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Objective: There are conflicting reports regarding the effects of cytochrome P450 (P450, CYP) genotypes on the plasma concentrations of risperidone and its pharmacologically active metabolite, 9-hydroxyrisperidone (paliperidone), in clinical patients. The aim of this study was to investigate individual differences in the metabolic clearance of risperidone in vitro and in vivo., Methods: In vitro liver microsomal risperidone 9-hydroxylation activities and in vivo plasma concentrations of risperidone and paliperidone were investigated in 15 male and 12 female Japanese subjects (mean age 52 years, range: 24-75 years) genotyped for CYP2D6 and CYP3A5., Results: CYP2D6 intermediate and poor metabolizers showed significantly lower liver microsomal risperidone 9-hydroxylation activities than extensive metabolizers did at 5 μM of risperidone; this difference was not evident at 50 μM of risperidone. The recombinant CYP3A5 Vmax/Km value for risperidone 9-hydroxylation was 30% that of CYP3A4, and liver microsomes from CYP3A5 expressers had similar risperidone 9-hydroxylation activities to those of CYP3A5 poor expressers. The plasma concentration/dose ratios for risperidone and paliperidone in 27 Japanese patients were not significantly influenced by the CYP2D6 or CYP3A5 genotypes., Conclusions: Individual differences in metabolic clearance of risperidone under the present conditions were not significantly influenced by the genotypes of CYP2D6 or CYP3A5., (Copyright © 2016 John Wiley & Sons, Ltd.)

Objective: The antipsychotic olanzapine is reportedly metabolized by inducible human cytochrome P450 (CYP) 1A2 and variable copy-number CYP2D6 and polymorphic flavin-containing monooxygenase 3 (FMO3) in different pathways. We investigated individual differences in the metabolite formation and clearance of olanzapine in vitro and in vivo., Methods: Human liver microsomal olanzapine oxidation activities were evaluated, and plasma concentrations of olanzapine were determined in 21 Japanese patients (mean age: 50 years, range: 32-69 years, 14 male and 7 female, including 6 smokers) genotyped for CYP2D6 (*1, *5, and *10) and FMO3 (E158K, C197fsX, R205C, V257M, E308G, and R500X)., Results: Furafylline (a CYP1A2 inhibitor), quinidine (a CYP2D6 inhibitor), and heat treatment (inactivates FMO3) suppressed liver microsomal metabolic clearance of olanzapine by approximately 30%. Olanzapine N-demethylation and N-oxygenation were found to be catalyzed by CYP1A2 and CYP2D6 and by CYP2D6 and FMO3, respectively, in experiments using liver microsomes and recombinant enzymes. Plasma concentrations and clearance of olanzapine were not affected by CYP2D6 or FMO3 genotypes or smoking behavior., Conclusions: Olanzapine clearance was not affected by CYP2D6 or FMO3 genotypes or smoking behavior as a single factor under the present conditions because olanzapine clearance is mediated by multiple enzymes involved in two major and one minor pathways., (Copyright © 2016 John Wiley & Sons, Ltd.)

Coplanar polychlorinated/brominated biphenyls (Co-PXBs) are environmental pollutants previously identified in market fish samples. In this study, we observed that mother's milk in Japan is contaminated with Co-PXBs. Based on assumption that the toxicity of the same congener of PXDDs/DFs and Co-PXBs is nearly equal to that of the corresponding PCDDs/DFs and Co-PCBs, respectively, the toxic equivalent (TEQ) concentration was 10% of the total TEQ concentration (∑PCDDs/DFs, ∑PXDDs/DFs, ∑Co-PCBs and ∑Co-PXBs) in the milk. This observation suggested that humans, and especially infants, are exposed to high levels of Co-PXBs, which might cause adverse effects. However, the toxicity of Co-PXBs has to date not been reported. We assessed the toxic potency of Co-PXBs by studying their effect on the activity of cytochrome P450. Only the mRNA level and activity of CYP1A increased in a dose-dependent manner upon exposure to Co-PXBs. Substitution of bromine for chlorine into Co-PCBs provided higher CYP1A activity in in vitro and in vivo experiments. The expression level of aryl hydrocarbon receptor (AhR) mRNA was not altered, but luciferase activity, an indicator of AhR transcriptional activity, increased following treatment with Co-PXBs. The results suggest that CYP1A induction by Co-PXBs depended on AhR transcriptional activity and not on AhR expression. Although the TEFs of Co-PXBs are not set, if Co-PXBs are included in these calculations because of their higher toxicity compared to Co-PCBs, exposure to Co-PXBs cannot be neglected when assessing human health risks., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

