Bioactivation of lapachol responsible for DNA scission by NADPH-cytochrome P450 reductase

The reduction of the naphthoquinone derivative, lapachol, which is responsible for its bioactivation was examined using microsomal preparations and NADPH-cytochrome P450 reductase (P450 reductase). Phenobarbital (PB) pretreatment resulted in an induction of enzyme activities for cytochrome c reduction (1.54 times) and lapachol reduction (1.20 times) by hepatic microsomal preparation of rats. The specific activity of lapachol reduction by purified P450 reductase showed 56-fold higher than that by untreated liver microsomes. Addition of antibody against P450 reductase (2 mg of IgG/mg of protein) to the microsomal incubation mixture caused an immunoinhibition of cytochrome c (32%) and lapachol (19%) reduction activities, suggesting that P450 reductase catalyzes lapachol reduction. Generation of superoxide anion radical (1321 nmol/mg per min) in approximately equivalent amounts of with NADPH consumption (941 nmol/mg per min) was detected during metabolism of lapachol by P450 reductase. Electron spin resonance (ESR) experiments confirmed generation of superoxide anion radical and hydroxyl radical as these 5,5′-dimethyl-1-pyrroline N-oxide (DMPO) adducts. Incubation of lapachol with P450 reductase caused a cleavage of DNA which was reduced in the presence of Cu,Zn-superoxide dismutase (Cu,Zn-SOD), catalase¹, and hydroxyl radical scavengers such as dimethyl sulfoxide (DMSO) and thiourea. Taken together, these results indicate that lapachol is bioactivated by P450 reductase to reactive species, which promote DNA scission through the redox cycling based generation of superoxide anion radical.     

Generation of free radicals during the reductive metabolism of nilutamide by lung microsomes: possible role in the development of lung lesions in patients treated with this anti-androgen.

The pulmonary metabolism of nilutamide, a nitroaromatic anti-androgen drug leading to pulmonary lesions in a few recipients, has been investigated in rats. Incubation of nilutamide (1 mM) with rat lung microsomes and NADPH under anaerobic conditions led to the formation of the nitro anion free radical, as indicated by ESR spectroscopy. The steady state concentration of this radical was not decreased by CO or SKF 525-A (two inhibitors of cytochrome P450), but was decreased by NADP+ (10 mM) or p-chloromercuribenzoate (0.47 mM) (two inhibitors of NADPH-cytochrome P450 reductase activity). Anaerobic incubations of [3H]nilutamide (0.1 mM) with rat lung microsomes and a NADPH-generating system resulted in the in vivo covalent binding of [3H]nilutamide metabolites to microsomal proteins; covalent binding required NADPH; it was decreased in the presence of NADP+ (10 mM), or in the presence of the nucleophile glutathione (10 mM), but was unchanged in the presence of carbon monoxide. Under aerobic conditions, in contrast, the nitro anion free radical was reoxidized by oxygen, and its ESR signal was not detected. Covalent binding was essentially suppressed. Instead, there was consumption of NADPH and oxygen, and production of superoxide anion and hydogen peroxide. We conclude that nilutamide is reduced by rat lung microsomes NADPH-cytochrome P450 reductase into a nitro anion free radical. In anaerobiosis, the radical is reduced further to covalent binding species. In the presence of oxygen, in contrast, this nitro anion free radical undergoes redox cycling, with the generation of reactive oxygen species.     

One-electron reductive bioactivation of 2,3,5,6-tetramethylbenzoquinone by cytochrome P450.

