Application of the Amplex red/horseradish peroxidase assay to measure hydrogen peroxide generation by recombinant microsomal enzymes

The formation of reactive oxygen species by the cytochrome P450 monooxygenase system is thought to be due to autoxidation of NADPH-cytochrome P450 reductase and the nonproductive decay of oxygen-bound cytochrome P450 intermediates. To characterize this process in recombinant microsomal enzymes, we used a highly sensitive hydrogen peroxide assay based on Amplex red oxidation. This assay is 20 times more sensitive (LLD = 5.0 pmol/assay and LLQ = 30 pmol/assay) than the standard ferrous thiocyanate assay for detection of hydrogen peroxide. We found low, but detectable, spontaneous generation of hydrogen peroxide by recombinant human NADPH-cytochrome P450 reductase complexes (0.09 nmol hydrogen peroxide/min/100 Units of NADPH-cytochrome P450 reductase). Significantly higher rates of hydrogen peroxide production were observed when recombinant cytochrome P450 enzymes were coexpressed with NADPH-cytochrome P450 reductase (0.31 nmol of hydrogen peroxide/min/100 Units of NADPH-cytochrome P450 reductase). This was independent of the addition of any exogenous cytochrome P450 substrates. These data demonstrate that cytochrome P450s are a major source of hydrogen peroxide in the recombinant cytochrome P450 monooxygenase system. Moreover, substrate binding is not required for the cytochrome P450s to generate reactive oxygen species.     

Enhanced decomposition of oxyferrous cytochrome P450CIA1 (P450cam) by the chemopreventive agent 3-t-butyl-4-hydroxyanisole

The efficacy of 2(3)-t-butyl-4-hydroxyanisole (BHA) and other chemicals as chemopreventive agents against chemically induced cancer or toxicity may involve direct modulation of cytochrome P450 activity. Direct interaction of BHA with cytochrome P450 was investigated using substrate-bound, oxyferrous cytochrome P450CIA1 either in a reconstituted system containing cytochrome P450CIA1, putidaredoxin, and putidaredoxin reductase with NADH as electron donor or in the absence of physiological electron donors. In the reconstituted system, BHA caused a concentration-dependent decrease in the production of 5-exo-hydroxycamphor and a substoichiometric increase in hydrogen peroxide production. However, BHA did not appreciably inhibit either NADH oxidation or oxygen utilization under conditions optimal for accumulation of oxyferrous cytochrome P450CIA1 during steady-state metabolism of camphor. In the absence of electron donor, BHA enhanced decomposition of the ternary oxyferrous substrate complex of cytochrome P450CIA1 without the formation of any apparent spectral intermediate(s). The rate of decomposition of the oxyferrous complex was pseudo-first order and was dependent upon the concentration of BHA present. Enhanced decomposition of the complex was not attributable to catalytic turnover of cytochrome P450CIA1 (i.e., acquisition of a second electron from an indeterminate source) since no appreciable metabolism of either camphor or BHA was observed. The enhanced decomposition was accompanied by a substoichiometric increase in hydrogen peroxide production, suggesting that BHA may facilitate four-electron reduction of molecular oxygen to water. These results indicate that BHA inhibits cytochrome P450 function, presumably by enhancing autoxidation of the substrate-bound oxyferrous complex.     

Intracellular acidification triggered by mitochondrial-derived hydrogen peroxide is an effector mechanism for drug-induced apoptosis in tumor cells

We recently showed that two photoproducts of merocyanine 540, C2 and C5, triggered cytochrome C release; however, C5 was inefficient in inducing caspase activity and apoptosis in leukemia cells, unlike C2. Here we show that HL60 cells acidified upon exposure to C2 but not C5. The intracellular drop in pH and caspase activation were dependent upon hydrogen peroxide production, and were inhibited by scavengers of hydrogen peroxide. On the contrary, caspase inhibitors did not block hydrogen peroxide production. In turn, increased intracellular hydrogen peroxide concentration was downstream of superoxide anion produced within 2 h of exposure to C2. Inhibitor of NADPH oxidase diphenyleneiodonium neither inhibited superoxide production nor caspase activation triggered by C2. However, exposure of purified mitochondria to C2 resulted in significantly increased superoxide production. Furthermore, cytochrome C release from isolated mitochondria induced by C2 was completely inhibited in the presence of scavengers of hydrogen peroxide. Contrarily, scavenging hydrogen peroxide had no effect on the cyclosporin A-sensitive mitochondrial permeability transition induced by C5. Our data suggest a scenario where drug-induced hydrogen peroxide production induces intracellular acidification and release of cytochrome C, independent of the inner membrane pore, thereby creating an intracellular environment permissive for caspase activation.     