During the course of sequencing for the CYP2D6 gene, we found a novel single nucleotide polymorphism of g.3318G>A (E383K) associated with CYP2D6*10, termed as CYP2D6*72. We also found a g.1611T>A (F120I) in the CYP2D6*49, which was previously identified as a CYP2D6*10-associated allele in an independent Japanese population. To clarify the effects of these novel CYP2D6*10 haplotypes on the functions of CYP2D6, kinetic analysis for dextromethorphan O-demethylation was performed using the Escherichia coli expression system and human liver microsomes. The V(max)/K(m) values for dextromethorphan O-demethylation catalyzed by recombinant CYP2D6 forms encoded by CYP2D6*10, CYP2D6*49, and CYP2D6*72 were 3.0, 0.5, and 1.3%, respectively, compared with that catalyzed by CYP2D6.1. Liver microsomes from a human subject genotyped as CYP2D6*10/*49 also showed a reduced dextromethorphan O-demethylase activity. CYP2D6.49 formed a 7-hydroxydextromethorphan, with a roughly similar V(max)/K(m) value to that of O-demethylation. These results suggest that these two CYP2D6*10 haplotypes are possible causes of interindividual variation in the activities and the substrate specificity of CYP2D6.

The aim of this study was to investigate the inter-individual variations in cytochrome P4502J2 (CYP2J2) and its typical drug oxidation activities in human liver microsomes in both Japanese and Caucasian populations. CYP2J2 contents were determined immunochemically in liver microsomes from 20 Japanese and 29 Caucasian samples using recombinant CYP2J2 commercially available as a standard. Ebastine hydroxylation and astemizole O-demethylation activities were compared. The CYP2J2 genotype was determined by direct sequencing of liver genomic DNA. The mean expression levels of CYP2J2 determined immunochemically in liver microsomes from Japanese and Caucasian samples were 2.0 +/- 1.5 and 1.2 +/- 2.1 pmol CYP2J2 mg-1 protein (mean +/- standard deviation), respectively, accounting for 1.8 +/- 1.1% and 0.52 +/- 0.65% of the total hepatic P450 content (0.15 +/- 0.19 and 0.27 +/- 0.14 nmol P450 mg-1 protein, respectively). The individual variation of the two marker drug oxidation activities could not be fully accounted for by the CYP2J2 contents or currently known CYP2J2 genotypes. The amounts of CYP2J2 in liver microsomes with the CYP2J2*7 allele (-76G>T) were decreased to 39% compared with those of liver microsomes from other individuals. The results indicate that CYP2J2 accounts for approximately 1-2% of total P450 in human liver microsomes. The information about large inter-individual variation of the CYP2J2 suggests that this enzyme plays a significant role in the metabolism of xenobiotics and may be useful in in-silico simulations of drug disposition.

The purpose of this study was to evaluate a contribution of polymorphic cytochrome P450 (CYP) 3A5 to the oxidation of diltiazem, midazolam and testosterone by liver microsomes from Japanese subjects. Twenty-seven liver samples were classified into three groups according to the CYP3A5 genotypes; CYP3A5(*)1/(*)1 (n=3), (*)1/(*)3 (n=12) and (*)3/(*)3 (n=12). The results of genotyping and immunochemical quantitation of CYP3A5 protein showed a good accordance between the CYP3A5 genotype and CYP3A5 content but not CYP3A4 content in liver microsomes. The expression levels of hepatic CYP3A5 protein ranged from 20 to 60% of the sum of CYP3A4 and CYP3A5 contents in subjects with at least one wild type allele ((*)1). The CYP3A5 contents correlated well with liver microsomal activities of diltiazem N-demethylation, midazolam 1'- and 4-hydroxylations and testosterone 6beta-hydroxylation among subjects carrying at least one (*)1 allele. In addition, the correlation coefficients of CYP3A5 contents with the rates of diltiazem N-demethylation, midazolam 1'-hydroxylation and testosterone 6beta- hydroxylation were higher than those of CYP3A4, although the value of CYP3A5 with the midazolam 4-hydroxylation rate was similar to that of CYP3A4. Kinetic analyses revealed a biphasic diltiazem N-demethylation in liver microsomes from subjects carrying the (*)1 allele. The apparent V(max)/K(m) values for recombinant CYP3A5 indicated the greater contributions to diltiazem N-demethylation and midazolam 1'-hydroxylation as compared with CYP3A4. These results suggest that polymorphic CYP3A5 contributes markedly to the drug oxidations, particularly diltiazem N-demethylation, midazolam 1'- hydroxylation and testosterone 6beta-hydroxylation by liver microsomes from Japanese subjects.

