A genetic analysis of meat compositions in Japanese Black cattle: Genetic parameters and sex influence.

Meat composition in beef is related to eating quality and food functionality. Genetic parameters for several meat compositions including free amino acid, peptide and sugar, however, remain poorly described. In this study, we estimated genetic parameters for 51 meat components, including free amino acids, peptides, sugars and fatty acid compositions, and two carcase traits in 1,354 heifers and 1,797 steers of Japanese Black cattle. Heritability estimates were generally equivalent to or moderately greater than those in previous studies of this breed. Genetic correlations between free amino acids, peptides and sugars and carcase traits were often negative, suggesting a trade-off between traits. Using two-trait animal models that treat records from the two sexes as different traits, we estimated sex-specific heritabilities and cross-sex genetic correlations which indicate the sex differences in genetic architecture. In these analyses, 12 traits showed significant heritability differences between sexes and cross-sex genetic correlations occasionally deviated from unity. These results could be used to inform future breeding schemes and investigations of the genetic architecture of meat compositions in beef.

Whole-genome prediction of fatty acid composition in meat of Japanese Black cattle.

Because fatty acid composition influences the flavor and texture of meat, controlling it is particularly important for cattle breeds such as the Japanese Black, characterized by high meat quality. We evaluated the predictive ability of single-step genomic best linear unbiased prediction (ssGBLUP) in fatty acid composition of Japanese Black cattle by assessing the composition of seven fatty acids in 3088 cattle, of which 952 had genome-wide marker genotypes. All sires of the genotyped animals were genotyped, but their dams were not. Cross-validation was conducted for the 952 animals. The prediction accuracy was higher with ssGBLUP than with best linear unbiased prediction (BLUP) for all traits, and in an empirical investigation, the gain in accuracy of using ssGBLUP over BLUP increased as the deviations in phenotypic values of the animals increased. In addition, the superior accuracy of ssGBLUP tended to be more evident in animals whose maternal grandsire was genotyped than in other animals, although the effect was small.

Genetic analysis of semen production traits of Japanese Black and Holstein bulls: genome-wide marker-based estimation of genetic parameters and environmental effect trends.

The semen production traits of bulls from 2 major cattle breeds in Japan, Holstein and Japanese Black, were analyzed comprehensively using genome-wide markers. Weaker genetic correlations were observed between the 2 age groups (1 to 3 yr old and 4 to 6 yr old) regarding semen volume and sperm motility compared with those observed for sperm number and motility after freeze-thawing. The preselection of collected semen for freezing had a limited effect. Given the increasing importance of bull proofs at a young age because of genomic selection and the results from preliminary studies, we used a multiple-trait model that included motility after freeze-thawing with records collected at young ages. Based on variations in contemporary group effects, accounting for both seasonal and management factors, Holstein bulls may be more sensitive than Japanese Black bulls to seasonal environmental variations; however, the seasonal variations of contemporary group effects were smaller than those of overall contemporary group effects. The improvement of motilities, recorded immediately after collection and freeze-thawing, was observed in recent years; thus, good management and better freeze-thawing protocol may alleviate seasonal phenotypic differences. The detrimental effects of inbreeding were observed in all traits of both breeds; accordingly, the selection of candidate bulls with high inbreeding coefficients should be avoided per general recommendations. Semen production traits have never been considered for bull selection. However, negative genetic trends were observed. The magnitudes of the estimated h were comparable to those of other economically important traits. A single-step genomic BLUP will provide more accurate predictions of breeding values compared with BLUP; thus, marker genotype information is useful for estimating the genetic merits of bulls for semen production traits. The selection of these traits would improve sperm viability, a component related to breeding success, and alleviate negative genetic trends.

Hepatic microsomal tolbutamide hydroxylation in Japanese: in vitro evidence for rapid and slow metabolizers.

