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PubMed1994

Hydroxylation of phenylalanine and salicylate by stimulated polymorphonuclear leukocytes and the accelerating effect of glutathione on their hydroxylation.

Incubation of phorbol-myristate acetate-stimulated human polymorphonuclear leukocytes (PMNs) with phenylalanine and salicylate induced significant levels of formation of o- and m-tyrosines, and 2,3- and 2,5-dihydroxybenzoates (DHBAs), respectively, dependent on reaction time. Aromatic hydroxylation reactions were not inhibited by desferrioxamine, nor were they affected by the removal of trace ion contamination from the buffer solution used by treatment with conalbumin. Hydroxylation reactions were largely blocked by superoxide dismutase and hydroxyl radical (OH.) scavengers. The results of the present study suggest that the generation of OH. by human PMNs occurs during the respiratory burst. Hydroxylation of both phenylalanine and salicylate by stimulated human PMNs were significantly accelerated by incubation in the presence of the reduced form of glutathione (GSH). Hydroxylation of phenylalanine by stimulated guinea pig PMNs in the presence of GSH was significantly inhibited by desferrioxamine, although the same hydroxylation in the absence of GSH was not affected. Hydroxylation of phenylalanine by the hypoxanthine (HX)-xanthine oxidase (XO) system by intact PMNs was significantly accelerated by the addition of GSH, although that in the absence of PMNs was largely inhibited. Desferrioxamine showed an inhibitory effect on hydroxylation by the HX-XO system in the presence, but not in the absence, of intact PMNs. The results suggest that the formation of OH. by stimulated PMNs is accelerated by GSH, based on the occurrence of the Harber-Weiss reaction catalyzed by transition metal ions liberated and reduced by GSH from PMNs, and by the effective accumulation of H2O2 by the GSH-induced inhibition of catalase.

Animals↗
PubMed1994

Induction of tamoxifen-4-hydroxylation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), beta-naphthoflavone (beta NF), and phenobarbital (PB) in avian liver: identification of P450 TCDDAA as catalyst of 4-hydroxylation induced by TCDD and beta NF.

Tamoxifen has been found to be metabolized by liver primarily into three metabolites, tamoxifen-N-oxide, formed by the flavin-containing monooxygenase, and N-desmethyl- and 4-hydroxytamoxifen, formed by cytochrome P450. The N-demethylation was demonstrated to be catalyzed by P4503A in rat and human liver; however, the P450s catalyzing the 4-hydroxylation have not been identified. Although 4-hydroxytamoxifen exhibits more potent estrogen agonist/antagonist activity than tamoxifen, the relative contributions of the parent drug and its 4-hydroxy metabolite(s) to the activity of tamoxifen in vivo have not been established. We report here that the rate of tamoxifen 4-hydroxylation is higher in livers of adult chicken and chick embryos than in livers of mammalian species. Tamoxifen 4-hydroxylation was increased by treatment of chick embryos with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), beta-naphthoflavone (beta NF), and to a lesser extent by phenobarbital (PB). The major effect of PB treatment was an increase in tamoxifen N-demethylation. Tamoxifen 4-hydroxylase activity of reconstituted purified chicken P450s was highest for TCDDAA, a P450 active in arachidonate epoxygenation and estradiol 2-hydroxylation, and one of the two major P450s induced by TCDD and beta NF in chick embryo liver. The second P450, TCDDAHH, which is active in aryl hydrocarbon hydroxylase and 7-ethoxyresorufin deethylase was inactive in tamoxifen 4-hydroxylation. Anti-TCDDAA IgG immunoinhibited tamoxifen 4-hydroxylation in microsomes from beta NF-treated embryos by over 80%, but was ineffective against this reaction in the controls. The immunochemical findings together with the reconstitution data identify TCDDAA as the P450 responsible for TCDD/beta NF-induced tamoxifen 4-hydroxylation in chick liver. In PB-treated livers, a P450 fraction containing CYP2H1/H2, the major PB-induced P450s, had the highest tamoxifen 4-hydroxylase and N-demethylase activities, a finding compatible with one or both of those P450s being responsible for the PB-induced tamoxifen 4-hydroxylation and N-demethylation. The findings reported here raise the possibility that exposure of women undergoing tamoxifen therapy to agents that induce human CYP1A2 or CYPB1/2 analogues may produce increased levels of 4-hydroxytamoxifen and that this may affect the therapeutic potency of tamoxifen.