Bioreductive activation of quinones in mammalian liver has generally been attributed to NADPH-cytochrome P450 reductase. However, in view of the 20-30-fold molar excess of cytochrome P450 over NADPH-cytochrome P450 reductase on the endoplasmic reticulum of the rat liver cell and the capability of cytochrome P450 to bind and reduce xenobiotics, it was considered of interest to investigate the possible role of cytochrome P450 in the bioreduction of quinones. In the present study, 2,3,5,6-tetramethyl-1,4-benzoquinone (TMQ) was chosen as a model quinone. First, TMQ was found to bind at the metabolic active site of phenobarbital (PB)-inducible cytochrome P450s of rat liver microsomes, indicating that TMQ is a potential substrate for cytochrome P450-mediated biotransformation. Second, with electron spin resonance, one-electron reduction of TMQ to a semiquinone free radical (TMSQ) was found to occur in these microsomal fractions. SK&F 525-A, a well-known inhibitor of cytochrome P450, strongly inhibited TMSQ formation in these subcellular fractions without affecting NADPH-cytochrome P450 reductase activity. One-electron reductive bioactivation of TMQ was further investigated with purified NADPH-cytochrome P450 reductase alone and in reconstituted systems of purified cytochrome P450-IIB1 and NADPH-cytochrome P450 reductase. As measured by ESR, purified cytochrome P450-IIB1 in the presence of NADPH-cytochrome P450 reductase was able to reduce TMQ to TMSQ at a much greater rate than in the presence of NADPH-cytochrome P450 reductase alone. Reduction of TMQ was also investigated by measuring the initial rate of NADPH oxidation by TMQ under anaerobic conditions. Inhibitors of cytochrome P450, namely SK&F 525-A and antibodies against PB-inducible cytochrome P450s, caused a substantial decrease in reductive metabolism in PB-treated microsomes. These antibodies were also effective in the inhibition of TMQ-induced NADPH oxidation in a complete reconstituted system of equimolar concentrations of cytochrome P450-IIB1 and NADPH-cytochrome P450 reductase, indicating that the reaction was specific for cytochrome P450-IIB1. Finally, initial rates of NADPH oxidation were determined in reconstituted systems containing varying amounts of NADPH-cytochrome P450 reductase and cytochrome P450-IIB1 to determine the contribution of either enzyme in the reduction of TMQ. As expected, NADPH-cytochrome P450 reductase was able to reduce TMQ to a small extent. However, reconstitution in the presence of increasing amounts of cytochrome P450-IIB1 (relative to NADPH-cytochrome P450 reductase) resulted in increasing rates of TMQ-induced NADPH oxidation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytochrome P450 reductase-mediated anaerobic biotransformation of ethanol to 1-hydroxyethyl-free radicals and acetaldehyde

The ability of cytochrome P450 reductase to metabolize ethanol (EtOH) to acetaldehyde (AC) and 1-hydroxyethyl free radicals (1HEt) in anaerobic media was studied. Determination of AC was made by GC-FID analysis of the head space of incubation mixtures. The formation of 1HEt was established by GC-MS analysis of the adduct formed between the radical and the spin trap PBN. Results showed that pure human P450 reductase is able to biotransform EtOH to AC and 1HEt in a NADPH-dependent process under an oxygen-free nitrogen atmosphere. Pure FAD in the presence of NADPH was also able to generate AC and 1HEt from the alcohol. Anaerobic incubation mixtures containing either rat liver microsomes or pure nuclei were also able to biotransform EtOH to AC and 1HEt in the presence of NADPH. These processes were inhibited by antibody against rat liver microsomal P450 reductase. Results suggest that semiquinone forms of the flavin in P450 reductase may biotransform EtOH. These reactions might be of some significance in tissues where the P450 reductase is present in the absence of specific forms of cytochrome P450 known to be involved in EtOH metabolism (e.g. CYP2E1). However the toxicological significance of this enzymatic process remains to be established.     

Generation of reactive oxygen species and 8-hydroxy-2'-deoxyguanosine formation from diesel exhaust particle components in L1210 cells.

The generation of the reactive oxygen species during the interaction of diesel exhaust particles (DEP) with NADPH-cytochrome P450 reductase (P450 reductase) was investigated by electron spin resonance using the spin-trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO). Addition of DEP extract to an incubation mixture of mouse lung microsomes in the presence of NADPH resulted in a time-dependent NADPH oxidation and acetylated-cytochrome c reduction. Using purified P450 reductase as the enzyme source, superoxide radicals which were detected as the spin adduct (DMPO-OOH) while metabolized by P450 reductase were dependent upon both DEP and enzyme concentrations. The ELISA method using a specific monoclonal antibody revealed that DEP produced 8-hydroxy-2'-deoxyguanosine (8-OHdG), which is formed from deoxyguanosine in DNA by hydroxyl radicals, in the culture medium of L1210 cells. Active oxygen scavengers such as superoxide dismutase and catalase effectively blocked the formation of 8-OHdG in culture medium, and deferoxamine, which inhibits hydroxyl radicals production by chelating iron, was also effective in inhibiting the DEP-produced 8-OHdG formation. These results indicate that DEP components produce 8-OHdG through the hydroxyl radical formation via superoxide by redox cycling of P450 reductase.     

