Salicylic acid-induced hepatotoxicity triggered by oxidative stress

Salicylic acid is a widely used nonsteroidal anti-inflammatory drug (NSAID). But it is known to cause serious liver damage occasionally. Mitochondrial dysfunction and oxidative stress are predicted to be the major factors of salicylic acid-induced liver injury. We investigated the influence of salicylic acid on ATP contents, oxygen consumption and lipid peroxidation in the presence of the same concentration of salicylic acid. Leakage of lactate dehydrogenase (LDH) was significantly higher in the presence of 5 mM salicylic acid than in its absence. Salicylic acid-induced thiobarbituric acid-reactive substance (TBARS) formation and spontaneous chemiluminescence (CL) in rat hepatocytes, whereas antioxidants, such as promethazine (PMZ) and N,N-diphenylphenylenediamine (DPPD), suppressed both TBARS formation and LDH leakage. TBARS formation in rat liver microsomes was suppressed by diethyldithiocarbamate (a specific inhibitor of cytochrome P450 (CYP)2E1) and diclofenac (a specific inhibitor of CYP2C11). These results suggest that salicylic acid-induced lipid peroxidation was related to oxidative metabolism mediated by CYP2E1 and CYP2C11.On the other hand, 5 mM salicylic acid induced a drastic decrease of ATP contents in rat isolated hepatocytes. Furthermore, mitochondrial respiration control ratio (RC ratio), calculated by State 3/State 4 also decreased with the increase of salicylic acid concentration. These findings suggest that salicylic acid would trigger mitochondrial dysfunction and cause ATP decrease, leading to lethal liver cell injury by lipid peroxidation, although this hypothesis remains to be elucidated in vivo.     

Weak chemiluminescence of bilirubin and its stimulation by aldehydes

Bilirubin in an alkaline solution exhibits a weak chemiluminescence (CL) under aerobic conditions. This spontaneous CL was markedly enhanced by the addition of various aldehydes. The fluorescent emission spectrum of bilirubin, excited by weak intensity light at 350 nm, coincided with its CL emission spectrum (peak at 670 nm). CL emission from bilirubin was not quenched by active oxygen scavengers. This suggests that triplet oxygen reacts with bilirubin, and forms an oxygenated intermediate (hydroperoxide) as a primary emitter (oxidative scission of tetrapyrrole bonds in bilirubin is not involved in this CL). The Ehrlich reaction (test for monopyrroles) and hydrolsulphite reaction (test for dipyroles) on the CL reaction mixture and unreacted bilirubin showed no differences. When the CL was initiated by singlet oxygen, rather than superoxide anion, monopyrrole, was detected in the reaction products by gel chromatography. The inhibitory effect of a scavenger of singlet oxygen on CL was eliminated in the presence of formaldehyde. Therefore, triplet carbonyl, formed by singlet oxygen through the dioxetane structure in bilirubin, is not an emitter. The reaction mechanism of bilirubin CL and the formation of a hydroperoxide intermediate is discussed in relation to the chemical structure of luciferin molecules from bioluminescent organisms.     

Oxygen radical formation during cytochrome P450-catalyzed cyclosporine metabolism in rat and human liver microsomes at varying hydrogen ion concentrations

The role of pH in uncoupling the electron-flux between oxidoreductase and cytochrome P450 (P450) or P450 and cyclosporine (CyA) and resulting in the generation of oxygen radicals was investigatedin vitro in rat and human liver microsomal preparations. Since the electron-flux from NADPH to cytochrome c via oxidoreductase showed a fairly constant reduction activity from pH 7.0–9.5, the generation of oxygen radicals at the level of P450-Cyclosporine (instead of oxidoreductase-P450) was investigated. The effects of increasing pH on oxygen radical formation was measured by the thiobarbituric acid assay (TBA) and the adrenochrome reaction. The trends in oxygen radical production were correlated with benzphetamine metabolism (production of formaldehyde) and CyA metabolism (analyzed by high performance liquid chromatography). The TBA assay showed increased MDA-detected lipid peroxidation (unrelated to autooxidation) at pH<8.0 and pH>8.0 (rat and human, respectively) while the adrenochrome reaction showed decreased oxygen radical production. When these results were compared to benzphetamine (a substrate of P450 2B and 3A) metabolism and CyA (a substrate of P450 3A) metabolism, increased metabolism followed the pH-dependent trend of MDA-detected lipid peroxidation. Benzphetamine metabolism with formaldehyde production and depletion of parent compound during CyA metabolism were increased at pH<8.0 in the rat samples and at pH>8.0 in the human samples. This parallel relation suggests that the increased metabolism of CyA at lower pH in rats and higher pH in humans may be the result of favorable interactions of P450 with Cyclosporine that also result in increased oxygen radical-related lipid peroxidation.Abbreviations CCl₄ carbon tetrachloride - CyA cyclosporin A - EDTA ethylenediaminetetraacetic acid - HPLC high performance liquid chromatography - MDA malondialdehyde - MFO mixed function oxidase - MICROS microsomes - NADPH nicotinamide adenine dinucleotide phosphate - TBA thiobarbituric acid This work was supported by Grant No. CA-53191 from the National Cancer Institute DHHW     