Microsomal hydroxylation of tolbutamide in Japanese livers was studied in vitro to ascertain the enzyme catalysing this reaction. Rates of tolbutamide hydroxylation differed individually 33-fold and 42-fold at 0.1 mM and 2.4 mM tolbutamide concentrations, respectively, and were segregated into two groups, rapid and slow metabolizers. An antibody raised against P450 human-2 (a form of CYP2C9) strongly inhibited the hydroxylation in livers of rapid metabolizers but only weakly inhibited in the slow metabolizer. Kinetic experiments further demonstrated a clear distinction in tolbutamide hydroxylation between two groups; the mean of apparent Km values for tolbutamide was 0.25 mM (n = 3) in the rapid group and 2.58 mM (n = 2) in the slow, respectively. These data suggest that different enzymes are involved in the hydroxylation in both metabolizer groups. Furthermore, CYP2C9 produced by cDNA expression in yeasts, catalysed tolbutamide hydroxylation at rates similar to the rapid metabolizer group at both the 0.1 mM and 2.4 mM concentrations. The apparent Km value of the expressed protein for tolbutamide, 0.26 mM, was similar to that determined for the rapid group of microsomal samples. Clear correlations were observed between the rate of microsomal tolbutamide hydroxylation at 0.1 mM and CYP2C9 protein content or the rate of S-mephenytoin 4'-hydroxylation in human liver. These results indicate that considerable portions of microsomal tolbutamide hydroxylation are catalysed by CYP2C9 or the closely related form in the rapid metabolizers.

Cytochrome P450 mediated metabolism of diazepam in human and rat: involvement of human CYP2C in N-demethylation in the substrate concentration-dependent manner.

Metabolism of diazepam (DZP) was studied in vitro to clarify the involvement of different forms of hepatic cytochrome P450 (CYP) in rats, and humans of Japanese and Caucasian origin. Microsomal 3-hydroxylation was the major pathway of DZP metabolism in rats and was inhibited by anti-CYP3A antibodies. Purified CYP3As and CYP2C11 catalysed 3-hydroxylation and N-demethylation, respectively, in the reconstituted systems. The rates of both reactions in human liver microsomes depended on the substrate concentration: the rate of 3-hydroxylation was 3-4 times higher than N-demethylation at 0.2 mM; the two activities were essentially the same at a lower substrate concentration (0.02 mM). Inhibitions of the N-demethylation by anti-CYP2C antibody and S-mephenytoin also depended on the substrate concentration and was detectable only at a low substrate concentration. Kinetic studies revealed the presence of two distinct catalytic activities for the N-demethylation; low Km and low Vmax, and high Km and high Vmax. The former activity seems to be mediated by a CYP2C P450 form. On the other hand, DZP 3-hydroxylation was rather selectively catalysed by a CYP3A P450 at the low and high substrate concentrations. These results were consistent with the observation in vivo that DZP N-demethylation and S-mephenytoin 4'-hydroxylation are closely correlated in humans. These results also suggest that the apparent discrepancy on the role of CYP forms in DZP metabolism in vitro and in vivo may reside in the difference in substrate concentration.

CYP2C9 Ile359 and Leu359 variants: enzyme kinetic study with seven substrates.

To assess the effects of Ile359 to Leu359 change on CYP2C9-mediated metabolism, we performed site-directed mutagenesis and cDNA expression in yeast for CYP2C9 and examined in detail the kinetics of seven metabolic reactions by wild-type CYP2C9 (Ile359) and its Leu359 variant. For the metabolism of all the substrates studied, the Leu359 variant exhibited smaller Vmax/Km values than did the wild-type. The differences in the Vmax/Km values between the wild-type and the Leu359 variant varied from 3.4-fold to 26.9-fold. The Leu359 variant had higher Km values than did the wild-type for all the reactions studied. Among the seven reactions studied, the greatest difference in the Vmax values between the wild-type and the Leu359 variant was for piroxicam 5'-hydroxylation (408 versus 19 pmol/min/nmol P450), whereas there were no differences in the Vmax values between the wild-type and the Leu359 variant for diclofenac 4'-hydroxylation and tolbutamide methylhydroxylation. These results indicate that the Ile359 to Leu359 change significantly decreases the catalytic activity of all the CYP2C9-mediated metabolisms studied, whereas the extent of the reduction in activity and changes of the kinetic parameters varies between substrates. Moreover, the amino acid substitution decreased the enantiomeric excess in the formation of 5-(4-hydroxyphenyl)-5-phenylhydantoin from phenytoin.

