spectra when added to oxidized (Fe3+) cytochrome P-450 (CYT P-450) in control, phenobarbital- (PB) induced, and beta-naphthoflavone- (BNF) induced male Sprague-Dawley rat liver microsomes. The spectral binding constants (Ks) for HCB in control and PB-induced microsomes are 180 microM and 83 microM, respectively, and correlate inversely with the specific content of CYT P-450 (0.9 and 2.1 nmol/mg) in the two microsomal preparations. BNF-induced microsomes show type 1 interaction only at low HCB concentration. Overall biotransformation of HCB, monitored by loss of [14C]HCB from the reaction medium, is dependent on NADPH and intact microsomes. Dimethyl sulfoxide (Me2SO), a potent hydroxyl radical scavenger and the solvent used for HCB dissolution, does not affect the biotransformation of HCB in aerobic reactions. Pentachlorobenzene (PCB) appears to be the initial and major isolatable CYT P-450 mediated dechlorination product of HCB with NADPH-fortified rat liver microsomes. Trace levels of pentachlorophenol (PCP) and an unidentified metabolite are also observed. PCB formation is enhanced under anaerobic conditions but is inhibited by metyrapone and carbon monoxide. PCB formation is also inhibited with aerobic reaction conditions, while PCP formation is observed. The data indicate that CYT P-450 in hepatic microsomes supports the reductive dechlorination of HCB to PCB.