Effect of nonsteroidal anti-inflammatory drugs on the microsomal monooxygenase system of rat liver

The effect of acetylsalicylic acid, ibuprofen, indomethacin, ketoprofen, naproxen, phenylbutazone, and salicylic acid on the microsomal oxidative drug metabolism of rat liver was studied. Pretreatment of the rats with pharmacologic doses of acetylsalicylic acid, indomethacin, and ketoprofen decreased both the demethylase and hydroxylase activities of rat liver microsomes. These effects were paralleled by decreases in microsomal cytochrome P-450 content. The rate of the microsomal reactions was increased after pretreatment with ibuprofen and naproxen but only the former increased the concentration of cytochrome P-450. Phenylbutazone and salicylic acid had no in vivo effect on the hepatic monooxygenase. The addition of 1 mM of ibuprofen, indomethacin, ketoprofen, naproxen, and phenylbutazone to rat liver microsomes inhibit both the aminopyrine N-demethylase and p-nitro-anisole O-demethylase activities. The extent of the inhibition varied between 21 and 73% of the control incubation. Indomethacin, naproxen, and phenylbutazone also decreased the aniline hydroxylase activity to roughly 60% of the control value. Acetylsalicylic acid and salicylic acid had no in vitro effect on the microsomal monooxygenase. The nonsteroidal anti-inflammatory drugs produced a reverse type I binding spectrum with oxidized cytochrome P-450; indomethacin and phenylbutazone were the strongest ligands. There is no correlation between the effect of addition of nonsteroidal anti-inflammatory drugs to the hepatic microsomal homogenate and their in vivo effect on the monooxygenase activity.     

HPLC study on Fenton-reaction initiated oxidation of salicylic acid. Biological relevance of the reaction in intestinal biotransformation of salicylic acid

Abstract

Fenton-reaction initiated in vitro oxidation and in vivo oxidative biotransformation of salicylic acid was investigated by HPLC-UV-Vis method. By means of the developed high performance liquid chromatography (HPLC) method salicylic acid, catechol, and all the possible monohydroxylated derivatives of salicylic acid can be separated. Fenton oxidations were performed in acidic medium (pH 3.0) with two reagent molar ratios: (1) salicylic acid: iron: hydrogen peroxide 1:3:1 and (2) 1:0.3:1. The incubation samples were analysed at different time points of the reactions. The biological effect of elevated reactive oxygen species concentration on the intestinal metabolism of salicylic acid was investigated by an experimental diabetic rat model. HPLC-MS analysis of the in vitro samples revealed presence of 2,3- and 2,5-dihydroxybenzoic acids. The results give evidence for nonenzyme catalysed intestinal hydroxylation of xenobiotics.     

Salicylate hydroxylation as an early marker of in vivo oxidative stress in diabetic patients.

In vivo metabolism of salicylic acid produces two main hydroxylated derivatives (2,5- and 2,3-dihydroxybenzoic acid). The former can be produced by enzymatic pathways through the cytochrome P-450 system, while the latter is reported to be solely formed by direct hydroxyl radical attack. Therefore, measurement of 2,3-dihydroxybenzoate, following oral administration of salicylate in its acetylated form (aspirin), has been proposed for assessment of oxidative stress. In this article we report plasma levels of 2,3- and 2,5-dihydroxybenzoates following the administration of 1 g aspirin and plasma levels of thiobarbituric acid-reactive material (TBARM) in well-controlled diabetic patients and in healthy subjects. 2,3-Dihydroxybenzoate levels were significantly higher (23%) in diabetic patients than in controls (63.4 +/- 20.1 versus 49.0 +/- 6.8 nM; p < .05). On the other hand, TBARM values were not significantly different between groups. These results suggest that the method is useful to reveal in vivo oxidative stress independently from the peroxidation of lipids, and they support the hypothesis that oxygen radicals are involved in the pathogenesis of chronic complications of diabetes.     

