Metabolism of 1,8-cineole by human cytochrome P450 enzymes: identification of a new hydroxylated metabolite

Human metabolism of the monoterpene cyclic ether 1,8-cineole was investigated in vitro and in vivo. In vitro, the biotransformation of 1,8-cineole was investigated by human liver microsomes and by recombinant cytochrome P450 enzymes coexpressed with human CYP-reductase in Escherichia coli cells. Besides the already described metabolite 2alpha-hydroxy-1,8-cineole we found another metabolite produced at high rates. The structure was identified by a comparison of its mass spectrum and retention time with the reference compounds as 3alpha-hydroxy-1,8-cineole. There was a clear correlation between the concentration of the metabolites, incubation time and enzyme content, respectively. CYP3A4/5 antibody significantly inhibited the 2alpha- and 3alpha-hydroxylation catalyzed by pooled human liver microsomes. Further kinetic analysis revealed that the Michaelis-Menten K(m) and V(max) for oxidation of 1,8-cineole in position three were 19 microM and 64.5 nmol/min/nmol P450 for cytochrome P450 3A4, and 141 microM and 10.9 nmol/min/nmol P450 for cytochrome P450 3A5, respectively. To our knowledge, this is the first time that 3alpha-hydroxy-1,8-cineole is described as a human metabolite of 1,8-cineole. We confirmed these in vitro results by the investigation of human urine after the oral administration of cold medication containing 1,8-cineole. In human urine we found by GC-MS analysis the described metabolites, 2alpha-hydroxy-1,8-cineole and 3alpha-hydroxy-1,8-cineole.     

Induction of cytochrome P-450 and b5 of the mouse liver by phenytoin during chronic hypoxia (Fi02 : 0,08)]

During chronic hypoxia at sea level (FiO2 : 0,08) the liver concentrations of cytochrome P 450 and b5 decreased. Phenytoin administration induces an increase in cytochrome P 450 and b5 liver content. The hydroxylation of phenytoin that was decreased at low level of cytochrome P 450 and oxygen, reached again a normal level after induction of cytochrome P 450 in spite of the persistency of a low level of available oxygen. Liver cytochrome P 450 concentration is the main limiting factor of phenytoin hydroxylation.     

Catalysis of the oxidation of steroid and stilbene estrogens to estrogen quinone metabolites by the beta-naphthoflavone-inducible cytochrome P450 IA family.

Diethylstilbestrol (DES) or catecholestrogens are metabolized by microsomal enzymes to quinones, DES Q or catecholestrogen quinones, respectively, which have been shown to bind covalently to DNA and to undergo redox cycling. The isoforms of cytochrome P450 catalyzing this oxidation of estrogens to genotoxic intermediates were not known and have been identified in this study by (a) using microsomes of rats treated with various inducers of cytochrome P450; (b) using purified cytochrome P450 isoforms; and (c) examining the peroxide cofactor concentrations necessary for this oxidation by microsomes or pure isoenzymes. The highest rate of oxidation of DES to DES Q was obtained using beta-naphthoflavone-induced microsomes (14.0 nmol DES Q/mg protein/min) or cytochrome P450 IA1 (6.4 pmol DES Q/min/pmol P450). Isosafrole-induced microsomes or cytochrome P450 IA2 oxidized DES to quinone at one-third or one-fifth of that rate, respectively. Low or negligible rates of oxidation were measured when oxidations were catalyzed by microsomal rat liver enzymes induced by phenobarbital, ethanol, or pregnenolone-16 alpha-carbonitrile or by pure cytochromes P450 IIB1, IIB4, IIC3, IIC6, IIE1, IIE2, IIG1, or IIIA6. Cytochrome P450 IA1 also catalyzed the oxidation of 2- or 4-hydroxyestradiol to their corresponding quinones. The beta-naphthoflavone-induced microsomes and cytochrome P450 IA1 had the highest "affinity" for cumene hydroperoxide cofactor (Km = 77 microM). Cofactor concentrations above 250 microM resulted in decreased rates of oxidation. The other cytochrome P450 isoforms required much higher cofactor concentrations and were not inactivated at high cofactor concentrations. The data demonstrate that beta-naphthoflavone-inducible cytochrome P450 IA family enzymes catalyze most efficiently the oxidation of estrogenic hydroquinones to corresponding quinones. This oxidation may represent a detoxification pathway to keep organic hydroperoxides at minimal concentrations. The resulting quinone metabolites may be detoxified by other pathways. However, in cells with decreased detoxifying enzyme activities, quinones metabolites may accumulate and initiate carcinogenesis or cell death by covalent arylation of DNA or proteins.     

