Biomedical subjects

Tsuyoshi Yokoi

Publications and source records attributed to Tsuyoshi Yokoi.

At least 37 records · Page 2Linked to original sources

PubMed2005

CYP2A6 AND CYP2B6 are involved in nornicotine formation from nicotine in humans: interindividual differences in these contributions.

Nornicotine is an N-demethylated metabolite of nicotine. In the present study, human cytochrome P450 (P450) isoform(s) involved in nicotine N-demethylation were identified. The Eadie-Hofstee plot of nicotine N-demethylation in human liver microsomes was biphasic with high-affinity (apparent K(m) = 173 +/- 70 microM, V(max) = 57 +/- 17 pmol/min/mg) and low-affinity (apparent K(m) = 619 +/- 68 microM, V(max) = 137 +/- 6 pmol/min/mg) components. Among 13 recombinant human P450s expressed in baculovirus-infected insect cells (Supersomes), CYP2B6 exhibited the highest nicotine N-demethylase activity, followed by CYP2A6. The apparent K(m) values of CYP2A6 (49 +/- 12 microM) and CYP2B6 (550 +/- 46 microM) were close to those of high- and low-affinity components in human liver microsomes, respectively. The intrinsic clearances of CYP2A6 and CYP2B6 Supersomes were 5.1 and 12.5 nl/min/pmol P450, respectively. In addition, the intrinsic clearance of CYP2A13 expressed in Escherichia coli (44.9 nl/min/pmol P450) was higher than that of CYP2A6 expressed in E. coli (2.6 nl/min/pmol P450). Since CYP2A13 is hardly expressed in human livers, the contribution of CYP2A13 to the nicotine N-demethylation in human liver microsomes would be negligible. The nicotine N-demethylase activity in microsomes from 15 human livers at 20 microM nicotine was significantly correlated with the CYP2A6 contents (r = 0.578, p < 0.05), coumarin 7-hydroxylase activity (r = 0.802, p < 0.001), and S-mephenytoin N-demethylase activity (r = 0.694, p < 0.005). The nicotine N-demethylase activity at 100 microM nicotine was significantly correlated with the CYP2B6 contents (r = 0.677, p < 0.05) and S-mephenytoin N-demethylase activities (r = 0.740, p < 0.005). These results as well as the inhibition analyses suggested that CYP2A6 and CYP2B6 would significantly contribute to the nicotine N-demethylation at low and high substrate concentrations, respectively. The contributions of CYP2A6 and CYP2B6 would be dependent on the expression levels of these isoforms in any human liver.

Adult↗

PubMed2005

Detection of autoantibody to aldolase B in sera from patients with troglitazone-induced liver dysfunction.

Troglitazone is a thiazolidinedione antidiabetic agent with insulin-sensitizing activities that was withdrawn from the market in 2000 due to its association with idiosyncratic hepatotoxicity. To address the suspected autoantibody production associated with troglitazone, we investigated autoantibodies in sera from patients with type II diabetes mellitus with troglitazone-induced liver dysfunction. Two female patients (47- and 70-year-old) ceased taking troglitazone (400 mg/day) after 23.5 and 16 weeks, respectively, due to increased serum ALT. Using two-dimensional electrophoresis and amino acid sequence analyses, aldolase B was identified as an autoantigen that reacted with antibodies in sera from both patients. The titer of anti-aldolase B remained high for several weeks after stopping troglitazone administration. The mean reactivity of autoantibodies to aldolase B determined by ELISA with sera of patients with chronic hepatitis (n = 40) and liver cirrhosis (n = 40) was significantly higher (p < 0.05 and p < 0.001, respectively) than with sera of healthy subjects (n = 80). These findings suggest that liver injury may cause the appearance of autoantibodies to aldolase B which may then aggravate the hepatitis. In addition, the anti-aldolase B titer might indicate the severity of liver dysfunction.

Aged↗

PubMed2005

Relationship between hepatic gene expression profiles and hepatotoxicity in five typical hepatotoxicant-administered rats.