Lack of low Km diazepam N-demethylase in livers of poor metabolizers for S-mephenytoin 4'-hydroxylation.

Metabolism of diazepam was studied in vitro to identify the forms of cytochrome P450 (CYP) responsible for N-demethylation (nordazepam formation) and 3-hydroxylation (temazepam formation), using liver microsomes obtained from extensive (EM) and poor metabolizers (PM) for S-mephenytoin 4'-hydroxylation. Involvement of at least two P450 forms in diazepam N-demethylation was suggested by a biphasic pattern in Lineweaver-Burk and Eadie-Hofstee plots from the EM, whereas a monophasic pattern was observed from the PM liver microsomes. The kinetic parameters for the N-demethylation in the EM group were: Km 1, 19.4 +/- 0.4 microM; Vmax 1, 0.27 +/- 0.04 nmol min-1 per mg protein; Km 2, 346 +/- 34 microM; Vmax2, 1.82 +/- 0.63 nmol min-1 per mg protein (n = 3, mean +/- SD). The PM group showed the mean values of Km and Vmax (Km, 319 +/- 30 microM; Vmax, 1.49 +/- 0.62 nmol min-1 per mg protein) (n = 3) similar to those of Km2 and Vmax2 in the EM group. An antibody raised against CYP2C9 (anti-human CYP2C) strongly inhibited diazepam N-demethylation in EM liver microsomes at a low substrate concentration (20 microM). However, the anti-human CYP2C showed no clear inhibition of N-demethylation in EM liver microsomes at a high substrate concentration (200 microM). Diazepam N-demethylation in PM liver microsomes was not clearly inhibited by the anti-human CYP2C at either the low or high substrate concentrations. These data suggest that different P450 forms mediated diazepam N-demethylation in EM and PM liver microsomes, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Polymorphism in hydroxylation of mephenytoin and hexobarbital stereoisomers in relation to hepatic P-450 human-2.

Stereoselective 4'-hydroxylations of R-(-)-mephenytoin and S-(+)-mephenytoin and 3'-hydroxylation of R-(-)-hexobarbital and S-(+)-hexobarbital were determined in liver microsomes of 14 Japanese subjects who were extensive metabolizers of mephenytoin and in five Japanese subjects who were poor metabolizers of mephenytoin. Content of P-450 human-2 assessed by Western blots was correlated to microsomal S-(+)-mephenytoin 4'-hydroxylation, R-(-)-hexobarbital 3' alpha-hydroxylation, and S-(+)-hexobarbital 3' beta-hydroxylation, and was less correlated to R-(-)mephenytoin 4'-hydroxylation, R-(-)-hexobarbital 3' beta-hydroxylation, and S-(+)-hexobarbital 3' alpha-hydroxylation. Antibodies raised against P-450 human-2 inhibited microsomal S-(+)-mephenytoin 4'-hydroxylation efficiently but was less efficient on R-(-)-mephenytoin 4'-hydroxylation in extensive metabolizers and on 4'-hydroxylation of mephenytoin enantiomers in poor metabolizers. The antibodies also inhibited R-(-)-hexobarbital 3' alpha-hydroxylation and S-(+)-hexobarbital 3' beta-hydroxylation but did not effectively inhibit the hydroxylation of the two other optical isomers of hexobarbital in extensive metabolizers and of four stereoisomers in poor metabolizers. These findings indicate the close relationship between polymorphic mephenytoin 4'-hydroxylation and two stereospecific hexobarbital hydroxylations, and they suggest that P-450 human-2 is a typical S-(+)-mephenytoin 4'-hydroxylase and a major hexobarbital 3'-hydroxylase in the livers of extensive metabolizers. The findings were further supported by the experiments that used P-450 human-2 complementary dexoyribonucleic acid-derived protein in yeast microsomes.

Potentiation of anticoagulant effect of warfarin caused by enantioselective metabolic inhibition by the uricosuric agent benzbromarone.