Animals↗
PubMed1976

Side chain hydroxylations in biosynthesis of cholic acid. 25- and 26-Hydroxylation of 5beta-cholestane-3alpha, 7alpha, 12alpha-triol by reconstituted systems from rat liver microsomes.

25- and 26-Hydroxylation of 5beta-cholestane-3alpha, 7alpha, 12alpha-triol was studied with reconstituted systems from rat liver microsomes consisting of partially purified cytochrome P -450, NADPH-cytochrome P -450 reductase, a phospholipid, and an NADPH -generating system. Cytochrome P -450 was prepared either by sodium cholate treatment and ammonium sulfate fractionation or by subtilisin and sodium deoxycholate treatment followed by DEAE-cellulose chromatography. No side chain hydroxylation was observed when cytochrome P-450 was omitted. With ammonium sulfate-fractionated cytochrome P-450 25- and 26-hydroxylation was stimulated 5- to 8-fold by addition of NADPH-cytochrome P-450 reductase. With subtilisin-treated cytochrome P-450 an almost absolute requirement for NADPH-cytochrome P-450 reductase was observed. Omission of lipid did not reduce the rate of hydroxylation. Centrifugation of the cytochrome P-450 preparation at 100,000 X g for 1 hour just before incubation increased markedly lipid dependency. A significant difference between 25- and 26-hydroxylation was observed with respect to substrate saturation. The stimulatory effect of phenobarbital treatment on 25-hydroxylation and the inhibitory effect of this treatment on 26-hydroxylation were associated with the cytochrome P-450 fraction. The use of increasing amounts of sodium cholate in the solubilization of cytochrome P -450 resulted in a gradual decrease of 25-hydroxylase activity and a gradual increase of 26-hydroxylase activity. 25- and 26-Hydroxylase activities were separated partially by chromatography of subtilisintreated cytochrome P-450 fraction on DEAE-cellulose. The question whether different species of cytochrome P-450 are involved in 25- and 26-hydroxylation is discussed.

Animals↗
PubMed2000

The salicylate hydroxylation assay to measure hydroxyl free radicals induced by local application of glutamate in vivo or induced by the Fenton reaction in vitro.

The direct measurement of hydroxyl radicals in vivo is extremely difficult. Therefore, the indirect determination of hydroxyl radicals using salicylate (2-hydroxybenzoate) is widely accepted. Reverse microdialysis with glutamate led to a dose-dependent production of hydroxyl free radicals indicated by the hydroxylation adduct of salicylate, namely 2,3-dihydroxybenzoic acid. The local stimulation of hydroxyl free radical formation seems to be suitable to investigate a radical-scavenging property of potential neuroprotective drugs. In vitro experiments using the Fenton reaction may be a helpful tool to assess whether or not a substance is able to act as a radical scavenger in a cell free environment, which is easy to handle and a simple screening method before in vivo experiments were performed. In the present study we present an in vivo approach using local application of glutamate into the striatum and an in vitro screening using the Fenton reaction to induce hydroxyl radical formation. The main goal is to reliable measure hydroxyl free radicals, which are the most reactive oxygen radicals in biology and medicine.

Animals↗
PubMed1997

Peroxynitrite-dependent aromatic hydroxylation and nitration of salicylate and phenylalanine. Is hydroxyl radical involved?