In the field of gene expression analysis, DNA microarray technology has a major impact on many different areas including toxicogenomics, such as in predicting the adverse effects of new drug candidates and improving the process of risk assessment and safety evaluation. In this study, we investigated whether there is relationship between the hepatotoxic phenotypes and gene expression profiles of hepatotoxic chemicals measured by DNA microarray analyses. Sprague-Dawley rats (6 weeks old) were administered five hepatotoxicants: acetaminophen (APAP), bromobenzene, carbon tetrachloride, dimethylnitrosamine, and thioacetamide. Serum biochemical markers for liver toxicity were measured to estimate the maximal toxic time of each chemical. Hepatic mRNA was isolated, and the gene expression profiles were analyzed by DNA microarray containing 1,097 drug response genes, such as cytochrome P450s, other phase I and phase II enzymes, nuclear receptors, signal transducers, and transporters. All the chemicals tested generated specific gene expression patterns. APAP was sorted to a different cluster from the other four chemicals. From the gene expression profiles and maximal toxic time estimated by serum biochemical markers, we identified 10 up-regulated genes and 10 down-regulated genes as potential markers of hepatotoxicity. By Quality-Threshold (QT) clustering analysis, we identified major up- and down-regulated expression patterns in each group. Interestingly, the average gene expression patterns from the QT clustering were correlated with the mean value profiles from the biochemical markers. Furthermore, this correlation was observed at any extent of hepatotoxicity. In this study, we identified 17 potential toxicity markers, and those expression profiles could estimate the maximal toxic time independently of the hepatotoxicity levels. This expression profile analysis could be one of the useful tools for evaluating a potential hepatotoxicant in the drug development process.

Acetaminophen↗

PubMed2005

Expression of human phase II enzymes in chimeric mice with humanized liver.

We clarified that major human cytochrome P450 (P450) enzymes were expressed in a chimeric mouse line established recently in Japan, in which the liver could be replaced by more than 80% with human hepatocytes. In this study, we investigated major human phase II enzymes such as UDP-glucuronosyltransferase (UGT), sulfotransferase (SULT), N-acetyltransferase (NAT), and glutathione S-transferase (GST) in the livers of chimeric mice by mRNA, protein, and enzyme activity using reverse transcription-polymerase chain reaction, Western blot analysis, and high-performance liquid chromatography, respectively. Human UGT, SULT, NAT, and GST mRNA were expressed in the liver of the chimeric mice, and UGT2B7, SULT1E1, SULT2A1, and GSTA1 proteins could be detected. The expression of mRNA and protein was correlated with the human albumin (hAlb) concentration in mouse blood, the replacement of which by human hepatocytes could be estimated by the hAlb concentration in the blood of the chimeric mice, because the chimeric mice produce human albumin. The enzyme activities, such as morphine 6-glucuronosyltransferase activity and estrone 3-sulfotransferase activity, activities that are specific to humans but not to mice, were increased in a hAlb concentration-dependent manner. The chimeric mice with humanized liver with nearly 90% replacement by human hepatocytes demonstrated almost the same protein contents of human phase II enzymes and enzyme activities as those of the donor. In conclusion, the chimeric mice exhibited an efficient capacity of drug conjugation similar to that in humans. These chimeric mice expressed human phase II enzymes as well as P450s, suggesting that they could be a useful animal model in drug development.

Animals↗

PubMed2005

Characterization of novel CYP2A6 polymorphic alleles (CYP2A618 and CYP2A619) that affect enzymatic activity.