Role of ethanol-inducible cytochrome P450 (P450IIE1) in catalysing the free radical activation of aliphatic alcohols

Incubation of rat liver microsomes with 1-propanol and 1-butanol in the presence of NADPH and of the spin trapping agent 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) allowed the detection of free radical intermediates tentatively identified as 1-hydroxypropyl and 1-hydroxybutyl radical, respectively. Microsomes isolated from rats treated chronically with ethanol (EtOH) or with the combination of starvation and acetone treatment (SA), exhibited a two-fold increase in the ESR signal intensity as compared to untreated controls, whereas no increase was observed in phenobarbital-induced (PB) microsomes. Consistently, in reconstituted membrane vesicles, ethanol-inducible cytochrome P450IIE1 was twice as active as phenobarbital-inducible P450IIB1 in producing 1-butanol free radicals. In the microsomal preparations from EtOH and SA pretreated rats the addition of antibodies against cytochrome P450IIE1, but not of preimmune IgGs, lowered the ESR signal of 1-butanol radicals by more than 50%. The same antibodies decreased the free radical production by untreated microsomes by 35-40%, but were ineffective on microsomes from PB-treated animals. This indicated that cytochrome P450IIE1 is the major enzyme responsible for the free radical activation of alcohols in control and ethanol-fed rats. The generation of 1-hydroxybutyl radicals by EtOH microsomes was inhibited by 40, 48 and 68%, respectively, by the addition of isoniazid, tryptamine and octylamine, compounds known to specifically affect the NADPH oxidase activity of this isoenzyme. This effect was not due to the scavenging of the alcohol radical since none of these compounds affected the ESR signals originated from 1-butanol in a xanthine-xanthine oxidase system. When added to reconstituted membrane vesicles isoniazid, tryptamine and octylamine also decreased 1-butanol radical formation by P450IIE1 by 54, 38 and 66%, respectively. Such an inhibition corresponded to the effect exerted by the same compounds on O2- release from P450IIE1 containing vesicles. These results indicate that the capacity of cytochrome P450IIE1 to reduce oxygen is related to its ability to generate alcohol free radicals and suggest that ferric cytochrome P450-oxygen complex might act as oxidizing species toward alcohols.     

Evidence for superoxide formation during hepatic metabolism of tamoxifen.

Spin trapping of free radicals during the hepatic metabolism of tamoxifen was investigated; the spin trap employed in this study was 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). The spin adduct 2-hydroxy-5,5-dimethyl-1-pyrrolidinyloxyl (DMPO-OH) was detected in an in vitro incubation mixture of phenobarbital-treated rat hepatocytes containing tamoxifen, dimethyl sulfoxide, and DMPO. However, since the spin adduct 2,5,5-trimethyl-1-pyrrolidinyloxyl (DMPO-CH3) was not observed, the DMPO-OH resulted from the cellular bioreduction of 2-hydroperoxy-5,5-dimethyl-1-pyrrolidinyloxyl (DMPO-OOH) by glutathione peroxidase. Addition of superoxide dismutase (SOD) to the in vitro system indicated that superoxide production was intracellular. When noninduced hepatocytes were utilized, free radical production was not evident. Thus, the cytochrome P450 monooxygenase system was responsible, in part, for the intermediacy of superoxide anion during hepatic metabolism.     

Enzymatic and nonenzymatic production of free radicals from the carcinogens 4-nitroquinoline N-oxide and 4-hydroxylaminoquinoline N-oxide.