Twenty-four genetic polymorphisms in the CYP2D6 gene were analysed in liver DNA samples of 39 Japanese and 44 Caucasians and compared with CYP2D6 protein levels and bufuralol 1'- and 6-hydroxylation activities in liver microsomes of these human samples. We detected 13 types of CYP2D6 genetic polymorphisms and classified these into 20 genotypes; nine types were found in Japanese and 14 types in Caucasian samples. CYP2D6*10B, but not CYP2D6*10A, was the most frequent (34.6%) in Japanese. In Caucasians, several CYP2D6 polymorphisms including CYP2D6*4, *4D, *4E, *4L, *3, *9, *5 and *2E (frequencies of 6.8, 3.4, 4.5, 9.1, 1.1, 2.3, 2.3 and 4.5%, respectively) were detected. A Caucasian having CYP2D6*3/*5 had a protein with slower gel mobility (immunoblotting with anti-CYP2D6 antibody) and very low activity for bufuralol 1'-hydroxylation. Five Caucasian samples (CYP2D6*4/*4, *4/*4L, or *4D/*4L) had no measurable CYP2D6 protein and very low bufuralol 1'-hydroxylation activities. Seven Japanese subjects with CYP2D6*10B/*10B had CYP2D6 protein at levels of approximately 20% of those present in humans with CYP2D6*1 and *2 and catalysed bufuralol 1'-hydroxylation at low rates. Kinetic analysis of bufuralol 1'- and 6-hydroxylation indicates that (i) the Km values for 1'-hydroxylation were lower in individuals with CYP2D6*1/*1, *1/*2, *1/*2X2, and *2/*2 than those with CYP2D6*4/*4, *4/*4L, *4D/*4L, or *10B/*10B and Vmax values tended to be higher in the former groups (*1, *2), and (ii) individuals with heterozygous CYP2D6*1/*4D, *1/*4L, and *1/*5 had relatively high Vmax/Km ratios, whereas individuals with heterozygous CYP2D6*1/*9, *2/4D, *2/*5, *2/*10B, *2E/*4E, *3/*5, *4L/*9, and *10B/*39 had lower Vmax/Km ratios for bufuralol 1'-hydroxylation. Quinidine inhibited bufuralol 1'-hydroxylation in liver microsomes, particularly at low substrate concentrations, in individuals with CYP2D6*1/*1, and 1/1*2, but not those with CYP2D6*4/*4 and very slightly in individuals with CYP2D6*10B/*10B. The latter two groups were found to be more sensitive to alpha-naphthoflavone than the former groups, indicative of the contribution of CYP1A2. These results support the view that CYP2D6*3, *4, *4D, and *4L are major genotypes producing poor metabolizer phenotypes in CYP2D6 in Caucasians, whereas CYP2D6*10B is a major factor in decreased CYP2D6 protein expression and catalytic activities in Japanese.

Genotypes of CYP2A6, namely CYP2A6(*)1 (wild-type), CYP2A6(*)2, and CYP2A6(*)3, were examined in liver DNA of 39 Japanese and 43 Caucasians using two-step polymerase chain reaction (PCR) methods. We first amplified a DNA fragment (1725 bp) located between near middle of exon 1 and end of exon 4 of the CYP2A6 gene and further amplified using a forward primer 't' or 'mut' (middle of exon 3) and a reverse primer 'E3R' (middle of intron 3) for the detection of CYP2A6(*)2-genetic polymorphism. The 1725 bp fragment was also used for the amplification between exon 3 and near middle of intron 3 of the CYP2A6 gene and the fragment thus obtained digested with XcmI or DdeI to detect and confirm the CYP2A6(*)2- and CYP2A6(*)3-types, respectively. Only one DNA sample from a Japanese origin (J18) was not amplified by CYP2A6-specific primers; liver microsomes from this individual had very low activity of coumarin 7-hydroxylation and were devoid of protein(s) immunoreactive to anti-CYP2A6 antibody. Thus, this individual was suggested to be due to the gene deletion in CYP2A6. By analyzing the remaining 38 Japanese and 43 Caucasians, we found that there were no cases of CYP2A6(*)3-type polymorphism in the samples examined in this study, and no cases of CYP2A6(*)2-type polymorphism in the Japanese samples. Of Caucasians studied two individuals were classified into heterozygous CYP2A6(*)1/(*)2-type. Liver microsomal coumarin 7-hydroxylation activities in these two Caucasians were found to be lower than those of the other 41 Caucasians. Kinetic analysis showed that two CYP2A6(*)1/(*)2 individuals had a very low ratio of V(max) to K(m) for nicotine C-oxidation as well as coumarin 7-hydroxylation in liver microsomes, compared with those of homozygous CYP2A6(*)1-type. These results suggest that among 39 Japanese and 43 Caucasians examined one Japanese is classified to be CYP2A6 gene deletion and two Caucasians are heterozygous CYP2A6(*)1/(*)2-genotype. Thus the race-related differences in the occurrence of CYP2A6 genetic polymorphisms were supported.