Rat liver microsomal enzyme catalyzed oxidation of 1-cyclopropyl-4-phenyl-1,2,3,6-tetrahydropyridine

NADPH supplemented rat liver microsomal enzyme preparations catalyze the conversion of 1-cyclopropyl-4-phenyl-1,2,3,6-tetrahydropyridine to the p-hydroxyphenyl (low yield), descyclopropyl (high yield) and 2,3-dihydropyridinium and, subsequently, pyridinium (intermediary yield) metabolites. When the methine proton of the cyclopropyl group was replaced with a deuteron, a normal deuterium isotope effect (1.4) was observed on the formation of the decyclopropylated metabolite and an inverse isotope effect (0.6) on the dihydropyridinium metabolite. A larger deuterium isotope effect (3.6) was observed on the ring -carbon oxidation pathway with the 2,2,6,6-d₄ analogue as substrate. These results and the observation that the ratios of the rates of these two -carbon oxidation pathways are independent of initial substrate concentrations suggest that both pathways are catalyzed by the same active site of one form of P450. These transformations are discussed in terms of metabolic pathways that have been proposed for the cytochrome P450 catalyzed -carbon oxidation of amines.     

Scoparone O-demethylase assay is not useful to differentiate the effects of model inducers of cytochrome P-450 in rabbit and guinea pig liver

Coumarin derivative, scoparone (6,7-dimethoxycoumarin), is regioselectively O-demethylated into isoscopoletin (I) and scopoletin (S). This oxidation is inversely influenced by cytochrome P-450 inducers in the rat such as 3-methylcholantrene (3-MC) and phenobarbital (PB). The I/S ratio is higher than 1.5 with 3-MC treatment whereas it is lower than 0.5 with PB treatment. With regards to this contrasting effect, it gas been suggested that the I/S ratio should be useful to differemtiate between the effects of these types of inducers. We studied the consequences of in vivo PB and 3-MC treatment on scoparone biotransformation in guinea pig and rabbit. In these two species, at the basal state, scoparone biotransformation was enhanced in comparison to the rat. Moreover, in these untreated animals, two other metabolites were formed. After 3-MC or PB treatment, scoparone metabolism is, in contrast to the rat, inappropriate to differentiate between the P-450 profile of other animals.     

Cyclopropylamine inactivation of cytochromes P450: role of metabolic intermediate complexes

The inactivation of cytochrome P450 enzymes by cyclopropylamines has been attributed to a mechanism involving initial one-electron oxidation at nitrogen followed by scission of the cyclopropane ring leading to covalent modification of the enzyme. Herein, we report that in liver microsomes N-cyclopropylbenzylamine (1) and related compounds inactivate P450 to a large extent via formation of metabolic intermediate complexes (MICs) in which a nitroso metabolite coordinates tightly to the heme iron, thereby preventing turnover. MIC formation from 1 does not occur in reconstituted P450 systems with CYP2B1/2, 2C11 or 2E1, or in microsomes exposed to gentle heating to inactivate the flavin-containing monooxygenase (FMO). In contrast, N-hydroxy-N-cyclopropylbenzylamine (3) and N-benzylhydroxylamine (4) generate MICs much faster than 1 in both reconstituted and microsomal systems. MIC formation from nitrone 5 (PhCH = N(O)cPr) is somewhat faster than from 1, but very much faster than the hydrolysis of 5 to a primary hydroxylamine. Thus the major overall route from 1 to a P450 MIC complex would appear to involve FMO oxidation to 3, further oxidation by P450 and/or FMO to nitrone 5' (C2H4C = N(O)CH2Ph), hydrolysis to 4, and P450 oxidation to alpha-nitrosotoluene as the precursor to oxime 2 and the major MIC from 1.     