The anion radicals of 4-nitroquinoline N-oxide (4-NQO) and 4-nitrosoquinoline N-oxide (4-NOQO) carcinogens were detected and characterized by electron spin resonance (ESR) spectroscopy. The structures of the radical intermediates were examined by density functional theory (DFT) at the level of hybrid unrestricted uBecke3LYP. The formation of superoxide anion radical catalyzed by flavin-containing enzymes such as cytochrome P450 reductase or xanthine oxidase in the presence of 4-NQO or 4-nitroquinoline N-oxide was studied by spin-trapping experiments. In this case, the ESR signal of the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-superoxide radical adduct was observed, and its formation was inhibited by superoxide dismutase (SOD). No ESR signal was detected when the two-electron-transferring flavoenzyme DT-diaphorase (NADPH-quinone oxidoreductase) was used. The above is consistent with a one-electron reduction in the metabolism of these nitro compounds to anion free radicals by various flavoenzyme reductases.     

Human enzymes involved in the metabolic activation of carcinogenic aristolochic acids: evidence for reductive activation by cytochromes P450 1A1 and 1A2

Aristolochic acid (AA), a naturally occurring nephrotoxin and rodent carcinogen, has recently been associated with the development of urothelial cancer in humans. Determining the capability of humans to metabolize AA and understanding, which human enzymes are involved in AA activation is important in the assessment of individual susceptibility. Using the nuclease P1-enhanced version of the (32)P-postlabeling assay, we compared the ability of human, minipig and rat hepatic microsomal samples to activate AA to metabolites forming DNA adducts. Human microsomes generated AA-DNA adduct profiles reproducing those found in renal tissues from humans exposed to AA. Identical patterns of AA-DNA adducts were also observed when AA was activated by minipig and rat microsomes. Therefore, microsomes of both animals are suitable in vitro systems mimicking the enzymatic activation of AA in humans. To define the role of specific P450 enzymes and NADPH:P450 reductase in the activation of AA by human microsomes we investigated the modulation of AA-DNA adduct formation by specific inducers or selective inhibitors of P450s and cofactors or inhibitors of NADPH:P450 reductase. The inducer of P450 1A1/2, beta-naphthoflavone, significantly stimulated the levels of AA-DNA adducts formed by rat microsomes, but inducers of P450 2B1/2 and 2E1 had no such effect. Furthermore, only inhibitors of the P450 1A subfamily (alpha-naphthoflavone, furafylline) significantly decreased the amount of adducts formed by microsomes from humans, minipigs and rats. alpha-Lipoic acid, an inhibitor of NADPH:P450 reductase, inhibited adduct formation too, but to a lower extent. On the basis of these results, we attribute most of the microsomal activation of AA to P450 1A1 and 1A2, although a role of NADPH:P450 reductase cannot be ruled out. With purified enzymes (recombinant P450 1A1/2 and NADPH:P450 reductase) and microsomes from baculovirus transfected insect cells expressing recombinant human P450 1A1/2 and NADPH:P450 reductase, the participation of these enzymes in the formation of AA-DNA adducts was confirmed. These results are the first report on the activation of AA by human enzymes and clearly demonstrate the role of P450 1A1, 1A2, and NADPH:P450 reductase in catalyzing the reductive activation of AA.     

In vivo rat hemoglobin thiyl free radical formation following phenylhydrazine administration

The reaction of oxyhemoglobin with phenylhydrazine has received considerable attention for many decades. The basis for this interest stems from the ability of phenylhydrazine and hydrazine-based drugs to induce hemolytic anemia. Considerable evidence obtained from in vitro ESR experiments implicates free radicals in the events leading to red blood cell hemolysis. However, until this report, no corroborating ESR evidence for in vivo free radical formation has been presented. We have successfully employed ESR to detect the formation of a radical adduct in the blood of rats which received an intragastric dose of phenylhydrazine followed by an intraperitoneal injection of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). An immobilized radical adduct was detected by ESR when phenylhydrazine was administered in a dosage comparable to that prescribed for currently employed hydrazine-based drugs. We were also able to detect this immobilized DMPO adduct when hydrazine was employed in place of phenylhydrazine in the rat studies. The results of a series of experiments led us to ascribe this DMPO radical adduct to the trapping of a hemoglobin-derived thiyl free radical.     