Biosynthesis of D-glucaric acid in mammals: a free-radical mechanism?

In the presence of iron salts and hydrogen peroxide, D-glucuronic acid was converted into D-glucaric acid. The reaction was strongly inhibited by free-radical scavengers and is ascribed to the action of the hydroxyl radical. The formation of D-glucarate was dependent upon pH and occurred in the presence of some iron-complexing agents. The first product of oxidation was a lactone that was a strong inhibitor of beta-D-glucuronidase and assumed to be D-glucaro-1,5-lactone. Microsomal preparations in the presence of NADPH also produced D-glucarate from D-glucuronic acid, presumably due to formation of hydrogen peroxide, and the product was an inhibitor of beta-D-glucuronidase. Superoxide did not produce D-glucarate from D-glucuronate. The cytochrome P450 system is more likely than "glucuronolactone dehydrogenase" to be responsible for the production of D-glucaric acid in vivo.     

Hydrogen peroxide-mediated inactivation of microsomal cytochrome P450 during monooxygenase reactions

Cytochrome P450 can undergo inactivation following monooxygenase reactions in liver microsomes of untreated, phenobarbital and 3-methylcholanthrene-treated rats and rabbits. The acceleration of cytochrome P450 loss in the presence of catalase inhibitors (sodium azide, hydroxylamine) indicates that hydrogen peroxide is involved in hemoprotein degradation. It was revealed that cytochrome P450 is inactivated mainly by H₂O₂ formed through peroxy complex breakdown, whereas H₂O₂ formed via the dismutation of superoxide anions produces a slight inactivating effect. The hydrogen peroxide added outside or formed by a glucose-glucose oxidase system has less of an inactivating effect than H₂O₂ produced within the cytochrome P450 active center. Self-inactivation of cytochrome P450 during oxygenase reactions is highly specific. Other components of the monooxygenase system, such as cytochrome b₅, NADH- and NADPH-specific flavorproteins, undergo no inactivation. The alterations in phospholipid content and in the rate of lipid peroxidation were not observed as well. The inactivation of cytochrome P450 by H₂O₂ is the result of heme loss or destruction without cytochrome P420 formation. Such. a mechanism operates with different substrates and cytochrome P450 species catalyzing the partially coupled monooxygenase reactions.     

A possible role of cAMP dependent phosphorylation of hepatic microsomal cytochrome P450: a mechanism to increase lipid peroxidation in response to hormone

Enzymatic lipid peroxidation in hepatocytes is believed to involve cytochrome P450. cAMP dependent phosphorylation of cytochrome P450 was found to increase the NADPH dependent production of malondialdehyde (lipid peroxidation) by about 30%. The cytochrome P450 inhibitor cyanide abolished this activity. The presence of spermine decreased the cytochrome P450 dependent lipid peroxidation in non-phosphorylated microsomes, phosphorylation partially reversed this effect. Thus, phosphorylation of cytochrome P450 and the associated increased lipid peroxidation may be a hormone dependent response to pathological conditions e.g. stress Phosphorylation was observed to subtly alter other properties of cytochrome P450. The rate of 7-ethoxycoumarin deethylase activity was reduced and the microwave power required to saturate the EPR spectrum of the low spin cytochrome P450 was decreased. It is hypothesized that phosphorylation of cytochrome P450 alters the interaction between the components of the cytochrome P450 system, which may enhance production of free radical species, initiating lipid peroxidation.     