Ethnic differences in the pharmacokinetics of nifedipine, a substrate of CYP3A, and in CYP3A7 expression have been reported. The aim of the present study was to measure the protein levels of CYP3A4, CYP3A5, and CYP3A7 and nifedipine oxidation activity in hepatic microsomes from 15 Caucasian and 15 Japanese patients for comparison between the two ethnic groups. Nifedipine oxidation activity and CYP3A4 protein level were well correlated. No significant difference between Caucasian and Japanese microsomal samples was found in nifedipine oxidation activity or in the CYP3A4 protein level. CYP3A5 was detected in 6 of 15 Caucasian samples and in 5 of 15 Japanese samples, but no ethnic difference was found in either the frequency of expression or its protein level. CYP3A7 was found in 10 of 15 Caucasian samples and in 14 of 15 Japanese samples. Although the estimated CYP3A7 protein level was higher in the Japanese than in the Caucasian samples, its protein level was much lower than that of CYP3A4. These results imply that the contribution of CYP3A5 or CYP3A7 to the purported Caucasian-Japanese ethnic difference in the overall CYP3A activity seems to be small.

1. Tolbutamide methyl hydroxylation and racemic warfarin 7-hydroxylation activities were determined in liver microsomes of 39 Japanese and 45 Caucasians genotyped for the cytochrome P450 (P450 or CYP) 2C9 gene into three groups, namely the wild-type (Arg144.Ile359), and two heterozygous Cys allele (Cys144.Ile359) and Leu allele (Arg144.Leu359) variants. 2. Good correlations were found between tolbutamide methyl hydroxylation and racemic warfarin 7-hydroxylation activities in liver microsomes of Japanese and Caucasians. Humans with the Cys allele CYP2C9 variant, which was detected in 22% of Caucasians, were found to have similar catalytic rates to those of the wild-type in the oxidations of tolbutamide and racemic warfarin, whereas humans with the Leu allele, which was detected in 8% Japanese and 7% Caucasian samples, had lower catalytic rates than those of other two groups. 3. The rates of 6- and 7-hydroxylation of racemic warfarin were correlated well with those of S-warfarin, but not R-warfarin, in human liver microsomes. 4. Both human liver microsomes and recombinant CYP2C9 catalysed 7-hydroxylation of S-warfarin more extensively than those of R-warfarin. K(m)'s for the 7-hydroxylation of S-warfarin were not very different in liver microsomes of humans with these three genotypes. Anti-CYP2C9 antibodies and sulphaphenazole inhibited the 6- and 7-hydroxylation of S-warfarin, but not R-warfarin, by > 90% and the methyl hydroxylation of tolbutamide by about 50%. 5. These results suggest that humans with Leu allele of CYP2C9 have lower Vmax's for S-warfarin 7-hydroxylation and tolbutamide methyl hydroxylation than those with wild-type and Cys allele CYP2C9, although the K(m)'s are not very different in liver microsomes of these three groups of humans. R-warfarin hydroxylation may be catalysed by P450 enzymes other than CYP2C9 in man.

1. Interethnic differences in cytochrome P4502A6 (CYP2A6) levels and coumarin 7-hydroxylation activities were determined in liver microsomes of 30 Japanese and 30 Caucasians. 2. Although CYP2A6 levels and coumarin 7-hydroxylation activities varied very significantly in the 60 human samples examined, both CYP2A6 levels and coumarin 7hydroxylation activities were found to be higher in Caucasian than Japanese population. 3. Interestingly, eight of the 30 Japanese examined showed very low or undetectable levels of coumarin 7-hydroxylation activities as well as of CYP2A6 in liver microsomes. All of the Caucasians, however, had significant CYP2A6 levels and variable 7-hydroxylation activities. 4. Kinetic analvsis of coumarin 7-hydroxylation activities in liver microsomes of various human samples suggested that although there were 260-fold differences in Vmax's in 10 human samples examined, the Km's were very similar (2.1 + or - 107 mu M); a value consistent with that obtained (1.2 mu M) with purified CYP2A6 in reconstituted system. 5. The results suggest that CYP2A6 is actually involved in the 7-hydroxylation of coumarin in human liver microsomes, and that interethnic differences in coumarin 7-hydroxylation activities in Japanese and Caucasian population may be ascribed to the differences in expression of CYP2A6 protein.