Irreversible binding of heme to microsomal protein during inactivation of cytochrome P450 by 4-alkyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine

The porphyrinogenicity of 4-alkyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) is related to the process of mechanism-based destruction of cytochrome P450 (P450) heme, accompanied by conversion of heme to N-alkylprotoporphyrins (N-alkylPPs). Certain DDC analogues (4-isopropyl, 4-isobutyl, 4-hexyl) are weakly porphyrinogenic in comparison to the potent porphyrinogen, 4-ethyl DDC. We have examined the abilities of these DDC analogues to promote irreversible binding of radiolabeled heme to protein in rat liver microsomal preparations. The goals of this study were to determine whether DDC analogues with different porphyrinogenicities differ in the extents to which they cause heme adduct formation, and whether P450 isozymes differ in their capacities to catalyze heme covalent binding. Incubation of microsomes with NADPH alone promoted heme covalent binding, while loss of spectral P450 heme was minimal or absent. In microsomal incubations containing NADPH, the 4-ethyl, 4-isopropyl, and 4-isobutyl analogues caused heme covalent binding to extents which paralleled their P450 destructive activities. In contrast, 4-hexyl DDC caused less heme covalent binding as a function of P450 loss than the other analogues in microsomes from untreated and beta-naphthoflavone (beta NF)-treated rats. Thus, the weakly porphyrinogenic DDC analogues do not cause greater heme covalent binding than 4-ethyl DDC. Weak porphyrinogenicity, therefore, cannot be explained by diversion of the heme moiety of P450 from conversion to N-alkylPPs towards utilization for formation of heme-derived protein adducts. Treatment of rats with P450 inducing agents altered the degree to which DDC analogues caused heme covalent binding. The greatest heme adduct formation occurred in microsomes from untreated and dexamethasone (DEX)-treated rats, whereas treatment with phenobarbital and especially beta NF reduced heme covalent binding as a function of P450 loss. Thus, these microsomal studies suggest that constitutive P450 isozymes and members of the DEX-inducible P450IIIA subfamily appear to catalyze heme covalent binding, while beta NF-inducible forms such as P450IA1 (P450c) seem to be relatively inactive in this regard.     

Free radical production from normal and adriamycin-treated rat cardiac sarcosomes

The production of hydroxyl radicals in rat myocardial sarcosomes treated with adriamycin was demonstrated by the electron spin resonance technique of spin trapping. Using the spin trapping agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the formation of a hydroxyl radical spin adduct was observed in adriamycin-treated rat heart sarcosomes with NADPH as co-factor. Oxygen, NADPH and sarcosomal protein were absolute requirements for hydroxyl radical production. Hydroxyl radical spin adduct formation was not inhibited by the metal ion chelators diethylenetriaminepenta-acetic acid (DETAPAC) or desferrioxamine, or by addition of superoxide dismutase but could be inhibited by addition of catalase and high concentration of the hydroxyl radical scavengers mannitol and N-acetylcysteine. Hydroxyl radical production in adriamycin-treated rat myocardial sarcosomes appears to arise from the reductive metabolism of adriamycin by an NADPH-dependent quinone reductase--NADPH: cytochrome P450 reductase; the reduced quinone (semiquinone) reduces oxygen to hydrogen peroxide, probably via superoxide, although this was not detected. The hydrogen peroxide appears to react directly with adriamycin semiquinone, although involvement of traces of iron in a Fenton type of reaction cannot be excluded. From the observations it is suggested that adriamycin-induced cardiotoxicity is an oxidative pathology arising from intracellular generation of relatively high levels of hydroxyl radicals.     

The role of cytochrome P450 and cytochrome P450 reductase in the reductive bioactivation of the novel benzotriazine di-N-oxide hypoxic cytotoxin 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233, WIN 59075) by mouse liver.