Hydroxyl radical-mediated, cytochrome P-450-dependent metabolic activation of benzene in microsomes and reconstituted enzyme systems from rabbit liver

The mechanism of benzene oxygenation in liver microsomes and in reconstituted enzyme systems from rabbit liver was investigated. It was found that the NADPH-dependent transformation of benzene to water-soluble metabolites and to phenol catalyzed by cytochrome P-450 LM2 in membrane vesicles was inhibited by catalase, horseradish peroxidase, superoxide dismutase, and hydroxyl radical scavengers such as mannitol, dimethyl sulfoxide, and catechol, indicating the participation of hydrogen peroxide, superoxide anions, and hydroxyl radicals in the process. The cytochrome P-450 LM2-dependent, hydroxyl radical-mediated destruction of deoxyribose was inhibited concomitantly to the benzene oxidation. Also the microsomal benzene metabolism, which did not exhibit Michaelis-Menten kinetics, was effectively inhibited by six different hydroxyl radical scavengers. Biphenyl was formed in the reconstituted system, indicating the cytochrome P-450-dependent production of a hydroxycyclohexadienyl radical as a consequence of interactions between hydroxyl radicals and benzene. The formation of benzene metabolites covalently bound to protein was efficiently inhibited by radical scavengers but not by epoxide hydrolase. The results indicate that the microsomal cytochrome P-450-dependent oxidation of benzene is mediated by hydroxyl radicals formed in a modified Haber-Weiss reaction between hydrogen peroxide and superoxide anions and suggest that any cellular superoxide-generating system may be sufficient for the metabolic activation of benzene and structurally related compounds.     

Cytochrome p450 epoxygenase metabolism of arachidonic acid inhibits apoptosis

The ubiquitous cytochrome P450 hemoproteins play important functional roles in the metabolism and detoxification of foreign chemicals. However, other than established roles in cholesterol catabolism and steroid hormone biosynthesis, their cellular and/or organ physiological functions remain to be fully characterized. Here we show that the cytochrome P450 epoxygenase arachidonic acid metabolite 14,15-epoxyeicosatrienoic acid (14,15-EET) inhibits apoptosis induced by serum withdrawal, H(2)O(2), etoposide, or excess free arachidonic acid (AA), as determined by DNA laddering, Hoechst staining, and fluorescein isothiocyanate-labeled annexin V binding. In the stable transfectants (BM3 cells) expressing a mutant bacterial P450 AA epoxygenase, F87V BM3, which was genetically engineered to metabolize arachidonic acid only to 14,15-EET, AA did not induce apoptosis and protected against agonist-induced apoptosis. Ceramide assays demonstrated increased AA-induced ceramide production within 1 h and elevated ceramide levels for up to 48 h, the longest time tested, in empty-vector-transfected cells (Vector cells) but not in BM3 cells. Inhibition of cytochrome P450 activity by 17-octadecynoic acid restored AA-induced ceramide production in BM3 cells. Exogenous C2-ceramide markedly increased apoptosis in quiescent Vector cells as well as BM3 cells, and apoptosis was prevented by pretreatment of Vector cells with exogenous 14,15-EET and by pretreatment of BM3 cells with AA. The ceramide synthase inhibitor fumonisin B1 did not affect AA-induced ceramide production and apoptosis; in contrast, these effects of AA were blocked by the neutral sphingomyelinase inhibitor scyphostatin. The pan-caspase inhibitor Z-VAD-fmk had no effect on AA-induced ceramide generation but abolished AA-induced apoptosis. The antiapoptotic effects of 14,15-EET were blocked by two mechanistically and structurally distinct phosphatidylinositol-3 (PI-3) kinase inhibitors, wortmannin and LY294002, but not by the specific mitogen-activated protein kinase kinase inhibitor PD98059. Immunoprecipitation followed by an in vitro kinase assay revealed activation of Akt kinase within 10 min after 14,15-EET addition, which was completely abolished by either wortmannin or LY294002 pretreatment. In summary, the present studies demonstrated that 14,15-EET inhibits apoptosis by activation of a PI-3 kinase-Akt signaling pathway. Furthermore, cytochrome P450 epoxygenase promotes cell survival both by production of 14,15-EET and by metabolism of unesterified AA, thereby preventing activation of the neutral sphingomyelinase pathway and proapoptotic ceramide formation.     

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.     