Genetic polymorphisms of CYP2A6 gene are known as a causal factor of the interindividual differences in nicotine metabolism. We found three novel CYP2A6 alleles. The CYP2A6(*)18A allele has a single nucleotide polymorphism (SNP) of A5668T (A1175T, Y392F) in exon 8. The CYP2A6(*)18B allele has synonymous SNPs of G51A (G51A), T5684C (T1191C), and T5702C (T1209C) in addition to A5668T (A1175T, Y392F). The CYP2A6(*)19 allele has the SNPs of A5668T (A1175T, Y392F), T6354C (intron 8), and T6558C (T1412C, I471T) as well as the conversion with the CYP2A7 sequence in the 3'-untranslated region, in which the latter two changes correspond to CYP2A6(*)7. Ethnic differences in the frequencies of these alleles were observed between whites, African-Americans, Japanese, and Koreans. Wild or variant CYP2A6 (CYP2A6(*)18, CYP2A6(*)19, and CYP2A6(*)7) were expressed in Escherichia coli. For coumarin 7-hydroxylation and 5-fluorouracil formation from tegafur, the K(m) values were increased, and V(max) values were decreased in CYP2A6.18 compared with those in CYP2A6.1, resulting in decreased clearance to 50 and 35% of that of the wild type, respectively. The K(m) and V(max) values for nicotine C-oxidation were both increased, resulting in no change of clearance. In CYP2A6.19, the effects on the coumarin 7-hydroxylation and 5-fluorouracil formation (increased K(m) and decreased V(max)) were prominent, resulting in decreased clearance to 8% of those of the wild type. For nicotine C-oxidation, the K(m) and V(max) values were both decreased, resulting in decreased clearance to 30% of that of the wild type. The changes of the kinetics in CYP2A6.19 were similar to those in CYP2A6.7. In vivo nicotine metabolism was evaluated in whites (n = 56) and Koreans (n = 40). Although the CYP2A6(*)18 and CYP2A6(*)19 alleles were found only heterozygously, a subject with CYP2A6(*)7/CYP2A6(*)19 showed a lower cotinine/nicotine ratio of the plasma concentration compared with homozygotes of the CYP2A6(*)1A, supporting the in vitro results that the CYP2A6(*)19 allele leads to decreased enzymatic activity.

Black or African American↗

PubMed2005

In vivo induction of human cytochrome P450 enzymes expressed in chimeric mice with humanized liver.

The induction and inhibition of human cytochrome P450 (P450) enzymes are clinically responsible for drug interactions. Although the induction of P450s is investigated using human hepatocytes in the drug development process, there are some disadvantages, such as the decline of the enzyme activity during culture. In the present study, we examined the in vivo induction potency in chimeric mice with humanized liver, which was recently established in Japan to clarify whether this chimeric mouse model would be more suitable for human induction studies. Rifampicin and 3-methylcholanthrene (3-MC) were used in vivo as typical P450 inducers in the chimeric mice. The expression levels of human CYP3A4 mRNA and CYP3A4 protein and dexamethasone 6-hydroxylase activity, specific for human CYP3A4, were increased 8- to 22-, 3- to 10-, and 5- to 12-fold, respectively, by treatment with rifampicin. In addition, the expression levels of human CYP1A2 mRNA and CYP1A2 protein were also increased 2- to 9- and 5-fold, respectively, by treatment with 3-MC. Although other human P450s are expressed in the chimeric mice, there were few effects by the treatment of rifampicin and 3-MC on the mRNA, protein, and enzyme activity of those P450s. It was demonstrated that human P450s expressed in the chimeric mice with humanized liver were induced by rifampicin and 3-MC. This chimeric mouse model may be a useful animal model to estimate and predict the in vivo induction of P450s in humans.

Animals↗

PubMed2005

Cancer pharmacogenomics: achievements in basic research.

Pharmacogenomics is the study of how genetic inheritance influences the responses to drugs. Genetic polymorphisms in drug-metabolizing enzymes result in altered pharmacokinetics in therapeutic drugs. In recent years, there has been great progress in our knowledge of the effects of cytochrome P450 (CYP) polymorphisms on the pharmacokinetics of therapeutic drugs. CYP enzymes catalyze the activation or detoxification of several anticancer drugs. Anticancer drugs generally have a narrow therapeutic margin. Therefore, the interindividual variability in their efficacy and toxicity is a major problem in clinical practice. In this review, genetic polymorphisms of CYP enzymes and their clinical relevance in cancer chemotherapy are discussed.