SR 4233 or WIN 59075 (3-amino-1,2,4-benzotriazine-1,4-dioxide) is a novel and highly selective hypoxic cell cytotoxin requiring reductive bioactivation for its impressive antitumour effects. Expression of appropriate reductases will contribute to therapeutic selectivity. Here we provide more detailed information on the role of cytochrome P450 and cytochrome P450 reductase in SR 4233 reduction by mouse liver microsomes. Reduction of SR 4233 to the mono-N-oxide SR 4317 (3-amino-1,2,4-benzotriazine-1-oxide) is NADPH, enzyme and hypoxia dependent. An inhibitory antibody to cytochrome P450 reductase decreased the microsomal SR 4233 reduction rate by around 20%. Moreover, studies with purified rat cytochrome P450 reductase showed unequivocally that this enzyme was able to catalyse SR 4233 reduction at a rate of 20-30% of that for microsomes with equivalent P450 reductase activity. Exposure to the specific cytochrome P450 inhibitor carbon monoxide (CO) inhibited microsomal reduction by around 70% and CO plus reductase antibody blocked essentially all activity. Additional confirmation of cytochrome P450 involvement was provided by the use of other P450 ligands: beta-diethylaminoethyl diphenylpropylacetate hydrochloride gave a slight stimulation while aminopyrine, n-octylamine and 2,4-dichloro-6-phenylphenoxyethylamine were inhibitory. Induction of SR 4233 reduction was seen with phenobarbitone, pregnenalone-16-alpha-carbonitrile and beta-napthoflavone, suggesting that cytochrome P450 subfamilies IIB, IIC and IIIA may be involved. Since cytochrome P450 and P450 reductase catalyse roughly 70 and 30%, of mouse liver microsomal SR 4233 reduction respectively, we propose that expression of these and other reductases in normal and tumour tissue is likely to be a major factor governing the toxicity and antitumour activity of the drug.     

Reductive metabolism of an alpha,beta-ketoalkyne, 4-phenyl-3-butyn-2-one, by rat liver preparations.

The reduction of the triple bond and carbonyl group of an alpha,beta-ketoalkyne, 4-phenyl-3-butyn-2-one (PBYO), by rat liver microsomes and cytosol was investigated. The triple-bond-reduced product trans-4-phenyl-3-buten-2-one (PBO) and the carbonyl-reduced product 4-phenyl-3-butyn-2-ol (PBYOL) were formed when PBYO was incubated with rat liver microsomes in the presence of NADPH. The triple bond of 1-phenyl-1-butyne, deprenyl, ethynylestradiol, ethinamate, and PBYOL, in which the triple bond is not adjacent to a carbonyl group, were not reduced by liver microsomes even in the presence of NADPH. PBO was further reduced to 4-phenyl-2-butanone (PBA) by liver cytosol with NADPH. PBYOL was also formed from PBYO by liver cytosol in the presence of NADPH or NADH. The microsomal triple-bond reductase activity was inhibited by disulfiram, 7-dehydrocholesterol, and 18 beta-glycyrrhetinic acid but not beta-diethylaminoethyldiphenylpropylacetate or carbon monoxide. The triple-bond reductase activity in liver microsomes was not enhanced by several inducers of the rat cytochrome P450 system. These results suggested that the triple-bond reduction is caused by a new type of reductase, not cytochrome P450. The microsomal and cytosolic carbonyl reductase activities were not inhibited by quercitrin, indomethacin, or phenobarbital. Only S-PBYOL was formed from PBYO by liver cytosol. In contrast, liver microsomes produced R-PBYOL together with the S-enantiomer to some extent. Ethoxyresorufin-O-dealkylase activity in rat liver microsomes was markedly inhibited by PBYO and PBO, partly by PBYOL, but not by PBA.     