Species difference in enantioselectivity for the oxidation of propranolol by cytochrome P450 2D enzymes

We examined and compared enantioselectivity in the oxidation of propranolol (PL) by liver microsomes from humans and Japanese monkeys (Macaca fuscata). PL was oxidized at the naphthalene ring to 4-hydroxypropranolol, 5-hydroxypropranolol and side chain N-desisopropylpropranolol by human liver microsomes with enantioselectivity of [R(+)>S(-)] in PL oxidation rates at substrate concentrations of 10 microM and 1 mM. In contrast, reversed enantioselectivity [R(+)相似文献   

Evidence for cytochrome P450-mediated oxidation of uroporphyrinogen by cell-free liver extracts from chick embryos treated with 3-methylcholanthrene

Cell-free preparations from liver of chick embryo treated with 3-methylcholanthrene catalyzed oxidation of uroporphyrinogen I in the presence of NADPH and 3,4,3',4'-tetrachlorobiphenyl. Extracts of untreated embryo liver or liver from embryo treated with glutethimide, a phenobarbital-like inducer of cytochrome P450 in this system, did not catalyse the oxidation. Direct involvement of cytochrome P450 was demonstrated by inhibition of the oxidation by CO, piperonyl butoxide and specific antisera to the methylcholanthrene-induced cytochrome P450. 2,4,2',4'-tetrachlorobiphenyl was inactive in the oxidation. These results may explain the role of induced cytochrome P450 in experimental uroporphyria. The oxidation may be useful as a simple assay for reactive O2 species.     

In silico pharmacogenetics of warfarin metabolism

Pharmacogenetic approaches can be instrumental for predicting individual differences in response to a therapeutic intervention. Here we used a recently developed murine haplotype-based computational method to identify a genetic factor regulating the metabolism of warfarin, a commonly prescribed anticoagulant with a narrow therapeutic index and a large variation in individual dosing. After quantification of warfarin and nine of its metabolites in plasma from 13 inbred mouse strains, we correlated strain-specific differences in 7-hydroxywarfarin accumulation with genetic variation within a chromosomal region encoding cytochrome P450 2C (Cyp2c) enzymes. This computational prediction was experimentally confirmed by showing that the rate-limiting step in biotransformation of warfarin to its 7-hydroxylated metabolite was inhibited by tolbutamide, a Cyp2c isoform-specific substrate, and that this transformation was mediated by expressed recombinant Cyp2c29. We show that genetic variants responsible for interindividual pharmacokinetic differences in drug metabolism can be identified by computational genetic analysis in mice.     

Biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a rabbit liver cytochrome P450: insight into the mechanism of RDX biodegradation by Rhodococcus sp. strain DN22

A unique metabolite with a molecular mass of 119 Da (C(2)H(5)N(3)O(3)) accumulated during biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Rhodococcus sp. strain DN22 (D. Fournier, A. Halasz, J. C. Spain, P. Fiurasek, and J. Hawari, Appl. Environ. Microbiol. 68:166-172, 2002). The structure of the molecule and the reactions that led to its synthesis were not known. In the present study, we produced and purified the unknown metabolite by biotransformation of RDX with Rhodococcus sp. strain DN22 and identified the molecule as 4-nitro-2,4-diazabutanal using nuclear magnetic resonance and elemental analyses. Furthermore, we tested the hypothesis that a cytochrome P450 enzyme was responsible for RDX biotransformation by strain DN22. A cytochrome P450 2B4 from rabbit liver catalyzed a very similar biotransformation of RDX to 4-nitro-2,4-diazabutanal. Both the cytochrome P450 2B4 and intact cells of Rhodococcus sp. strain DN22 catalyzed the release of two nitrite ions from each reacted RDX molecule. A comparative study of cytochrome P450 2B4 and Rhodococcus sp. strain DN22 revealed substantial similarities in the product distribution and inhibition by cytochrome P450 inhibitors. The experimental evidence led us to propose that cytochrome P450 2B4 can catalyze two single electron transfers to RDX, thereby causing double denitration, which leads to spontaneous hydrolytic ring cleavage and decomposition to produce 4-nitro-2,4-diazabutanal. Our results provide strong evidence that a cytochrome P450 enzyme is the key enzyme responsible for RDX biotransformation by Rhodococcus sp. strain DN22.     