Role of cytochrome P450 reductase in nitrofurantoin-induced redox cycling and cytotoxicity

The one-electron reduction of redox-active chemotherapeutic agents generates highly toxic radical anions and reactive oxygen intermediates (ROI). A major enzyme catalyzing this process is cytochrome P450 reductase. Because many tumor cells highly express this enzyme, redox cycling of chemotherapeutic agents in these cells may confer selective antitumor activity. Nitrofurantoin is a commonly used redox-active antibiotic that possesses antitumor activity. In the present studies we determined whether nitrofurantoin redox cycling is correlated with cytochrome P450 reductase activity and cytotoxicity in a variety of cell lines. Recombinant cytochrome P450 reductase was found to support redox cycling of nitrofurantoin and to generate superoxide anion, hydrogen peroxide, and, in the presence of redox-active iron, hydroxyl radicals. This activity was NADPH dependent and inhibitable by diphenyleneiodonium, indicating a requirement for the flavin cofactors in the reductase. Nitrofurantoin-induced redox cycling was next analyzed in different cell lines varying in cytochrome P450 reductase activity including Chinese hamster ovary cells (CHO-OR) constructed to overexpress the enzyme. Nitrofurantoin-induced hydrogen peroxide production was 16-fold greater in lysates from CHO-OR cells than from control CHO cells. A strong correlation between cytochrome P450 reductase activity and nitrofurantoin-induced redox cycling among the cell lines was found. Unexpectedly, no correlation between nitrofurantoin-induced ROI production and cytotoxicity was observed. These data indicate that nitrofurantoin-induced redox cycling and subsequent generation of ROI are not sufficient to mediate cytotoxicity and that cytochrome P450 reductase is not a determinant of sensitivity to redox-active chemotherapeutic agents.     

Oxygen reduction in chloroplast thylakoids results in production of hydrogen peroxide inside the membrane

Hydrogen peroxide production in isolated pea thylakoids was studied in the presence of cytochrome c to prevent disproportionation of superoxide radicals outside of the thylakoid membranes. The comparison of cytochrome c reduction with accompanying oxygen uptake revealed that hydrogen peroxide was produced within the thylakoid. The proportion of electrons from water oxidation participating in this hydrogen peroxide production increased with increasing light intensity, and at a light intensity of 630 micromol quanta m(-2) s(-1) it reached 60% of all electrons entering the electron transport chain. Neither the presence of a superoxide dismutase inhibitor, potassium cyanide or sodium azide, in the thylakoid suspension, nor unstacking of the thylakoids appreciably affected the partitioning of electrons to hydrogen peroxide production. Also, osmolarity-induced changes in the thylakoid lumen volume, as well as variation of the lumen pH induced by the presence of Gramicidin D, had negligible effects on such partitioning. The flow of electrons participating in lumen hydrogen peroxide production was found to be near 10% of the total electron flow from water. It is concluded that a considerable amount of hydrogen peroxide is generated inside thylakoid membranes, and a possible mechanism, as well as the significance, of this process are discussed.     

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.     

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.     

In vivo effect of diallyl sulfide and cimetidine on phenacetin metabolism and bioavailability in rat

Numerous cytochrome P450 inhibitors have been described as effective modulators of cytochrome P450 isoforms activity in vitro. Their inhibitory efficiency may be considerably modified after in vivo application. The aim of this study was to examine the effect of oral administration of diallyl sulfide--a cytochrome P450 2E1 inhibitor and cimetidine--a cytochrome P450 2C6 and 2C11 inhibitor on rat serum concentration of phenacetin and its metabolite acetaminophen. Both inhibitors increased area under the curve (AUC(0-4 h)) for phenacetin by 50%. Only cimetidine reduced AUC(0-4 h) for acetaminophen indicating inhibition of O-deethylation activity. Quinidine--a cytochrome P450 2D subfamily and P-glycoprotein inhibitor did not change significantly phenacetin bioavailability. These results suggest that diallyl sulfide inhibits the deacetylation pathway catalysed by arylamine N-acetyl transferase. Beside cytochrome P450 1A2 other cytochrome P450 isoforms (2A6 and/or 2C11) are involved in phenacetin O-deethylation in rat.     