OBJECTIVE: To clarify the mechanism(s) for the interaction between warfarin and benzbromarone, a uricosuric agent, and to predict changes in the in vivo pharmacokinetics of (S)-warfarin from in vitro data. METHODS: Warfarin enantiomers and benzbromarone in serum, 7-hydroxywarfarin in urine, and serum unbound fractions of warfarin enantiomers were measured in patients with heart disease given warfarin with (n = 13) or without (n = 18) oral benzbromarone (50 mg/d). In vitro inhibition constants (K(i)) of benzbromarone for (S)-warfarin 7-hydroxylation were determined with use of human CYP2C9 and liver microsomes. The magnitude of changes in the formation clearance for 7-hydroxylation (CLf), the unbound oral clearance (CL(oral,u)), and the oral clearance (CL(oral)) for (S)-warfarin were predicted by equations incorporating the in vitro Ki, the theoretical maximum unbound hepatic benzbromarone concentration, and the fractions of warfarin eliminated through metabolism and of CYP2C9-mediated metabolic reaction susceptible to inhibition by benzbromarone. RESULTS: The patients given warfarin with benzbromarone required a 36% less (P < .01) warfarin dose than those given warfarin alone (2.5 versus 3.9 mg/d) to attain similar international normalized ratios (2.1 and 2.2, respectively), and the former had 65%, 53%, and 54% lower (P < .05 or P < .01) CLf, CL(oral),u, and CL(oral) for (S)-warfarin than the latter, respectively. In contrast, no significant differences were observed for (R)-warfarin kinetics between the groups. Benzbromarone was found to be a potent competitive inhibitor (Ki < 0.01 micromol/L) for (S)-warfarin 7-hydroxylation mediated by CYP2C9. The average changes in the in vivo CLf, CL(oral),u, and CL(oral)values for (S)-warfarin induced by benzbromarone were largely predictable by the proposed equations. CONCLUSION: Benzbromarone would intensify anticoagulant response of warfarin through an enantioselective inhibition of CYP2C9-mediated metabolism of pharmacologically more potent (S)-warfarin. The magnitude of changes in the in vivo warfarin kinetics may be predicted by in vitro data.

Human CYP2C-mediated stereoselective phenytoin hydroxylation in Japanese: difference in chiral preference of CYP2C9 and CYP2C19.

Regio- and stereoselective hydroxylation of phenytoin was determined in liver microsomes of nine extensive (EM) and three poor metabolizers (PM) of mephenytoin. Hydroxyphenytoins (HPPH) were isolated and quantified after separation into four regio- and stereoisomers. The total rates of microsomal phenytoin 4'- hydroxylation were approximately 3-fold higher than those of 3'-hydroxylation, and not significantly different in EM and PM. Formation of 4'-(R)-HPPH was 4.4-fold higher in EM than in PM, whereas no clear differences between EM and PM were detected in the formation of 4'-(S)-, 3'-(R)-, and 3'-(S)-HPPH. Cytochrome P450 (CYP)2C9, expressed in a fission yeast, Schizosaccharomyces pombe, catalyzed the formation of 4'-(R)- and 4'-(S)-HPPH stereoselectively, as observed with EM, in which predominantly 4'-(S)-HPPH was formed. Recombinant CYP2C19 was more stereoselective for 4'-(R)-HPPH formation. These results, in addition to inhibition experiments with anti-human CYP2C antibody, indicate that phenytoin hydroxylation is mainly catalyzed by CYP2C9. Furthermore, CYP2C19 showed limited contribution to phenytoin 4'-hydroxylation with a different chiral preference from CYP2C9.

Cytochrome P-450 human-2 (P-450IIC9) in mephenytoin hydroxylation polymorphism in human livers: differences in substrate and stereoselectivities among microheterogeneous P-450IIC species expressed in yeasts.