Hepatic enzyme systems of drug metabolism and antioxidation were investigated in rats fed the complete human diets consumed in the two Japanese prefectures, Akita and Okinawa, where the incidence of cancers was quite different: Okinawa had the lowest and Akita the highest age-adjusted mortality rate. In rats fed the human diet consumed in Okinawa, hepatic glutathione S-transferase activity was higher and lipid peroxide content was lower than in rats fed the diet consumed in Akita. These data might indicate that the number and/or quantity of the dietary components attributed to the detoxification of carcinogens and the scavenging reactive carcinogen species was much higher in the foods consumed in the population having lower cancer mortality rate.

Interindividual variations in the level and activity of cytochrome P-450 enzymes were investigated in the liver microsomes of 30 Japanese and 30 Caucasian patients. The P-450 enzymes used in this study included P-450 1A2, 2A6, 2B6, 2C, 2D6, 2E1 and 3A, and the monooxygenase activities determined were 13 typical P-450 substrates and 9 procarcinogens. Although the total P-450 content was higher in Caucasian (mean, 0.43 nmol/mg of protein) than in Japanese populations (mean, 0.26 nmol/mg of protein), the relative levels (percent of total P-450) of individual forms of P-450 determined immunochemically were not very different except that P-450 2A6 and 2B6 levels were higher in the Caucasians. About 70% of liver P-450 could be accounted for by P-450 1A2, 2A6, 2B6, 2C, 2D6, 2E1 and 3A proteins, and P-450 3A (about 30% of total P-450) and 2C (about 20%) enzymes were found to be the major forms. Considerable levels of P-450 1A2 (about 13%) and 2E1 (about 7%) could be determined, whereas the P-450 2A6 (about 4%), 2D6 (about 2%) and 2B6 (< 1%) were the minor P-450 forms. Differences in some of the P-450 1A2-, 2A6-, 2D6-, 2E1- and 3A4-dependent activities were observed in Japanese and Caucasian populations. No clear sex-related differences in individual P-450 contents and drug- and carcinogen-metabolizing activities were detected in 60 human samples, except that P-450 1A2-dependent activities were found to be higher in mean than in women in the Caucasian population only. A single neonate sample tended to be lower in P-450 1A2-, 2A6- and 2E1-dependent activities. In contrast to rat counterparts, we could not detect apparent developmental changes in P-450 content and activity in humans between 12 and 73 years old. Thus, the results presented in this study provide useful information for the study of drug biotransformation in humans and for the basis of drug toxicities, carcinogenesis and teratogenesis.

The substrate specificity and other properties of a fatty acid hydroxylase system in liver microsomes of the Japanese house musk shrew, Suncus murinus, were examined. Shrew liver microsomes catalyzed the hydroxylation of various saturated fatty acids (C8-C18) to the corresponding omega- and (omega-1)-hydroxy derivatives. The relative activities of the hydroxylase with these substrates were as follows: C12 (100, actual conversion: 7.05 nmol/mg of microsomal protein/min), C10 (90), C14 (87), C16 (23), C8 (19), and C18 (11). The specific activity of the fatty acid hydroxylase in shrew liver was much higher than that in other species. The omega/omega-1-hydroxylation ratio decreased with increasing chain length of fatty acid substrates (C10-C18), but it was 0 for the C8 fatty acid. Both NADPH and O2 were required for hydroxylase activity, and NADH had little effect. The apparent Km value for laurate was 1.6 X 10(-5) M. The hydroxylase activity was 92% inhibited by CO at a CO-O2 ratio of 9. p-Chloromercuribenzoate (0.1 mM) inhibited hydroxylation by 94% whereas iodoacetate (0.1 mM) inhibited it by only 8%. SKF 525-A (1 mM) and menadione (0.01 mM), respectively, caused 41% and 29% inhibition of the activity. It is concluded that the hydroxylase catalyzing fatty acid hydroxylation in shrew liver microsomes is a typical cytochrome P-450-linked monooxygenase.