Antineoplastic Agents↗

PubMed2005

Pharmacokinetics of paclitaxel in ovarian cancer patients and genetic polymorphisms of CYP2C8, CYP3A4, and MDR1.

Interindividual differences in the pharmacokinetics of paclitaxel and its metabolites in Japanese ovarian cancer patients were investigated in relation to genetic polymorphisms of the CYP2C8, CYP3A4, and MDR1 genes. The area under the concentration-time curve (AUC) ratios of paclitaxel/6alpha-hydroxypaclitaxel and paclitaxel/3 -p-hydroxypaclitaxel calculated as the metabolic index of CYP2C8 and CYP3A4 showed 13- and 12-fold interindividual variations, respectively. No patient had any CYP2C8 variants, while 2 patients were heterozygotes of CYP3A4*16. For the MDR1 gene, the frequencies of -129C, 1236C, 2677T, 2677A, and 3435T alleles were 2.2%, 8.7%, 56.5%, 4.4%, and 52.2%, respectively. Subjects possessing the 3435T allele had a significantly (P < .05) higher AUC of 3'- p-hydroxypaclitaxel compared to those possessing the 3435C allele. Leukocytopenia was significantly (P < .05) related to the AUC of paclitaxel. Genotyping of the CYP2C8, CYP3A4, and MDR1 genes might not be essential to predict adverse effects of paclitaxel in Japanese patients, although an allelic variant of MDR1 may functionally affect the pharmacokinetics of its metabolite.

Area Under Curve↗

PubMed2005

Inhibitory effects of nicardipine to cytochrome P450 (CYP) in human liver microsomes.

To anticipate drug-drug interactions by nicardipine in vivo, cytochrome P450 (CYP) forms responsible for the metabolism of nicardipine and inhibition of CYP-dependent drug metabolism by nicardipine were investigated. Microsomes of human B-lymphoblastoid cells expressing each human CYP form were used for the metabolism of nicardipine. Inhibitory effects of nicardipine on drug metabolism were studied using human liver microsomes. CYP2C8, CYP2D6 and CYP3A4 were identified as major CYP forms for the metabolism of nicardipine in human liver microsomes. Nicardipine strongly inhibited two-pathways of triazolam hydroxylation both catalyzed by CYP3A4. Comparison of three Ca(2+) antagonists, nicardipine, nifedipine, and diltiazem revealed that only nicardipine showed such a strong inhibitory potency on the typical CYP2D6-catalyzed drug metabolism. Furthermore, nicardipine inhibited other reactions catalyzed by CYP1A, CYP2A6, CYP2C8, CYP2C9 and CYP2C19 with K(i) values ranging from 1.1 to 29.4 microM. In conclusion, nicardipine was a relatively potent inhibitor of human CYP2D6, CYP3A4 and CYP2C (especially for CYP2C8 and CYP2C19) in vitro, suggesting that drug-drug interactions between nicardipine and other drugs metabolized mainly by these CYP forms appear to occur in vivo.

Cell Line↗

PubMed2005

Induction of human CYP1A2 and CYP3A4 in primary culture of hepatocytes from chimeric mice with humanized liver.