Defluorination of 4-fluorophenol by cytochrome P450BM3-F87G: activation by long chain fatty aldehydes

Cytochrome P450(BM3)-F87G catalyzed the oxidative defluorination of 4-fluorophenol, followed by reduction of the resulting benzoquinone to hydroquinone via the NADPH P450-reductase activity of the enzyme. The k (cat) and K (m) for this reaction were 71?±?5?min(-1) and 9.5?±?1.3?mM, respectively. Co-incubation of the reaction mixture with long chain aldehydes stimulated the defluorination reaction, with the 2,3-unsaturated aldehyde, 2-decenal producing a 12-fold increase in catalytic efficiency. At 150?μM aldehyde, k (cat) increased to 158?±?4, while K (m) decreased to 1.8?±?0.2. The effects of catalase, glutathione and ascorbate on the reaction were all consistent with a direct oxygen insertion mechanism, as opposed to a radical mechanism. The study demonstrates the potential use of P450(BM3) mutants in oxidative defluorination reactions, and characterizes the novel stimulatory action of straight chain aldehydes on this activity.     

Bromine derivatives of amino acids as intermediates in the peroxidase-catalyzed formation of singlet oxygen

Recently, J. R. Kanofsky et al. (1988, J. Biol. Chem. 263, 9692-9696) reported that human eosinophils generated modest amounts of singlet oxygen. In the mechanism proposed, hypobromous acid (made from the peroxidase-catalyzed oxidation of bromide ion) reacted with hydrogen peroxide to form singlet oxygen. In contrast, human neutrophils, which generate both hypochlorous acid and hydrogen peroxide, do not make singlet oxygen. The failure of human neutrophils to generate singlet oxygen is due in part to the trapping of hypochlorous acid by endogenous amines. In this paper, I show that amino acids are much more effective traps for hypochlorous acid than for hypobromous acid. Glycine totally inhibits singlet oxygen generation from a model enzyme system composed of chloroperoxidase, hydrogen peroxide, and chloride ion, but causes only a 35% reduction in singlet oxygen generation from an analogous enzyme system containing bromide ion instead of chloride ion. The products of the reaction of hypobromous and glycine (presumably an equilibrium mixture of N-bromoglycine, N,N-dibromoglycine, and hypobromous acid) retain the ability to react with hydrogen peroxide to form singlet oxygen. In contrast, the products of the reaction of hypochlorous acid and glycine do not react with hydrogen peroxide to produce singlet oxygen. Similar results were obtained for L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine, L-glutamic acid, L-glutamine, L-histidine, L-lysine, L-phenylalanine, L-proline, L-serine, and L-tyrosine. Thus, bromine derivatives of amino acids may act as intermediates in the peroxidase-catalyzed generation of singlet oxygen.     

Dimerumic acid protected oxidative stress-induced cytotoxicity in isolated rat hepatocytes

Dimerumic acid (DMA) is contained in Monascus anka and Monascus pilosus fermented products. The purpose of this study was to evaluate the effect of DMA against salicylic acid (SA)- and tert-butylhydroperoxide (t-BHP)-induced oxidative stress and cytotoxicity in the liver, using rat liver microsomes and isolated rat hepatocytes. DMA was extracted from monascus-garlic-fermented extract using M. pilosus. In rat liver microsomes, 1 microM DMA decreased SA-induced lipid peroxidation but did not affect the production of the oxidative metabolite of SA via CYP. In isolated rat hepatocytes, 1 microM DMA decreased SA-induced lipid peroxidation and chemiluminescence (CL) generation and the intracellular glutathione-reduced form/oxidized form (GSH/GSSG) ratio in the presence of 1 microM DMA was higher than that without DMA; however, 100 microM DMA suppressed the leakage of lactate dehydrogenase (LDH). On the other hand, t-BHP-induced lipid peroxidation, CL generation, and LDH leakage were prevented by 100 microM DMA. Thus, DMA showed an antioxidative effect in hepatocytes and protected against hepatotoxicity by suppressing oxidative stress without affecting CYP enzymes.     