Reduction of 7-alkoxyresorufins by NADPH-cytochrome P450 reductase and its differential effects on their O-dealkylation by rat liver microsomal cytochrome P450

Antibody-inhibition experiments established that the induction of cytochrome P450c is largely responsible for the marked increase in liver microsomal 7-ethoxyresorufin O-dealkylation in rats treated with 3-methylcholanthrene, whereas the induction of cytochrome P450b and/or P450e is largely responsible for the marked increase in 7-pentoxy- and 7-benzyloxyresorufin O-dealkylation in rats treated with phenobarbital. When reconstituted with NADPH-cytochrome P450 reductase and lipid, purified cytochrome P450c catalyzed the O-dealkylation of 7-ethoxyresorufin at a rate of approximately 30 nmol/nmol P450/min, which far exceeded the rate catalyzed by either purified cytochromes P450b and P450e or microsomal cytochrome P450c. In contrast, purified cytochrome P450b and P450e were poor catalysts of the O-dealkylation of 7-pentoxy- and 7-benzyloxyresorufin. However, purified cytochrome P450b is an excellent catalyst of several other reactions, such as the N-demethylation of benzphetamine, the hydroxylation of testosterone, and the O-dealkylation of 7-ethoxycoumarin. The low rate of 7-pentoxyresorufin O-dealkylation catalyzed by purified cytochrome P450b did not reflect a requirement for cytochrome b5, and could not be ascribed to an artifact of the method used to measure the formation of resourufin. The catalytic activity of purified cytochrome P450b toward 7-pentoxyresorufin was consistently low over a range of substrate and lipid concentrations, and was not stimulated by sodium deoxycholate (which stimulates the N-demethylation of benzphatamine by purified cytochrome P450b). Evidence is presented which indicates that cytochrome P450c catalyzes the O-dealkylation of both the oxidized and reduced forms of 7-ethoxyresorufin, with perhaps a slight preference for the reduced form. In contrast, cytochrome P450b preferentially catalyzes the O-dealkylation of the oxidized form of 7-pentoxyresorufin. Conditions that favored formation of the reduced form of 7-ethoxyresorufin tended to stimulate its O-dealkylation by purified cytochrome P450c, whereas conditions that favored formation of the reduced form of 7-pentoxyresorufin decreased its rate of O-dealkylation by purified cytochrome P450b. Such conditions included a molar excess of NADPH-cytochrome P450 reductase over cytochrome P450, the presence of superoxide dismutase, and the presence of DT-diaphorase (liver cytosol).(ABSTRACT TRUNCATED AT 400 WORDS)     

Reconstitution of testosterone oxidation by purified rat cytochrome P450p (IIIA1)

Cytochrome P450p (IIIA1) has been purified from rat liver microsomes by several investigators, but in all cases the purified protein, in contrast to other P450 enzymes, has not been catalytically active when reconstituted with NADPH-cytochrome P450 reductase and dilauroylphosphatidylcholine. We now report the successful reconstitution of testosterone oxidation by cytochrome P450p, which was purified from liver microsomes from troleandomycin-treated rats. The rate of testosterone oxidation was greatest when purified cytochrome P450p (50 pmol/ml) was reconstituted with a fivefold molar excess of NADPH-cytochrome P450 reductase, an equimolar amount of cytochrome b5, 200 micrograms/ml of a chloroform/methanol extract of microsomal lipid (which could not be substituted with dilauroylphosphatidylcholine), and the nonionic detergent, Emulgen 911 (50 micrograms/ml). Testosterone oxidation by cytochrome P450p was optimal at 200 mM potassium phosphate, pH 7.25. In addition to their final concentration, the order of addition of these components was found to influence the catalytic activity of cytochrome P450p. Under these experimental conditions, purified cytochrome P450p converted testosterone to four major and four minor metabolites at an overall rate of 18 nmol/nmol P450p/min (which is comparable to the rate of testosterone oxidation catalyzed by other purified forms of rat liver cytochrome P450). The four major metabolites were 6 beta-hydroxytestosterone (51%), 2 beta-hydroxytestosterone (18%), 15 beta-hydroxytestosterone (11%) and 6-dehydrotestosterone (10%). The four minor metabolites were 18-hydroxytestosterone (3%), 1 beta-hydroxytestosterone (3%), 16 beta-hydroxytestosterone (2%), and androstenedione (2%). With the exception of 16 beta-hydroxytestosterone and androstenedione, the conversion of testosterone to each of these metabolites was inhibited greater than 85% when liver microsomes from various sources were incubated with rabbit polyclonal antibody against cytochrome P450p. This antibody, which recognized two electrophoretically distinct proteins in liver microsomes from troleandomycin-treated rats, did not inhibit testosterone oxidation by cytochromes P450a, P450b, P450h, or P450m. The catalytic turnover of microsomal cytochrome P450p was estimated from the increase in testosterone oxidation and the apparent increase in cytochrome P450 concentration following treatment of liver microsomes from troleandomycin- or erythromycin-induced rats with potassium ferricyanide (which dissociates the cytochrome P450p-inducer complex). Based on this estimate, the catalytic turnover values for purified, reconstituted cytochrome P450p were 4.2 to 4.6 times greater than the rate catalyzed by microsomal cytochrome P450p.     