Characterization of hydroxyl radical formation by microsomal enzymes using a water-soluble trap, terephthalate

Using terephthalic acid as a water-soluble trap, we characterized hydroxyl radicals (HO?) formation by liver microsomal enzymes from isoniazid-treated rats. We found that HO? formation was entirely dependent on intact microsomal enzymes, the presence of NADPH, and iron complexed with EDTA. In contrast to the other radical traps, we found no evidence that terephthalate is a substrate for cytochrome P450. Cumene hydroperoxide, an artificial supporter of cytochrome P450-catalyzed oxidation, failed to maintain HO(.-) formation. HO(.-) formation in liver microsomes was inhibited by the HO(.-) radical scavengers: dimethyl sulfoxide (DMSO), mannitol, and citrulline. It was abolished by catalase, but not superoxide dismutase (SOD), indicating that hydrogen peroxide was the sole precursor of the HO(.-). Therefore, the generation of hydroxyl radicals by microsomal enzymes appears to be dependent on two processes: (1) the rate of hydrogen peroxide production; and (2) the availability of iron ions or other transition metals for Fenton type reactions.     

Inhibition of NADPH cytochrome P450 reductase by the model sulfur mustard vesicant 2-chloroethyl ethyl sulfide is associated with increased production of reactive oxygen species

Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES), is a potent inhibitor of human recombinant cytochrome P450 reductase, as well as native cytochrome P450 reductase from liver microsomes of saline and β-naphthoflavone-treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from β-naphthoflavone-treated rats, CEES was found to inhibit CYP 1A1 activity. This inhibition was overcome by microsomal cytochrome P450 reductase from saline-treated rats, which lack CYP 1A1 activity, demonstrating that the CEES inhibitory activity was selective for cytochrome P450 reductase. Cytochrome P450 reductase also generates reactive oxygen species (ROS) via oxidation of NADPH. In contrast to its inhibitory effects on the reduction of cytochrome c and CYP1A1 activity, CEES was found to stimulate ROS formation. Taken together, these data demonstrate that sulfur mustard vesicants target cytochrome P450 reductase and that this effect may be an important mechanism mediating oxidative stress and lung injury.     

Cytochrome b(5) plays a key role in human microsomal chromium(VI) reduction

The reduction of chromium(VI) to Cr(III) results in the formation of reactive intermediates that contribute to the cytotoxicity, genotoxicity, and carcinogenicity of Cr(VI)-containing compounds. Previous studies suggest that human microsomal Cr(VI) reduction likely proceeds through cytochrome b(5). In order to better understand Cr(VI) toxicity in humans, the role of cytochrome b(5) in combination with P450 reductase was examined in the reductive transformation of Cr(VI). Proteoliposomes containing human recombinant cytochrome b(5) and P450 reductase were constructed. The ability of P450 reductase to mediate efficient electron transfer from NADPH to cytochrome b(5) was confirmed by spectral analysis. The NADPH-dependent Cr(VI) reduction rate mediated by proteoliposomes was then compared to that of human microsomes. When these rates were normalized to equivalent cytochrome b(5) concentrations, the NADPH-dependent Cr(VI) reduction rates mediated by human microsomes were essentially identical to those for proteoliposomes containing cytochrome b(5) plus P450 reductase. Proteoliposomes containing only P450 reductase or cytochrome b(5) exhibited poor Cr(VI) reducing capabilities. Since it had been previously shown that trace amounts of iron (Fe) could dramatically stimulate microsomal Cr(VI) reduction, the ability of Fe to stimulate Cr(VI) reduction by proteoliposomes was examined. Both ferric chloride (FeCl(3)) and ferric adenosine-5'-diphosphate (FeADP) were shown to stimulate Cr(VI) reduction; this stimulation could be abolished by the addition of deferoxamine, a specific Fe(III) chelator. The NADPH-dependent reduction rates of various ferric complexes by proteoliposomes were sufficient to account for the increased Cr(VI) reduction rates seen with the addition of FeCl(3) or FeADP. Cr(V) was detected by electron paramagnetic resonance (EPR) spectroscopy as a transient intermediate formed during NADPH-dependent Cr(VI) reduction mediated by proteoliposomes containing cytochrome b(5) and P450 reductase. Overall, cytochrome b(5) in combination with P450 reductase can account for the majority of the NADPH-dependent Cr(VI) reduction seen with human microsomes.     