There is considerable dispute about whether the hydroxylating ability of peroxynitrite (ONOO-)- derived species involves hydroxyl radicals (OH.). This was investigated by using salicylate and phenylalanine, attack of OH. upon which leads to the formation of 2,3- and 2,5-dihydroxybenzoates, and o- m- and p- tyrosines respectively. On addition of ONOO- to salicylate, characteristic products of hydroxylation (and nitration) were observed in decreasing amounts with rise in pH, although added products of hydroxylation of salicylate were not recovered quantitatively at pH 8.5, suggesting further oxidation of these products and underestimating of hydroxylation at alkaline pH. Hydroxylation products decreased in the presence of several OH. scavengers, especially formate, to extents similar to those obtained when hydroxylation was achieved by a mixture of iron salts, H2O2 and ascorbate. However, OH. scavengers also inhibited formation of salicylate nitration products. Ortho, p- and m-tyrosines as well as nitration products were also observed when ONOO- was added to phenylalanine. The amount of these products again decreased at high pH and were decreased by addition of OH. scavengers. We conclude that although comparison with Fenton systems suggests OH. formation, simple homolytic fission of peroxynitrous acid (ONOOH) to OH. and NO2. would not explain why OH. scavengers inhibit formation of nitration products.

Chromatography, High Pressure Liquid↗
PubMed2002

In vivo production of hydroxyl radical by Enterococcus faecalis colonizing the intestinal tract using aromatic hydroxylation.

Enterococcus faecalis is an intestinal commensal that produces extracellular superoxide (O(2)(*-)) through autoxidation of membrane-associated demethylmenaquinone. To assess free radical production by E. faecalis in vivo, intestinal tracts of rats were colonized using wild-type E. faecalis or a mutant strain with attenuated O(2)(*-) production. Ex vivo electron paramagnetic resonance spin trapping study of colonic contents (mean +/- SD) showed 1.4 +/- 1.5 and 0.094 +/- 0.24 microM 5,5-dimethyl-1-pyrroline-N-oxide-hydroxyl radical adduct/gm stool for rats colonized with wild-type and mutant strains, respectively (p = .002). In vivo hydroxyl radical production was further assayed by aromatic hydroxylation using phenyl N-tert-butylnitrone (PBN) and D-phenylalanine. Hydroxylated PBN and D-phenylalanine products were recovered from stool (microM/gm colonic contents/10(9) colony forming units) and urine (microM/h/ml), respectively, and quantified using electrochemical detection. Hydroxylated (OH) PBNs and isomeric tyrosines (hydroxylated phenylalanine) were significantly increased (mean +/- SD) for rats colonized with wild-type E. faecalis (2-OH PBN, 63 +/- 58; 3-OH PBN, 63 +/- 84; ortho-tyrosine, 31 +/- 27; meta-tyrosine, 17 +/- 14) compared to the mutant strain (2-OH PBN, 2.5 +/- 7.3 (p < .001); 3-OH PBN, 3.9 +/- 12.3 (p = .01); ortho-tyrosine, 1.9 +/- 6.0 (p < .001); meta-tyrosine, 1.5 +/- 3.4 (p = .03)). Similar differences were observed following in vitro incubations of these bacteria with aromatic targets. These results confirm in vivo production of hydroxyl radical by E. faecalis colonizing the intestine, and indicate this bacterium may be a potent source of oxidative stress on the intestinal epithelium.

Animals↗
PubMed1981

E- and Z-10-hydroxylation of nortriptyline: relationship to polymorphic debrisoquine hydroxylation.

Eight healthy subjects [who were phenotyped with a debrisoquine (D) hydroxylation test] were selected to cover a wide range in the ratio between D and 4-hydroxydebrisoquine (4-OH-D) in the urine. After a single oral dose of nortriptyline (NT) the metabolic clearance by 10-hydroxylation in the E-position, but not in the Z-position, correlated closely to the metabolic ratio D/4-OH-D (rs = -0.88, p less than 0.01). This indicates that common enzymatic mechanisms are involved in the hydroxylation of D and the E- but not the Z-10-hydroxylation of NT. Slow hydroxylators of NT and D excreted less 10-hydroxynortriptyline in urine and had lower plasma clearance of NT than the rapid hydroxylators. The strong correlation (r = 0.96) between the total plasma clearance of NT and the metabolic clearance by E-10-hydroxylation shows that this metabolic reaction is important in the disposition of the drug.