The cDNA of a P-450 human-2 and the two other closely related cDNAs, MP-8 (two deduced amino acids substituted) and lambda hPA6 (two deduced amino acids deleted) were expressed in Saccharomyces cerevisiae cells, and their catalytic and chemical properties were compared to identify which cDNA encodes a major S-mephenytoin 4'-hydroxylase in human livers. In immunoblots, P-450 human-2 cDNA-derived protein in yeasts was stained at the position identical with P-450 human-2 purified from liver and a major protein in microsomes of 19 Japanese livers. MP-8- and lambda hPA6-derived proteins were immunostained at positions near, but distinct from P-450 human-2, and were not detected in those 19 livers. All three proteins expressed in yeasts catalyzed hydroxylation of mephenytoin, hexobarbital, benzo[a]pyrene and tolbutamide, although the rates of the hydroxylation of most of the drugs by P-450 human-2 were higher than those of the two others. In addition, these expressed proteins showed clear differences in the hydroxylation of chiral substrates: P-450 human-2 catalyzed the hydroxylation of S-mephenytoin five times faster than that of the R-enantiomer. Similar high enantioselectivities were also observed on the hydroxylation of R- and S-hexobarbital. However, MP-8- and lambda hPA6-derived proteins catalyzed hydroxylation of these two drugs with less or almost no stereoselectivity. These results indicate that only a few amino acid alterations cause dramatic changes in both the chemical and catalytic properties of P-450 human-2.

Purification of human liver cytochrome P-450 catalyzing testosterone 6 beta-hydroxylation.

Two forms of cytochrome P-450 (P-450 human-1 and P-450 human-2) have been purified from human liver microsomes to electrophoretic homogeneity. P-450 human-1 and P-450 human-2 differ in their apparent molecular weights (52,000 and 56,000, respectively) and Soret peak maxima in the CO-binding reduced difference spectrum (447.6 and 450.3 nm, respectively). In the reconstituted system using rat liver NADPH-cytochrome c (P-450) reductase, P-450 human-2 more effectively oxidized benzo(a)pyrene (80-fold), ethylmorphine (2-fold), and 7-ethoxycoumarin (2-fold) than did P-450 human-1. However, P-450 human-1 showed higher testosterone 6 beta-hydroxylase activity, and the activity was markedly increased by the inclusion of cytochrome b5 or spermine in the reconstituted system. Antibodies raised against P-450 human-1 inhibited more than 80% of microsomal testosterone 6 beta-hydroxylase activity in human liver. Immunoblotting analysis using anti-P-450 human-1 IgG revealed a single immuno-staining band near Mr 52,000 in all human liver samples examined. The amount of immunochemically determined P-450 human-1 varied in parallel with the testosterone 6 beta-hydroxylase activity in human liver. These results indicate that P-450 human-1 is a major form of cytochrome P-450 responsible for microsomal testosterone 6 beta-hydroxylation. Thus, this paper is the first report on human cytochrome P-450 responsible for testosterone 6 beta-hydroxylation, which is the major hydroxylation pathway in human liver microsomes.

Nucleotide sequence of a human liver cytochrome P-450 related to the rat male specific form.

P-450 human-2 is a human cytochrome P-450 that is immunochemically related to a constitutive male-specific cytochrome P-450 (P-450-male) and the phenobarbital-inducible P-450b/e in rat liver. By screening a human liver cDNA library in bacteriophage lambda gt11, we isolated a clone with an insert length of 1,847 bases (pHY13). The clone was sequenced and shown to code for a protein of 487 amino acids. The N-terminal 11-amino-acid sequence was in agreement with the protein sequence of P-450 human-2. The nucleotide sequence of pHY13 showed less than 50% similarity with those of human cytochrome P-450s, pHP-450(1), HLp, P-450NF, P1-450 4, and P3(450), but the nucleotide sequence of pHY13 is 80% similar to the reported sequence of rat cytochrome P-450, P-450(M-1). In addition, the coding sequence of pHY13 showed close similarity to that of MP-8, which was recently reported as the sequence corresponding to human cytochrome P-450MP, although no apparent similarity was observed in their 3' non-coding sequences except for the first 75 bases and the expected length of the complete sequences. These results, together with the immunochemical data, indicate that P-450 human-2 is closely related, but not identical, to P-450MP, and may belong to the category of developmentally regulated constitutive cytochrome P-450s.

Genomic prediction in Japanese Black cattle: application of a single-step approach to beef cattle.