Production of three O-methhylated esculetins with Escherichia coli expressing O-methyltransferase from poplar

O-Methyltransferase, POMT-9 was expressed in Escherichia coli. HPLC analysis of reaction products revealed three peaks corresponding to isoscopoletin, scopoletin, and scoparone, and their structures were determined using NMR. Biotransformation of esculetin with E. coli expressing POMT-9 generated scopoletin, isoscopoletin, and scoparone at 30.3, 21, and 31 microM respectively. POMT-9 is the first O-methyltransferase that produces three different O-methylated products.     

Tolbutamide and phenytoin hydroxylations by cDNA-expressed human liver cytochrome P4502C9

A human cytochrome P4502C9 cDNA clone has been isolated from a human liver bacteriophage Lambda gt11 library using oligonucleotide probes. Expression of the 1762 base pair cDNA in COS cells demonstrated that the encoded enzyme has a molecular mass of 55 kDa as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The expressed enzyme catalysed the methylhydroxylation of tolbutamide with an apparent Km of 131.7 microM, similar to that observed in human liver microsomes. P4502C9 also catalysed the 4-hydroylation of phenytoin, and inhibition experiments demonstrated that phenytoin was a competitive inhibitor of tolbutamide hydroxylation with an apparent Ki of 19.1 microM. Sulphaphenazole was a potent inhibitor of the expressed enzyme with respect to both tolbutamide and phenytoin hydroxylations. These data demonstrate that a single isozyme can catalyse the hydroxylations of both tolbutamide and phenytoin, and suggest that both reactions are mediated by the same isozyme(s) of cytochrome P450 in human liver.     

Biotransformation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) by a Rabbit Liver Cytochrome P450: Insight into the Mechanism of RDX Biodegradation by Rhodococcus sp. Strain DN22

A unique metabolite with a molecular mass of 119 Da (C₂H₅N₃O₃) accumulated during biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Rhodococcus sp. strain DN22 (D. Fournier, A. Halasz, J. C. Spain, P. Fiurasek, and J. Hawari, Appl. Environ. Microbiol. 68:166-172, 2002). The structure of the molecule and the reactions that led to its synthesis were not known. In the present study, we produced and purified the unknown metabolite by biotransformation of RDX with Rhodococcus sp. strain DN22 and identified the molecule as 4-nitro-2,4-diazabutanal using nuclear magnetic resonance and elemental analyses. Furthermore, we tested the hypothesis that a cytochrome P450 enzyme was responsible for RDX biotransformation by strain DN22. A cytochrome P450 2B4 from rabbit liver catalyzed a very similar biotransformation of RDX to 4-nitro-2,4-diazabutanal. Both the cytochrome P450 2B4 and intact cells of Rhodococcus sp. strain DN22 catalyzed the release of two nitrite ions from each reacted RDX molecule. A comparative study of cytochrome P450 2B4 and Rhodococcus sp. strain DN22 revealed substantial similarities in the product distribution and inhibition by cytochrome P450 inhibitors. The experimental evidence led us to propose that cytochrome P450 2B4 can catalyze two single electron transfers to RDX, thereby causing double denitration, which leads to spontaneous hydrolytic ring cleavage and decomposition to produce 4-nitro-2,4-diazabutanal. Our results provide strong evidence that a cytochrome P450 enzyme is the key enzyme responsible for RDX biotransformation by Rhodococcus sp. strain DN22.     

Functional Coupling of ATP-binding Cassette Transporter Abcb6 to Cytochrome P450 Expression and Activity in Liver

Although endogenous mechanisms that negatively regulate cytochrome P450 (P450) monooxygenases in response to physiological and pathophysiological signals are not well understood, they are thought to result from alterations in the level of endogenous metabolites, involved in maintaining homeostasis. Here we show that homeostatic changes in hepatic metabolite profile in Abcb6 (mitochondrial ATP-binding cassette transporter B6) deficiency results in suppression of a specific subset of hepatic P450 activity. Abcb6 null mice are more susceptible to pentobarbital-induced sleep and zoxazolamine-induced paralysis, secondary to decreased expression and activity of Cyp3a11 and Cyp2b10. The knock-out mice also show decrease in both basal and xeno-inducible expression and activity of a subset of hepatic P450s that appear to be related to changes in hepatic metabolite profile. These data, together with the observation that liver extracts from Abcb6-deficient mice suppress P450 expression in human primary hepatocytes, suggest that this mouse model may provide an opportunity to understand the physiological signals and the mechanisms involved in negative regulation of P450s.     