The effect of valproic acid on hepatic and plasma levels of 15-F2t-isoprostane in rats

The mechanism by which valproic acid (VPA) induces liver injury remains unknown, but it is hypothesized to involve the generation of toxic metabolites and/or reactive oxygen species. This study's objectives were to determine the effect of VPA on plasma and hepatic levels of the F(2)-isoprostane, 15-F(2t)-IsoP, a marker for oxidative stress, and to investigate the influence of cytochrome P450- (P450-) mediated VPA biotransformation on 15-F(2t)-IsoP levels in rats. In rats treated with VPA (500 mg/kg), plasma 15-F(2t)-IsoP was increased 2.5-fold at t(max) = 0.5 h. Phenobarbital pretreatment (80 mg/kg/d for 4 d) in VPA-treated rats increased plasma and liver levels of free 15-F(2t)-IsoP by 5-fold and 3-fold, respectively, when compared to control groups. This was accompanied by an elevation in plasma and liver levels of P450-mediated VPA metabolites. Pretreatment with SKF-525A (80 mg/kg) or 1-aminobenzotriazole (100 mg/kg), which inhibited P450-mediated VPA metabolism, did not attenuate the increased levels of plasma 15-F(2t)-IsoP in VPA-treated groups. Plasma and hepatic levels of 15-F(2t)-IsoP were further elevated after 14 d of VPA treatment compared to single-dose treatment. Our data indicate that VPA increases plasma and hepatic levels of 15-F(2t)-IsoP and this effect can be enhanced by phenobarbital by a mechanism not involving P450-catalyzed VPA biotransformation.     

Production of singlet oxygen-derived hydroxyl radical adducts during merocyanine-540-mediated photosensitization: analysis by ESR-spin trapping and HPLC with electrochemical detection.

Activated oxygen species produced during merocyanine 540 (MC540)-mediated photosensitization have been examined by electron spin resonance (ESR) spin trapping and by trapping reactive intermediates with salicylic acid using HPLC with electrochemical detection (HPLC-EC) for product analysis. Visible light irradiation of MC540 associated with dilauroylphosphatidylcholine liposomes in the presence of the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) gave an ESR spectrum characteristic of the DMPO-hydroxyl radical spin adduct (DMPO/.OH). Addition of ethanol or methanol produced additional hyperfine splittings due to the respective hydroxyalkyl radical adducts, indicating the presence of free.OH.DMPO/.OH formation was not significantly inhibited by Desferal, catalase, or superoxide dismutase (SOD). Production of DMPO/.OH was strongly inhibited by azide and enhanced in samples prepared with deuterated phosphate buffer (PB-D2O), suggesting that singlet molecular oxygen (1O2) was an important intermediate. When MC540-treated liposomes were irradiated in the presence of salicylic acid (SA), HPLC-EC analysis indicated almost exclusive formation of 2,5-dihydroxybenzoic acid (2,5-DHBA), with production of very little 2,3-DHBA, in contrast to .OH generated by uv photolysis of H2O2, which gave nearly equimolar amounts of the two products. 2,5-DHBA production was enhanced in PB-D2O and inhibited by azide, again consistent with 1O2 intermediacy. 2,5-DHBA formation was significantly reduced in samples saturated with N2 or argon, and such samples showed no D2O enhancement. Ethanol had no effect on 2,5-DHBA production, even when present in large excess. Catalase and SOD also had no effect, and only a small inhibition was observed with Desferal. DMPO inhibited 2,5-DHBA production in a concentration-dependent fashion and enhanced formation of 2,3-DHBA. We propose that 1O2 reacts with DMPO to give an intermediate which decays to form DMPO/.OH and free.OH, and that the reaction between 1O2 and SA preferentially forms the 2,5-DHBA isomer. This latter process may provide the basis for a sensitive analytical method to detect 1O2 intermediacy. Singlet oxygen appears to be the principle activated oxygen species produced during MC540-mediated photosensitization.     

Effect of psoralens on Fenton-like reaction generating reactive oxygen species

Psoralens (psoralen, 5-methoxypsoralen, 8-methoxypsoralen, khellin, and visnagin) in 1 mM doses were shown to enhance the generation of reactive oxygen species, such as the hydroxyl radical (HO*), the superoxide anion radical (O2(-)), and singlet oxygen ((1)O(2)), from the system generating chemiluminescence (CL), as well as free radicals in the absence of light. The system that generated CL was made up of CoCl(2) and H(2)O(2). Incubation of psoralens in 0.2 mM doses with the generating system showed that only 8-methoxypsoralen and khellin have antioxidative effects. Antioxidative effects were also observed in the case of visnagin but in low concentration (0.05 mM). High doses of psoralens (1 mM) showed prooxidative effects. Measurements were done using a deoxyribose assay, the CL method, and spin-trapping with 5,5-dimethyl-1-pyrroline-N-oxide and 2,2,6,6-tetramethylpiperidine combined with electron spin resonance spectroscopy and spectrophotometry methods.     