Redox regulation of proliferation of lens epithelial cells in culture

Both oxidants and antioxidants have been shown to modulate cell proliferation. We studied the effects of hydrogen peroxide and two antioxidants on the rate of proliferation of lens epithelial cells in culture. Hydrogen peroxide at concentrations higher than 32 microM caused a significant inhibition of proliferation. However, in the concentration range of 0.01-0.5 microM, hydrogen peroxide stimulated the rate of proliferation. The effect of hydrogen peroxide was dependent on the amount of cells in an individual culture well, indicating decomposition of hydrogen peroxide by cellular enzymes. In order to eliminate the possibility of decomposition of the dose of hydrogen peroxide given as a bolus, we induced continual production of hydrogen peroxide by adding glucose oxidase to the incubation medium. We found that hydrogen peroxide, generated by 1-50 microU x ml(-1) of glucose oxidase significantly increased the rate of cell proliferation. This effect was most apparent at the beginning of the exponential phase of cellular growth. Glucose oxidase alone (100-500 microU x ml(-1)) did not produce any effect. The effects of pro-oxidative hydrogen peroxide were compared with the effects of two biologically important antioxidants, alpha-tocopherol and retinol. Both antioxidants completely inhibited proliferation at concentrations of 30 microM and higher. In contrast to retinol, the effect of alpha-tocopherol was dependent on the amount of cells, indicating cellular decomposition of alpha-tocopherol. The results document the possibility of redox regulation of cellular proliferation at physiologically relevant reactant concentrations.     

Reductive metabolism of nitroprusside in rat hepatocytes and human erythrocytes.

The metabolism of nitroprusside by hepatocytes or subcellular fractions involves a one-electron reduction of nitroprusside to the corresponding metal-nitroxyl radical. Thiol compounds also reduced nitroprusside to the metal-nitroxyl radical apparently via a thiol adduct. The nitroprusside reduction by microsomes was shown to be due to cytochrome P450 reductase as an antibody to cytochrome P450 reductase inhibits the microsomal reduction of nitroprusside, and the inhibitors of cytochrome P450 such as carbon monoxide or metyrapone had no effect. The reduction of nitroprusside by mitochondria in the presence of NADH or NADPH also produced the metal-nitroxyl radical. In hepatocytes, both mitochondria and the cytochrome P450 reductase are involved in the reduction of nitroprusside. The reductive metabolism of nitroprusside was found to produce toxic by-products, namely, free cyanide anion and hydrogen peroxide. We have also detected thiyl radicals formed in the thiol compound reduction of NP. We propose that cyanide and hydrogen peroxide are important toxic species formed in the metabolism of nitroprusside. The rate of reductive metabolism of nitroprusside by rat hepatocytes was much higher than with human erythrocytes. Therefore the major site of nitroprusside metabolism in vivo may be liver and not blood as originally proposed.     

Hydroxylation of Phenol to Hydroquinone Catalyzed by A Human Myeloperoxidase-Superoxide Complex: Possible Implications In Benzene-Induced Myelotoxicity

Benzene, a known human rnyelotoxin and leukemogen is metabolized by liver cytochrome P-450 mono-oxygenase to phenol. Further hydroxylation of phenol by cytochrome P-450 monooxygenase results in the formation of mainly hydroquinone, which accumulates in the bone marrow. Bone marrow contains high levels of myeloperoxidase. Here we report that phenol hydroxylation to hydroquinone is also catalyzed by human myeloperoxidase in the presence of a superoxide anion radical generating system, hypoxanthine and xanthine oxidase. No hydroquinone formation was detected in the absence of myeloperoxidase. At low concentrations superoxide disniutase stimulated, but at high concentrations inhibited, the conversion of phenol to hydroquinone. The inhibitory effect at high superoxide dismutase concentrations indicates that the active hydroxylating species of myeloperoxidase is not derived from its interaction with hydrogen peroxide. Furthermore, catalase a hydrogen peroxide scavenger, was found to have no significant effect on hydroxylation of phenol to hydroquinone, supporting the lack of hydrogen peroxide involvement. Mannitol (a hydroxyl radical scavenger) was found to have no inhibitory effect, but histidine (a singlet oxygen scavenger) inhibited hydroquinone formation. Based on these results we postulate that a myeloperoxidase-superoxide complex spontaneously rearranges to generate singlet oxygen and that this singlet oxygen is responsible for phenol hydroxylation to hydroquinone. These results also suggest that myeloperoxidase dependent hydroquinone formation could play a role in the production and accumulation of hydroquinone in bone marrow, the target organ of benzene-induced myelotoxicity.     