Debrisoquin↗
PubMed1993

Selective inhibition of mitochondrial 27-hydroxylation of bile acid intermediates and 25-hydroxylation of vitamin D3 by cyclosporin A.

It was demonstrated recently that cyclosporin A blocks bile acid synthesis in cultured rat and human hepatocytes by specific inhibition of chenodeoxycholic acid formation. The site of inhibition was found to be the 27-hydroxylation of cholesterol catalysed by the liver mitochondrial 27-hydroxylase [Princen, Meijer, Wolthers, Vonk and Kuipers (1991) Biochem J. 275, 501-505]. In this paper the mechanism by which cyclosporin A blocks mitochondrial 27-hydroxylation was further investigated. It is shown that cyclosporin A inhibited 27-hydroxylation of bile acid intermediates, depending on their polarity. In isolated rat liver mitochondria, 27-hydroxylation of cholesterol was dose-dependently blocked by the drug, giving half-maximal inhibition at 4 microM, whereas 27-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol was not affected. A similar observation was made using electrophoretically homogeneous cytochrome P-450(27) isolated from rabbit liver mitochondria, excluding the possibility that cyclosporin A interfered with transport of substrates into the mitochondrion. Kinetic studies showed that inhibition of the 27-hydroxylation of cholesterol by cyclosporin A was of a non-competitive type. The drug also inhibited the 25-hydroxylase activity towards vitamin D3, catalysed by the same enzyme preparation, to the same extent as 27-hydroxylation of cholesterol. These results suggest that cyclosporin A may interfere with binding of cholesterol, but not of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, to the active site of the enzyme. These data provide an explanation for the selective inhibition of chenodeoxycholic acid synthesis.

Animals↗
PubMed1988

Immunochemical evidence for the catalysis of vitamin D3 25-hydroxylation and testosterone 16 alpha-hydroxylation by homologous forms of cytochrome P-450 in rat liver microsomes.

Polyclonal antibody elicited in a rabbit against purified cytochrome P-450cc25, which catalyzes 25-hydroxylation of vitamin D3, inhibited not only 25-hydroxylation of cholecalciferol and 1 alpha-hydroxycholecalciferol, but also 16 alpha- and 2 alpha-hydroxylation of testosterone catalyzed by the purified P-450cc25 preparation. Antibody inhibition experiments with microsomes revealed that most 16 alpha- and 2 alpha-hydroxylation of testosterone and most 25-hydroxylation of cholecalciferol by male rat liver microsomes were catalyzed by P-450cc25. In order to examine the identity of cholecalciferol 25-hydroxylase and testosterone 16 alpha-hydroxylase, monoclonal antibodies recognizing three different epitopes of P-450cc25 were prepared from hybridoma clones produced by fusion of mouse myeloma cells (P3X63Ag8U1) with the spleen cells of immunized BALB/c mouse. All of these monoclonal antibodies inhibited both 25-hydroxylation of 1 alpha-hydroxycholecalciferol and 16 alpha-hydroxylation of testosterone by purified P-450cc25. These observations suggested that immunochemically indistinguishable form(s) of cytochrome P-450 catalyzed both reactions.

Animals↗
PubMed1996

Effect of plant phenolics on the formation of the spin-adduct of hydroxyl radical and the DNA strand breaking by hydroxyl radical.

The effect of plant phenolics, including flavonoids and green tea polyphenolics, on hydroxyl radical was examined by a common method using an electron spin resonance (ESR) technique with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trapping agent. The intensity of the ESR signals of DMPO-OH adduct formed by the interaction of DMPO with Fenton reagent was reduced in the presence of each phenolic in a dose-dependent manner. However, the decrease in the intensity of the signals was due partly to the enhanced disappearance of the spin adduct by the phenolics, as has been previously shown. This spin trapping method was unreliable for evaluation of the effect of the phenolics against hydroxyl radical. Hydroxyl radical induced-DNA single-strand breaks may be a better index for evaluation of the activity of the phenolics regarding hydroxyl radical. The effect of the phenolics on DNA single-strand breaks induced by Fenton reagent was examined. While sesamol and esculetin were inhibitory, most polyphenolics, especially (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCG), were rather stimulatory. The results indicate that sesamol and esculetin scavenged hydroxyl radical, and EGC and EGCG generated hydroxyl radical under the conditions where hydroxyl radical was generating.