Singlet oxygen production from the peroxidase-catalyzed oxidation of indole-3-acetic acid

The aerobic oxidation of indole-3-acetic acid catalyzed by horseradish peroxidase produces 1268 nm emission characteristic of singlet oxygen. Lactoperoxidase also oxidizes indole-3-acetic acid to produce singlet oxygen, but in contrast to horseradish peroxidase, this enzyme system requires hydrogen peroxide. In both of these systems, the intensity of the 1268 nm emission is small due to quenching of the singlet oxygen by indole-3-acetic acid and by reaction products derived from indole-3-acetic acid. The biomolecular reaction of peroxyl radicals via a Russell mechanism is a plausible mechanism for the singlet oxygen generation in these systems. Under typical conditions of p2H 4.0, 1 microM horseradish peroxidase, 1 mM indole-3-acetic acid, and 240 microM oxygen, the singlet oxygen yield was 15 +/- 1 microM or 13% of the amount predicted by the Russell mechanism.     

Bilirubin chemiluminescence induced by the attack of active oxygen species

Ultraweak chemiluminescence (CL) from bilirubin occurs in the presence of triplet oxygen and is stimulated by the addition of aldehydes. Active oxygen species also enhance bilirubin CL, in the absence of aldehydes. An inhibitory effect of active oxygen scavengers on the CL indicated that active oxygens generated from the decomposition of added hydrogen peroxide or from the xanthine-xanthine oxidase reaction contributed to the CL from bilirubin molecules. However, the contribution of singlet oxygen to the CL disappeared in the presence of formaldehyde. This suggested that the scission of tetrapyrrole bonds via a dioxetane intermediate or the production of triplet carbonyls from the oxidation of aldehydes by singlet oxygen was not involved in the CL, at least in the presence of formaldehyde. The spectrum of CL induced by the generation of active oxygen was the same as that from the aldehyde-enhanced CL reaction. We propose that the formation of a hydroperoxide (and/or hydroxide) bilirubin intermediate, but not a dioxetane, may be involved in the excitation of bilirubin molecules for CL.     

The role of superoxide and singlet oxygen in lipid peroxidation promoted by xanthine oxidase

The peroxidative oxidation of extracted rat liver microsomal lipid, assayed as malondialdehyde production, can be promoted by milk xanthine oxidase in the presence of 0.2 mM FeCl₃ and 0.1 mM EDTA. The reaction is inhibited by the superoxide dismutase activity of erythrocuprein. The reaction is also inhibited by 1,3-diphenylisobenzofuran, which reacts with singlet oxygen to yield dibenzoylbenzene. During inhibition of the lipid peroxidation reaction by 1,3-diphenylisobenzofuran, o-dibenzoylbenzene was produced. The rate of superoxide production by xanthine oxidase was not affected by 1,3-diphenylisobenzofuran. Lipid peroxidation promoted by ascorbic acid is not inhibited by either erythrocuprein or 1,3-diphenylisobenzofuran. Therefore it is suggested that the peroxidative oxidation of unsaturated lipid promoted by xanthine oxidase involves the formation of singlet oxygen from superoxide, and the singlet oxygen reacts with the lipid to form fatty acid hydroperoxides.     

Antioxidant activities of dihydroxylated salicylic acid derivatives

SUMMARY

The ability of hydroxylated metabolites of salicylic acid to scavenge reactive oxygen species and to inhibit arachidonic acid metabolism was investigated. The tested trihydroxybenzoic acids (THBAs) were potent scavengers of hydroxyl and superoxide anion radicals produced by Fenton reaction and xanthine/xanthine oxidase system or activated macrophages respectively. In the same tests, salicylic acid possessed moderate O₂^? and low OH'scavenging activities.

Our results demonstrate that adding two hydroxyl groups to salicylic acid strongly increases the reactive oxygen species (ROS) scavenging activities. Adding two hydroxyl groups at position 4 and 5 (2,4,5-THBA) affords the most active ROS scavenging activity probably due to the ortho unsubstituted catechol moiety. In fact, we can consider that the ROS scavenging properties of salicylic acid are essentially due to its metabolic products such as 2,3- and 2,5-DHBAs, catechol and also to THBAs.     