Radical mechanism of aminopyrine oxidation by cumene hydroperoxide catalyzed by purified liver microsomal cytochrome P-450

Under identical experimental conditions, purified preparations of rabbit liver microsomal cytochrome P-450 and beef heart metmyoglobin were equally effective at stimulating the oxidation of aminopyrine to a free radical species by cumene hydroperoxide. Mannitol had no effect on radical levels produced with either hemeprotein-hydroperoxide system; however, specific ligands of the two hemeproteins, substrates of cytochrome P-450, and phospholipid affected the two systems quite differently. Only the metmyo-globindependent oxidation of aminopyrine was significantly inhibited by fluoride and cyanide. Metyrapone, a specific ligand of cytochrome P-450, and benzphetamine, which was N-demethylated by cumene hydroperoxide only in the presence of cytochrome P-450, inhibited only the cytochrome P-450-stimulated oxidation of aminopyrine. Moreover, only with the solubilized liver hemeprotein was aminopyrine radical generation markedly stimulated by phospholipid. Similar properties of aminopyrine N-demethylation and radical formation by the cytochrome P-450-cumene hydroperoxide system have strongly implicated the radical as a requisite intermediate in product formation. Micromolar concentrations of metyrapone caused parallel inhibition, by at least 50%, of both radical generation and formaldehyde production. These results support a radical pathway of N-demethylation proposed for other hemeprotein-hydroperoxide systems (B. W. Griffin and P. L. Ting, 1978, Biochemistry, 17, 2206–2211), in which the substrate undergoes two successive one-electron abstractions, followed by hydrolysis of the iminium cation intermediate. Thus, for this class of substrates, the experimental data are consistent with the oxygen atom of the product arising from H₂O and not directly from the hydroperoxide, which has been previously proposed as a general mechanism for cytochrome P-450 peroxidatic activities.     

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-.     

Role of cytochrome P450 in phospholipase A2- and arachidonic acid-mediated cytotoxicity

Phospholipases A2 (PLA2) comprise a set of extracellular and intracellular enzymes that catalyze the hydrolysis of the sn-2 fatty acyl bond of phospholipids to yield fatty acids and lysophospholipids. The PLA2 reaction is the primary pathway through which arachidonic acid (AA) is released from phospholipids. PLA2s have an important role in cellular death that occurs via necrosis or apoptosis. Several reports support the hypothesis that unesterified arachidonic acid in cells is a signal for the induction of apoptosis. However, most of the biological effects of arachidonic acid are attributable to its metabolism by mainly three different groups of enzymes: cytochromes P450, cyclooxygenases, and lipoxygenases. In this review we will focus on the role of cytochrome P450 in AA metabolism and toxicity. The major pathways of arachidonic acid metabolism catalyzed by cytochrome P450 generate metabolites that are subdivided into two groups: the epoxyeicosatrienoic acids, formed by CYP epoxygenases, and the arachidonic acid derivatives that are hydroxylated at or near the omega-terminus by CYP omega-oxidases. In addition, autoxidation of AA by cytochrome P450-derived reactive oxygen species produces lipid hydroperoxides as primary oxidation products. In some cellular models of toxicity, cytochrome P450 activity exacerbates PLA2- and AA-dependent injury, mainly through the production of oxygen radicals that promote lipid peroxidation or production of metabolites that alter Ca2+ homeostasis. In contrast, in other situations, cytochrome P450 metabolism of AA is protective, mainly by lowering levels of unesterified AA and by production of metabolites that activate antiapoptotic pathways. Several lines of evidence point to the combined action of phospholipase A2 and cytochrome P450 as central in the mechanism of cellular injury in several human diseases, such as alcoholic liver disease and myocardial reperfusion injury. Inhibition of specific PLA2 and cytochrome P450 isoforms may represent novel therapeutic strategies against these diseases.