Chimeric mice with near-completely humanized liver were constructed by transplantating hepatocytes from a Japanese and Caucasian donor. In the present study, we investigated the induction of human CYP1A2 and CYP3A4 mRNA in a primary culture of the cryopreserved chimeric mouse hepatocytes. beta-naphthoflavone (beta-NF) and rifampicin (Rif) were used as typical cytochrome P450 (CYP) inducers for CYP1A2 and CYP3A4, respectively. Analysis was performed by the real-time reverse-transcription polymerase chain reaction method. CYP1A2 mRNA in the primary culture of chimeric mouse hepatocytes in mice No. 1, 2, and 3 was significantly increased 3.8-, 6.3-, and 3.3-fold by 5 microM beta-NF exposure, respectively, compared with the 0.1% DMSO treated control (p<0.01). CYP3A4 mRNA in the primary culture of chimeric mouse hepatocytes in mice No. 1, 2, and 3 was significantly increased 8.4-fold (p<0.001), 2.2-fold (p<0.01), and 2.3-fold (p<0.05) by 50 microM Rif exposure, respectively, compared with the 0.1% DMSO treated control. The present study demonstrated that a primary culture of cryopreserved hepatocytes from chimeric mice with humanized liver could be used for evaluating the induction of drug metabolizing enzymes in human. This in vitro method may be a useful method for screening the induction potency of new drug candidates on drug metabolizing enzymes.

Adolescent↗

PubMed2005

Urinary excretion of phenytoin metabolites, 5-(4'-hydroxyphenyl)-5-phenylhydantoin and its O-glucuronide in humans and analysis of genetic polymorphisms of UDP-glucuronosyltransferases.

The anticonvulsant agent phenytoin (5,5-diphenylhydantoin) is mainly excreted as 5-(4'-hydroxyphenyl)-5-phenylhydantoin (4'-HPPH) O-glucuronide in humans. Previously, we demonstrated that the glucuronidation of 4'-HPPH is catalyzed by multiple UDP-glucuronosyltransferases (UGTs) of UGT1A1, UGT1A4, UGT1A6, and UGT1A9. Since 4'-HPPH may be bioactivated to a reactive metabolite by peroxidase, the glucuronidation in considered to be a detoxification pathway. In the present study, we investigated the relationship between the extent of interindividual variability in the urinary excretion levels of 4'-HPPH and its O-glucuronide and genotyping of CYP2C9, CYP2C19, UGT1A1, UGT1A6, and UGT1A9. 4'-HPPH and its glucuronide in urine samples from 15 patients to whom phenytoin was administered were measured by liquid chromatography-tandem mass spectrometry. When the molar ratio of 4'-HPPH O-glucuronide/4'-HPPH was calculated as an index of glucuronidation, a large interindividual variability (11 fold) was observed in the 15 patients. Phenytoin is metabolized to 4'-HPPH by CYP2C9 and CYP2C19 in which there are genetic polymorphisms. Although 5 patients were genotyped as heterozygotes of mutated alleles of CYP2C9 or CYP2C19 genes, no relationship with the interindividual difference in the total excretion levels of 4'-HPPH and its O-glucuronide was observed. The UGT1A1*6, UGT1A1*28, UGT1A1*60 and UGT1A6*2 alleles were found in 1, 3, 6, and 8 patients, respectively. Although there was no relationship between the genetic polymorphisms of UGT1As and the interindividual difference in the 4'-HPPH glucuronidation, the large interindividual variability of 4'- HPPH glucuronidation may contribute to interindividual differences in toxic reactions to phenytoin.

Adolescent↗

PubMed2005

Interindividual variability in nicotine metabolism: C-oxidation and glucuronidation.

Nicotine has roles in the addiction to smoking, replacement therapy for smoking cessation, as a potential medication for several diseases such as Parkinson's disease, Alzheimer's disease, and ulcerative colitis. The absorbed nicotine is rapidly and extensively metabolized and eliminated to urine. A major pathway of nicotine metabolism is C-oxidation to cotinine, which is catalyzed by CYP2A6 in human livers. Cotinine is subsequently metabolized to trans-3'-hydroxycotinine by CYP2A6. Nicotine and cotinine are glucuronidated to N-glucuronides mainly by UGT1A4 and partly by UGT1A9. Trans-3'-hydroxycotinine is glucuronidated to O-glucuronide mainly by UGT2B7 and partly by UGT1A9. Approximately 90% of the total nicotine uptake is eliminated as these metabolites and nicotine itself. The nicotine metabolism is an important determinant of the clearance of nicotine. Recently, advances in the understanding of the interindividual variability in nicotine metabolism have been made. There are substantial data suggesting that the large interindividual differences in cotinine formation are associated with genetic polymorphisms of the CYP2A6 gene. Interethnic differences have also been observed in the cotinine formation and the allele frequencies of the CYP2A6 alleles. Since the genetic polymorphisms of the CYP2A6 gene have a major impact on nicotine clearance, its relationships with smoking behavior or the risk of lung cancer have been suggested. The metabolic pathways of the glucuronidation of nicotine, cotinine, and trans-3'-hydroxycotinine in humans would be one of the causal factors for the interindividual differences in nicotine metabolism. This review mainly summarizes recent results from our studies.