The use of 3-amino-1,2,4-triazole to investigate the short-term effects of oxygen toxicity on carbon assimilation by Pisum sativum seedlings

To study the in vivo short-term effect of hydrogen peroxide on plant metabolism, 2 mol m^?3 3-amino-1,2,4-triazole, a catalase inhibitor, was applied through the transpiration stream to Pisum sativum seedlings, and gas exchange characteristics, ascorbate peroxidase, glutathione reductase and catalase activities, and levels of hydrogen peroxide and formate were determined. Carbon dioxide assimilation rates were inhibited after the addition of aminotriazole: photorespiratory conditions exacerbated this inhibition. Carbon dioxide response curves showed that aminotriazole reduced both the RuBP regeneration rate and the efficiency of the carboxylation reaction of Rubisco. Catalase activity was completely inhibited 200 min after the application of this inhibitor, but no concomitant increase in H₂O₂ concentration was found. Under enhanced photorespiratory conditions, H₂O₂ concentrations increased. This suggests that under normal environmental conditions hydrogen peroxide is metabolized via alternative mechanisms. The aminotriazole treatment had no effect on the ascotbate peroxidase and glutathione reductase activities, but caused a substantial increase in the formate pool size. These results suggest that hydrogen peroxide is metabolized by reacting with glyoxylate to produce formate and CO₂. The increased production of formate may reduce the flow of carbon through the normal photorespiratory pathway and may also be used anaplerotically as a precursor of products of 1-C metabolism other than serine. This would prevent the return of photorespiratory carbon to the RPP pathway, leading to a smaller RuBP pool size which would in turn result in a decrease in carboxylation conductance (carboxylation efficiency) and regeneration rate of RuBP.     

Hydroxylation of Phenol to Hydroquinone Catalyzed by A Human Myeloperoxidase-Superoxide Complex: Possible Implications In Benzene-Induced Myelotoxicity

Benzene, a known human rnyelotoxin and leukemogen is metabolized by liver cytochrome P-450 mono-oxygenase to phenol. Further hydroxylation of phenol by cytochrome P-450 monooxygenase results in the formation of mainly hydroquinone, which accumulates in the bone marrow. Bone marrow contains high levels of myeloperoxidase. Here we report that phenol hydroxylation to hydroquinone is also catalyzed by human myeloperoxidase in the presence of a superoxide anion radical generating system, hypoxanthine and xanthine oxidase. No hydroquinone formation was detected in the absence of myeloperoxidase. At low concentrations superoxide disniutase stimulated, but at high concentrations inhibited, the conversion of phenol to hydroquinone. The inhibitory effect at high superoxide dismutase concentrations indicates that the active hydroxylating species of myeloperoxidase is not derived from its interaction with hydrogen peroxide. Furthermore, catalase a hydrogen peroxide scavenger, was found to have no significant effect on hydroxylation of phenol to hydroquinone, supporting the lack of hydrogen peroxide involvement. Mannitol (a hydroxyl radical scavenger) was found to have no inhibitory effect, but histidine (a singlet oxygen scavenger) inhibited hydroquinone formation. Based on these results we postulate that a myeloperoxidase-superoxide complex spontaneously rearranges to generate singlet oxygen and that this singlet oxygen is responsible for phenol hydroxylation to hydroquinone. These results also suggest that myeloperoxidase dependent hydroquinone formation could play a role in the production and accumulation of hydroquinone in bone marrow, the target organ of benzene-induced myelotoxicity.     

Simultaneous protective and damaging effects of cysteamine on intracellular DNA of leukocytes

Incubation of human leukocytes with cysteamine can lead to the induction of DNA strand breaks. The induction of breaks is biphasic with increasing concentration of scavenger. The number of breaks increases in a dose-dependent manner to a maximum and then decreases at higher concentrations. Catalase has been shown to prevent the production of breaks, indicating an involvement of hydrogen peroxide. Cysteamine reacts with oxygen to generate hydrogen peroxide but at higher concentrations it also reacts with hydrogen peroxide. Thus, the biphasic effect of cysteamine on leukocyte DNA may be due to the sum of two separate reaction pathways. (i) Cysteamine reacts with oxygen to generate hydrogen peroxide which leads to DNA strand breakage. (ii) At higher concentrations, it eliminates hydrogen peroxide by reacting with it, thereby protecting the cellular DNA. Other antioxidant scavengers such as WR2721, acetylcysteine and ascorbate can also autooxidize to produce strand breaks. Thiourea and tetramethylurea do not. When tested for their ability to protect cells against DNA damage from added H2O2, the agent which most damaging by itself, cysteamine, was also the most protective.