Specificity of a phenobarbital-induced cytochrome P-450 for metabolism of carbon tetrachloride to the trichloromethyl radical

Evidence is presented which demonstrates that the first polypeptide to disappear in liver microsomes of phenobarbital-induced rats treated with CCl₄ was the 52,000 dalton P-450 cytochrome. Data are also presented which show that this form of cytochrome P-450 was capable of generating the trichloromethyl radical from CCL₄ in a reconstituted system containing the purified cytochrome, NADPH-cytochrome P-450 reductase, NADPH, CCl₄, and the spin-trapping agent, phenyl-t-butyl nitrone. Other cytochrome P-450 fractions not containing the 52,000 dalton form did not produce this radical. The formation of this highly reactive radical may have resulted in localized damage to the cytochrome, causing the cytochrome either to be released from the microsomal membrane or to form large aggregates which did not migrate in the gel electrophoretic procedures employed.     

The effect of the anthrapyrazole antitumour agent CI941 on rat liver microsome and cytochrome P-450 reductase mediated free radical processes. Inhibition of doxorubicin activation in vitro

The anthrapyrazole CI941 is one of a new series of DNA complexing drugs which displays high level broad spectrum antitumour activity in mice. In view of the proposed role of drug free radical formation, superoxide generation and lipid peroxidation in anthracycline and anthraquinone induced toxicities, the redox biochemistry of CI941 has been investigated. Studies have been performed in vitro using rat liver microsomes and purified cytochrome P-450 reductase. In addition, the ability of CI941 to undergo chemical reduction has been examined. Pulse radiolysis of CI941 demonstrated that the drug can undergo chemical reduction with a one electron reduction potential of E1(7) = -538 +/- 10 mV. However, electron spin resonance (ESR) spectroscopy studies using either NADPH fortified microsomes or cytochrome P-450 reductase, failed to detect a drug free radical signal. Unlike doxorubicin, CI941 (150 microM) inhibited basal rate microsomal NADPH consumption by 45%. Furthermore, CI941 (50-200 microM) antagonised doxorubicin stimulated (1.8-fold) NADPH oxidation by over 50%. CI941 also antagonised the formation of a doxorubicin free radical ESR signal in a concentration dependent manner. CI941 induced minimal superoxide generation in the presence of either microsomes or cytochrome P-450 reductase and inhibited doxorubicin induced (50 microM) superoxide formation by up to 80% (50-200 microM CI941). Importantly, CI941 inhibits both basal rate and doxorubicin (100 microM) stimulated lipid peroxidation (52% inhibition at 5 microM CI941). These data suggest that CI941 is unlikely to induce free radical mediated tissue damage in vivo. On the contrary, CI941 may have a protective role if used in combination with doxorubicin.     

Effects of mansonones on lipid peroxidation, P450 monooxygenase activity, and superoxide anion generation by rat liver microsomes

Several structurally related ortho-naphthoquinones isolated from Mansonia altissima Chev (mansonones C, E and F) (a) inhibited NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (b) prevented NADPH-dependent cytochrome P450 destruction; (c) inhibited NADPH-supported aniline 4-hydroxylase activity; (d) inhibited Fe(III)ADP reduction by NADPH-supplemented microsomes; (e) stimulated superoxide anion generation by NADPH-supplemented microsomes; and (f) stimulated ascorbate oxidation. ESR investigation of ascorbate-reduced mansonone F demonstrated semiquinone formation. Mansonone C had a greater effect than mansonones E and F on NADPH-dependent lipid peroxidation, O2- production and ascorbate oxidation, whereas mansonone E was more effective than mansonones C and F on aniline 4-hydroxylase activity. Mansonones E and F did not inhibit hydroperoxide-dependent lipid peroxidation, cytochrome P450 destruction or microsomal aniline 4-hydroxylase activity. Mansonone C inhibited to a limited degree tert-butyl hydroperoxide-dependent lipid peroxidation, this inhibition being increased by NADPH. Mansonone A, a tetrahydro orthonapthoquinone derivative, was in all respects relatively less effective than mansonones C, E and F. It is postulated that mansonones C, E and F inhibited microsomal lipid peroxidation and cytochrome P450 catalyzed reactions by diverting reducing equivalents from NADPH to dioxygen, but mansonone C (including its reduced form) may also exert direct antioxidant activity.