Black or African American↗

PubMed2004

Cytochrome P450-mediated metabolism of estrogens and its regulation in human.

Estrogens are eliminated from the body by metabolic conversion to estrogenically inactive metabolites that are excreted in the urine and/or feces. The first step in the metabolism of estrogens is the hydroxylation catalyzed by cytochrome P450 (CYP) enzymes. Since most CYP isoforms are abundantly expressed in liver, the metabolism of estrogens mainly occurs in the liver. A major metabolite of estradiol, 2-hydroxyestradiol, is mainly catalyzed by CYP1A2 and CYP3A4 in liver, and by CYP1A1 in extrahepatic tissues. However, CYP1B1 which is highly expressed in estrogen target tissues including mammary, ovary, and uterus, specifically catalyzes the 4-hydroxylation of estradiol. Since 4-hydroxyestradiol generates free radicals from the reductive-oxidative cycling with the corresponding semiquinone and quinone forms, which cause cellular damage, the specific and local formation of 4-hydroxyestradiol is important for breast and endometrial carcinogenesis. Changes in the expression level of estrogen-metabolizing CYP isoforms not only alter the intensity of the action of estrogen but may also alter the profile of its physiological effect in liver and target tissues. Generally, many CYP isoforms are induced by the substrates themselves, resulting in enhanced metabolism and elimination from the body. Of particular interest is a novel finding that human CYP1B1 is regulated by estradiol via the estrogen receptor. This fact suggests that the regulation of CYP enzymes involved in estrogen metabolism by estrogen itself would be physiologically significant for the homeostasis of estrogens at local organs. In this mini-review, we discuss the CYP-mediated metabolism of estrogens and the regulation of the estrogen-metabolizing CYP enzymes in relation to the risk of cancer.

Animals↗

PubMed2004

Chaperone proteins involved in troglitazone-induced toxicity in human hepatoma cell lines.

Troglitazone (TRO), an effective thiazolidinedione antidiabetic agent, was reported to produce idiosyncratic hepatotoxic effects in some individuals. In contrast, rosiglitazone (RSG), in the same group of agents, has no significant toxic effects and now is widely used. In this study, human hepatoma (HepG2) cell lines were exposed to various doses of TRO as well as RSG (0, 25, 50, and 75 microM) for 48 h. Cell lysates were separated by two-dimensional electrophoresis, and the gels were stained with coomassie brilliant blue to compare the spot profiles. The greatest protein expression at a MW of 75 kDa and isoelectric point of 5 was specifically increased with TRO treatments of 50 and 75 microM. The spot was identified as a mixture of immunoglobulin heavy chain binding protein (BiP) and, to a lesser extent, protein disulfide isomerase-related protein (PDIrp). Immunoblot analyses showed that the BiP protein was dose-dependently increased by TRO treatment and, to a lower degree, by RSG. These effects were also correlated with the high induction of BiP mRNA by TRO (50 and 75 microM) and the lower induction by RSG. However, both treatments showed no significant effects on PDIrp expression. The toxic effects of TRO in relation to the overexpression of BiP were also demonstrated in HLE cells, another human hepatoma cell line. In HLE cells, the inhibition of BiP expression by small interference RNA rendered cells more susceptible to the toxic effects of TRO. These results suggest that the overexpression of BiP is a defense mechanism of the endoplasmic reticulum in response to TRO-induced toxicity.