Catechin↗
PubMed2005

Mechanistic pathways of the hydroxyl radical reactions of quinoline. 1. Identification, distribution, and yields of hydroxylated products.

The mechanistic details of the hydroxyl radical-induced transformations of quinoline have been elucidated. The nature and distribution of the final products have provided insight into the preferential attack of the hydroxyl radicals at different sites on the aromatic rings. Hydroxylated products at all of the carbon atoms but one, C2, have been observed and quantified following controlled radiolysis of N2O-purged aqueous quinoline solutions. The difference in the growth pattern and the lifetime of the monohydroxylated products under radiolytic conditions, as well as the formation of high-molecular-weight products (e.g., quinoline dimers), shows the complexity of the OH reaction pathways. The radiolytic yields (G values) for the degradation of the quinoline and the formation of the hydroxylated products are calculated in the absence and in the presence of an oxidant, K3Fe(CN)6. The addition of K3Fe(CN)6 changes only the distribution of the hydroxylated products. These experiments indicate that the nature of the hydroxylated products is determined in the initial addition step of the reaction of the hydroxyl radical with quinoline, whereas the chemistry of the OH adducts is relevant to the distribution of the final products. The discrepancy between the products of -radiolysis and the photo-Fenton reaction of quinoline is also discussed.

Journal Article↗
PubMed2001

Fe(II)-EDTA chelate-induced aromatic hydroxylation of terephthalate as a new method for the evaluation of hydroxyl radical-scavenging ability.

The investigation of Fe(II)-EDTA chelate-induced aromatic hydroxylation of terephthalate in pH 7.4 phosphate buffer solution and a new method for the evaluation of hydroxyl radical-scavenging ability are reported. The method is based on attack of the hydroxyl radical on the terephthalate to produce highly fluorescent 2-hydroxyterephthalate, which is detected fluorimetrically. The formation of hydroxyl radical is believed to be the result of the reduction of molecular oxygen by Fe(II)-EDTA to form superoxide radical, which in turn dismutates to hydrogen peroxide, and then Fe(II)-EDTA catalyzes the decomposition of hydrogen peroxide to produce hydroxyl radical. The mechanism of the generation of hydroxyl radical in the proposed system was confirmed. This study established a simple and inexpensive method for the evaluation of the scavenging ability of some compounds on hydroxyl radicals.

Journal Article↗
PubMed2006

Estimation of hydroxyl radical generation by salicylate hydroxylation method in multiple organs of mice exposed to whole-body X-ray irradiation.

Appropriate experimental conditions for the estimation of hydroxyl radical generation by salicylate hydroxylation were determined for multiple organs of X-irradiated mice in vivo. The in vitro experiments showed that there were significant correlations between the salicylic acid (SA) concentration, the amount of 2,3-dihydroxy benzoic acid (2,3-DHBA) and the X-ray exposure dose, and we obtained two linear-regression equations to calculate the amounts of hydroxyl radicals generated by the X-irradiation. The optimum dosage of SA and the appropriate sampling time for in vivo experiments was determined, and significant increases in the ratio of 2,3-DHBA to SA were detected in several organs of mice after X-irradiation. The hydroxyl radical equivalents of the 2,3-DHBA increases were also calculated. Our results clearly demonstrated the usefulness of the salicylate hydroxylation method in estimating hydroxyl radical generation in multiple organs in vivo.