The implementation of genomic selection for Japanese Black cattle, known for rich marbling of their meat, is now being explored. Although multiple-step methods are often adopted for dairy cattle, they present shortcomings such as bias and loss of information in addition to operational complexity. These can be avoided using single-step genomic BLUP (ssGBLUP) based on the relationship matrix H, which is constructed from the numerator relationship matrix (A) augmented by the genomic relationship matrix (G). This study assessed the use of ssGBLUP for 3 economically important traits in Japanese Black cattle. Three aspects of ssGBLUP that are important for practical use were examined specifically: the mixing proportions of blending G with A, selection of subsets of genotyped animals used for constructing H, and prediction ability for ungenotyped animals. Different mixing proportions were tested to assess the influence of these proportions on variance component estimation and prediction accuracy. For all traits, the highest or nearly highest accuracy was obtained when the adopted mixing proportion provided heritability closest to that inferred based on A. However, the accuracy did not increase greatly under adjustment of the mixing proportion, thereby suggesting that the influence of the mixing proportion on the accuracy was limited. Genotype data of influential bulls showed a greater contribution to accuracy than that of bulls that were less influential. Genotyping animals with phenotypic records increased the accuracy. It can be prioritized over genotyping bulls that are not influential on the population. These results are expected to present good guides to the future expansion of genotyped populations. Even for animals without genotype data but with genotyped sires, ssGBLUP provided more accurate prediction than BLUP did. For both phenotype and breeding value prediction, ssGBLUP provides more accurate prediction than BLUP, suggesting its usefulness in genomic selection in Japanese Black cattle.

Finasteride 1 mg has no inhibitory effect on omeprazole metabolism in extensive and poor metabolizers for CYP2C19 in Japanese.

OBJECTIVE: To examine the inhibitory effect of finasteride 1 mg on the metabolism of omeprazole in genetically determined extensive (EMs) and poor metabolizers (PMs) for CYP2C19 in young healthy Japanese male subjects. METHODS: Twenty-four volunteers participated in this study, among whom 12 were homozygous EMs and 12 were PMs for CYP2C19. A single center, controlled, randomized, open, crossover study with a 5 day washout between the two study periods was performed. Each of the six EMs and PMs received a single oral 20 mg dose of omeprazole on day 1 (treatment I). After a 5 day washout period, these subjects received 1 mg of finasteride once a day for three consecutive days, and a single oral 20 mg dose of omeprazole was co-administered on day 3 (treatment II). The 12 other EMs and PMs received treatments I and II in reverse. Plasma samples were collected for up to a 12 hours postdose of omeprazole, and the pharmacokinetic parameters of omeprazole were determined. RESULTS: The geometric mean ratio (GMR) for the AUC((0-12 hr)) of omeprazole when co-administered with finasteride/omeprazole alone is 1.13 (90%CI, 1.03, 1.25) and 0.96 (0.88, 1.05) in EMs and PMs, respectively. Finasteride did not significantly alter C(max), T(max) and t(1/2) in both genotypes. CONCLUSION: Finasteride 1 mg, widely used for the treatment of androgenetic alopecia in men, did not meaningfully increase omeprazole exposure (20 mg) in both EMs and PMs for CYP2C19. These results indicate that finasteride does not meaningfully inhibit CYP2C19 activity in vivo at the dose of 1 mg.

Polymorphism in stereoselective hydroxylations of mephenytoin and hexobarbital by Japanese liver samples in relation to cytochrome P-450 human-2 (IIC9).