Chemiluminescence Emission during Reactions between Superoxide and Selected Aliphatic and Aromatic Halocarbons in Aprotic Media

The reactions between superoxide free radical anion (.O⁻₂) with the halocarbons CCl₄, CHCl₃, BrCH₂CH₂Br(EDB), decachloro-biphenyl (DCBP), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in dimethyl sulphoxide (DMSO) results in the emission of chemiluminescence (CL). The chemiluminescence reactions are characterized as having biphasic second order kinetics, CL wavelengths between 350 nm and 650 nm, and exhibiting perturbation by chemicals reactive with singlet oxygen. These data suggest that singlet oxygen species are the excited state responsible for the light emissions. Polarographic studies confirm .O⁻₂ consumption and halide release in the reactions, while gas liquid chromatography and NBT reduction demonstrate the decomposition of the halocarbons into products. A chemiluminescent reaction mechanism is proposed involving reductive dehalogenation of the halocarbons and the generation of singlet oxygen. The significance of singlet oxygen generation is discussed with respect to a general mechanism for explaining the rapid initiation of lipid peroxidative membrane damage in halocarbon toxigenicity in animal and plant tissues.     

A chemiluminescence assay specific for the microsomal metabolite, benzo[a]pyrene-7,8-dihydrodiol

It is possible to assay for trans-7,8-dihydroxy 7,8-dihydrobenzo[a]-pyrene (BP-7,8-dihydrodiol) in complex metabolite mixtures produced during microsomal metabolism of benzo[a]pyrene (BP) because only the BP-7,8-dihydrodiol metabolite will produce significant chemiluminescence (CL) in the NaOCl-H2O2 singlet oxygen-generating system. The limiting CL sensitivity is 30 pmol in a 1-ml CL reaction mixture. CL assays for BP-7,8-dihydrodiol in microsomal reaction solutions gave concentrations identical with those determined by calibrated high-performance liquid chromatography.     

Determination of salicylate hydroxylation products as an in vivo oxidative stress marker

The in vivo measurement of highly reactive free radicals, such as the z.rad OH radical, is very difficult. New specific markers, which are based on the ability of z.rad OH to attack the benzene rings of aromatic molecules, are currently under investigation. The produced hydroxylated compounds can be measured directly. In vivo, radical metabolism of salicylic acid produces two main hydroxylated derivatives (2,3- and 2,5-dihydroxybenzoic acids). The latter acid can be also produced by enzymatic pathways through the cytochrome P-450 system, while the former acid is reported to be solely formed by direct hydroxyl radical attack. Therefore, measurement of 2, 3-DHBA, following oral administration of the drug acetyl salicylate, could be proposed for assessment of oxidative stress in vivo. In this paper, a sensitive method for the identification and quantification of hydroxylation products from the reaction of z. rad OH with salicylate in vivo is presented. It employs a high performance liquid chromatography and electrochemical detection system. A detection limit of < 1 pmol for the hydroxylation products has been achieved with linear response over at least five orders of magnitude. Using this technique, we measured plasma levels of 2,3- and 2,5-DHBA dihydroxylated derivatives and salicylic acid and determined the ratios following administration of 1 g acetyl salicylate in 20 healthy subjects.     

Metabolism of retinol and retinoic acid by human liver cytochrome P450IIC8

Liver microsomes obtained from nine subjects were found to metabolize retinol to polar metabolites, including 4-hydroxyretinol. In a reconstituted monooxygenase system containing human liver P450IIC8, retinol was converted to 4-hydroxyretinol and other polar metabolites, with a Km of 0.071 mM and a Vmax of 1.73 nmol/min/nmol P450. Neither P450IIC9 nor P450IIE1, two other purified human P450s, displayed significant retinol hydroxylase activity. Immunoblots performed with a monospecific antibody directed against human P450IIC8 revealed that appreciable amounts of this enzyme were present in human liver microsomes. The same antibody significantly inhibited retinol metabolism in liver microsomes and in the system reconstituted with P450IIC8. The system reconstituted with P450IIC8 also converted retinoic acid to polar metabolites. Thus, this study shows, for the first time, metabolism of two physiologic substrates by a human liver cytochrome P450 related to a group of "constitutive" rodent P450s believed to participate in the metabolism of endogenous compounds. Through its involvement in vitamin A metabolism, P450IIC8 may participate in maintaining the balance between those vitamin A concentrations that promote cellular integrity (and oppose the development of cancer) and those concentrations that cause cellular toxicity.