Animals↗
PubMed1991

Hydroxylation and formation of electrophilic metabolites of tienilic acid and its isomer by human liver microsomes. Catalysis by a cytochrome P450 IIC different from that responsible for mephenytoin hydroxylation.

Tienilic acid (TA) is metabolized by human liver microsomes in the presence of NADPH with the major formation of 5-hydroxytienilic acid (5-OHTA) which is derived from the hydroxylation of the thiophene ring of TA. Besides this hydroxylation, TA is oxidized into reactive metabolites which covalently bind to microsomal proteins. Oxidation of an isomer of tienilic acid (TAI), bearing the aroyl substituent on position 3 (instead of 2) of the thiophene ring, by human liver microsomes, gives a much higher level of covalent binding to proteins. Both covalent binding of TA and TAI metabolites are almost completely suppressed in the presence of glutathione. These three activities of human liver microsomes (TA 5-hydroxylation, covalent binding of TA and TAI metabolites) seem dependent on the same cytochrome P450 of the IIC subfamily, since (i) antibodies against human liver cytochromes P450 IIC strongly inhibit these three activities, (ii) there is a clear correlation between these activities in various human liver microsomes, and (iii) TA acts as a competitive inhibitor for TAI activation into electrophilic metabolites (Ki approximately equal to 25 microM) and TAI inhibits TA 5-hydroxylation. However cross inhibition experiments indicate that tienilic acid hydroxylation and mephenytoin hydroxylation, a typical reaction of some human liver P450 IIC isoenzymes, are not catalysed by the same member of the P450 IIC subfamily.

Biotransformation↗
PubMed1994

Dapsone N-acetylation, metoprolol alpha-hydroxylation, and S-mephenytoin 4-hydroxylation polymorphisms in an Indonesian population: a cocktail and extended phenotyping assessment trial.

We examined dapsone N-acetylation and metoprolol alpha-hydroxylation and S-mephenytoin 4-hydroxylation phenotypings using the respective test probes (dapsone and racemic metoprolol and mephenytoin) administered separately and in a cocktail manner to an Indonesian subject group (n = 30). After ascertaining that the separate and cocktail phenotyping tests of the probe drugs correlated with each other (all rs values > 0.84; p < 0.001), the cocktail phenotyping assessment was extended to the other 74 Indonesians. In a total of 104 Indonesians phenotyped with the cocktail test, a visual antimode was apparent only in the dapsone N-acetylation and S-mephenytoin 4-hydroxylation polymorphisms: the frequencies of slow acetylators and poor hydroxylators were 43.3% (95% confidence interval, 33.7% to 52.8%) and 15.4% (95% confidence interval, 8.5% to 22.3%), respectively. The distribution histogram and probit plots of the metabolic ratio of metoprolol gave no clear evidence for bimodality, and therefore no poor alpha-hydroxylator of metoprolol was considered to exist in the present sample size. The findings indicate that the Indonesian subjects have a greater incidence of slow acetylator phenotype compared with Japanese and Chinese, as well as a frequency of poor metabolizer phenotype of S-mephenytoin similar to that of Korean and Chinese subjects. They resemble an African population (Nigerians) in metoprolol alpha-hydroxylation polymorphism, with no apparent antimode derived from white populations.

Acetylation↗
PubMed2003

CYP2D6-mediated catalysis of tamoxifen aromatic hydroxylation with an NIH shift: similar hydroxylation mechanism in chicken, rat and human liver microsomes.