1. Stereoselective 4'-hydroxylations of R-(--)-mephenytoin and S-(+)-mephenytoin were determined in liver microsomes of 19 Japanese subjects. 2. The content of P-450 human-2 assessed by Western-blots correlated with microsomal S-(+)-mephenytoin 4'-hydroxylation. Antibody raised against P-450 human-2 effectively inhibited microsomal S-(+)-mephenytoin 4'-hydroxylation, but was less efficient for inhibition of R-(--)-mephenytoin 4'-hydroxylation in extensive metabolizers, and 4'-hydroxylation of both mephenytoin enantiomers in poor metabolizers. 3. Similar results were observed on the stereoselective hydroxylations of R-(--)- and S-(+)-hexobarbital. Clear correlations were observed for the content of P-450 human-2 and microsomal R-(--)-hexobarbital 3'alpha-hydroxylation and S-(+)-hexobarbital 3'beta-hydroxylation. 4. Moreover, yeast microsomes expressing P-450 human-2 cDNA showed high stereoselectivities for hydroxylations of mephenytoin and hexobarbital similar to those observed in human liver. 5. Two other cytochromes P-450(IIC 9/10) expressed in yeast, whose cDNA were synthesized by site-directed mutagenesis from human-2 cDNA, showed no stereoselectivity for the hydroxylations of mephenytoin and hexobarbital, in spite of the modification of only two amino acid substitutions or deletions in the whole sequence. 6. Only a cytochrome derived from P-450 human cDNA corresponding to P-450 human-2 was expressed in human livers, the two cytochromes of the three related IIC9/10 forms were not expressed. 7. These findings indicate that P-450 human-2 is the major cytochrome P-450 responsible for the polymorphisms in stereoselective hydroxylations of mephenytoin and hexobarbital.

Pharmacogenetics and polymorphism of human P-450 which activates and detoxicates xenobiotics and carcinogens.

Stereoselective hydroxylation of mephenytoin is expressed polymorphically in the Japanese population: the occurrence of the poor metabolizer was approximately 20% in Japanese, which was 5 to 8 times higher than that in Caucasians. Microsomal 4'-hydroxylation of S-mephenytoin, but not of R-mephenytoin, was correlated to the content of cytochrome P-450 human-2. P-450 human-2 cDNA was cloned and expressed in yeast cells. The expressed P-450 showed stereoselectivities similar to those in human livers for hydroxylations of mephenytoin and hexobarbital enantiomers. The P-450 catalyzed benzo[a]pyrene and tolbutamide hydroxylations and cyclophosphamide oxidation, but showed low activity for the mutagenic activation of IQ (2-amino-3-methylimidazo [4, 5-f]quinoline). Two cDNAs (MP-8 and lambda hPA6), which are two amino acids mutated and deleted, respectively, from P-450 human-2 cDNA were expressed in yeasts. These two expressed P-450s revealed complete loss of the stereoselectivity in the hydroxylation of mephenytoin and hexobarbital. These results indicate that many xenobiotics and carcinogens are detoxicated and activated by the human liver in quite different manners among individuals by reason of genetic differences.

Species differences in stereoselective metabolism of mephenytoin by cytochrome P450 (CYP2C and CYP3A).

Stereoselective involvement of hepatic cytochrome P450 in the metabolism of mephenytoin was investigated in vitro by using livers of five different experimental animal species and humans. The rates of microsomal 4'-hydroxylation were 2 to 6 times higher with the R-enantiomer than the S-enantiomer in rabbits, dogs and rats, whereas the rates of the 4'-hydroxylation in female mice were not different between R- and S-enantiomers. Preferential S-mephenytoin 4'-hydroxylation was observed in monkeys as similar to that in humans. The rates of microsomal mephenytoin N-demethylation were approximately 2 times higher with the R-enantiomer than the S-enantiomer in male rats and both sexes of dogs. Antibodies raised against CYP2C11 (anti-CYP2C) clearly inhibited microsomal 4'-hydroxylation of S-mephenytoin and N-demethylation of R-mephenytoin in rats, monkeys and humans. Antibodies raised against CYP3A2 (anti-CYP3A) clearly inhibited microsomal 4'-hydroxylation of R-mephenytoin, but marginally S-mephenytoin, in rats. Anti-CYP3A, however, showed no clear inhibition on microsomal 4'-hydroxylation and N-demethylation of both enantiomers in monkeys and humans, except for slight inhibition of R-mephenytoin 4'-hydroxylation in male monkeys. The results suggest that stereoselective involvement of rat CYP3A and scant involvement of human CYP3A in R-mephenytoin 4'-hydroxylation are major determinants of the species differences between rats and humans in stereoselective mephenytoin 4'-hydroxylation.