1. 4-Tritiated-tamoxifen (4-[(3)H]-tamoxifen) and 4-deuterated-tamoxifen (4-[(2)H]-tamoxifen) were synthesized to examine tamoxifen metabolism by human P450 (CYP) forms and also for the possibility of determining tamoxifen-4-hydroxylation in humans in vivo. 2. Liver microsomes from several species and cDNA-expressed human P450s were incubated with 4-[(3)H]-tamoxifen and the reaction monitored by assaying 4-hydroxytamoxifen (4-OH-tam) and (3)H(2)O formed. However, tamoxifen-4-hydroxylation did not generate stoichiometric amounts of (3)H(2)O and the expected unlabelled 4-OH-tam but instead yielded radiolabelled 4-OH-tam, apparently from [(3)H]-migration to the ortho-position, referred to as the NIH shift. 3. CYP2D6 was the prime catalyst of tam-4-hydroxylation, whereas CYP2B6, 2C9 and 2C19 yielded only low levels of 4-OH-tam; nevertheless, in all cases the 4-OH-tam was radioactive, apparently resulting from reactions involving an NIH shift. 4. Chicken liver microsomal preparation, being catalytically the most active in tamoxifen-4-hydroxylation, was incubated with deuterated tamoxifen (4-[(2)H]-tamoxifen) in order to determine whether an NIH shift occurs. Ion-trap mass-spectrometry of the HPLC-purified 4-OH-tam, from that incubation, indicated about 60% of [(2)H]-retention in 4-OH-tam, signifying an NIH shift. These findings indicate that the aromatic hydroxylation of tamoxifen does not entail hydroxyl insertion with an Sn2-displacement of hydrogen or a hydrogen isotope ((2)H or (3)H), but apparently involves epoxidation followed by migration of the (3)H, (2)H or (1)H to the ortho-position, and dissociation of the (1)H in preference to (3)H or (2)H, i.e. retention of the hydrogen isotope appears to be related to the bond strengths: C-(3)H>C-(2)H>C-(1)H.

Animals↗
PubMed1997

Relationship between CYP2C9 and 2C19 genotypes and tolbutamide methyl hydroxylation and S-mephenytoin 4'-hydroxylation activities in livers of Japanese and Caucasian populations.

Genomic DNA was isolated from livers of 39 Japanese and 45 Caucasians and the genotypes of CYP2C9 and 2C19 genes were determined with PCR methods using synthetic oligonucleotide primers. Liver microsomes were also prepared from these human samples and activities for tolbutamide methyl hydroxylation and S-mephenytoin 4'-hydroxylation were determined. The single base mutation of C416T (Arg144Cys) in CYP2C9 was detected in 22% of Caucasians but not in Japanese samples. Another single base mutation at A1061C (Ile359Leu) in the CYP2C9 gene was found with frequencies of about 8% in both races. We did not detect any individuals who have either homozygous Cys144/Cys144 or Leu359/Leu359 CYP2C9 variant nor both heterozygous Cys144-Ile359 and Arg144-Leu359 CYP2C9 variant in the human samples examined. The CYP2C19m2 genetic polymorphism was found only in Japanese people, while CYP2C19m1 type was determined in both races, with higher incidence in Japanese than in Caucasian population. Immunoblotting analysis of human liver microsomes suggested that CYP2C9 is a major component of the human CYP2C enzyme pool; it accounted for approximately 20% of total P450 in liver microsomes of both human populations. The levels of CYP2C19 protein were determined to be about 0.8% and 1.4% of total P450 (mean) in Japanese and Caucasians, respectively. We did not detect CYP2C19 protein in liver microsomes of humans who were genotyped for CYP2C19 gene as m1/m1, m1/m2, and m2/m2 variants but detected CYP2C9 protein in all of the samples examined. Good correlations were found between levels of CYP2C9 and activities of tolbutamide methyl hydroxylation (r = 0.77) and between levels of CYP2C19 and activities of S-mephenytoin 4'-hydroxylation (r = 0.86) in liver microsomes of the human samples examined. Tolbutamide methyl hydroxylation activities were lower in human samples with the Leu359 allele of CYP2C9 than those with the Cys144 allele and wild-type (Arg144-Ile359); the former type showed slightly higher K(m) values. When calculated on P450 basis, liver microsomes of individuals having m1/m1, m1/m2, and m2/m2 types of CYP2C19 had very low catalytic activities for S-mephenytoin 4'-hydroxylation. These results provide useful comparisons for pharmacokinetic and toxicokinetic models of some of the clinically used drugs that are oxidized by CYP2C proteins in humans.

Aryl Hydrocarbon Hydroxylases↗