Amino Acid Sequence↗

PubMed2004

Trans-3'-hydroxycotinine O- and N-glucuronidations in human liver microsomes.

Trans-3'-hydroxycotinine is a major metabolite of nicotine in humans and is mainly excreted as O-glucuronide in smoker's urine. Incubation of human liver microsomes with UDP-glucuronic acid produces not only trans-3'-hydroxycotinine O-glucuronide but also N-glucuronide. The formation of N-glucuronide exceeds the formation of O-glucuronide in most human liver microsomes, although N-glucuronide has never been detected in human urine. Trans-3'-hydroxycotinine N-glucuronidation in human liver microsomes was significantly correlated with nicotine and cotinine N-glucuronidations, which are catalyzed mainly by UDP-glucuronosyltransferase (UGT)1A4 and was inhibited by imipramine and nicotine, which are substrates of UGT1A4. Recombinant UGT1A4 exhibited substantial trans-3'-hydroxycotinine N-glucuronosyltransferase activity. These results suggest that trans-3'-hydroxycotinine N-glucuronidation in human liver microsomes would be mainly catalyzed by UGT1A4. In the present study, trans-3'-hydroxycotinine O-glucuronidation in human liver microsomes was thoroughly characterized, since trans-3'-hydroxycotinine O-glucuronide is one of the major metabolites of nicotine. The kinetics were fitted to the Michaelis-Menten equation with a K(m) of 10.0 +/- 0.8 mM and a V(max) of 85.8 +/- 3.8 pmol/min/mg. Among 11 recombinant human UGT isoforms expressed in baculovirus-infected insect cells, UGT2B7 exhibited the highest trans-3'-hydroxycotinine O-glucuronosyltransferase activity (1.1 pmol/min/mg) followed by UGT1A9 (0.3 pmol/min/mg), UGT2B15 (0.2 pmol/min/mg), and UGT2B4 (0.2 pmol/min/mg) at a substrate concentration of 1 mM. Trans-3'-hydroxycotinine O-glucuronosyltransferase activity by recombinant UGT2B7 increased with an increase in the substrate concentration up to 16 mM (10.5 pmol/min/mg). The kinetics by recombinant UGT1A9 were fitted to the Michaelis-Menten equation with K(m) = 1.6 +/- 0.1 mM and V(max) = 0.69 +/- 0.02 pmol/min/mg of protein. Trans-3'-hydroxycotinine O-glucuronosyltransferase activities in 13 human liver microsomes ranged from 2.4 to 12.6 pmol/min/mg and were significantly correlated with valproic acid glucuronidation (r = 0.716, p < 0.01), which is catalyzed by UGT2B7, UGT1A6, and UGT1A9. Trans-3'-hydroxycotinine O-glucuronosyltransferase activity in human liver microsomes was inhibited by imipramine (a substrate of UGT1A4, IC(50) = 55 microM), androstanediol (a substrate of UGT2B15, IC(50) = 169 microM), and propofol (a substrate of UGT1A9, IC(50) = 296 microM). Interestingly, imipramine (IC(50) = 45 microM), androstanediol (IC(50) = 21 microM), and propofol (IC(50) = 41 microM) also inhibited trans-3'-hydroxycotinine O-glucuronosyltransferase activity by recombinant UGT2B7. These findings suggested that trans-3'-hydroxycotinine O-glucuronidation in human liver microsomes is catalyzed by mainly UGT2B7 and, to a minor extent, by UGT1A9.

Animals↗

PubMed2004

Expression of human cytochromes P450 in chimeric mice with humanized liver.

Recently, a chimeric mouse line in which the liver could be replaced by more than 80% with human hepatocytes was established in Japan. Because the chimeric mouse produces human albumin (hAlb), replacement by human hepatocytes could be estimated by the hAlb concentration in the blood of chimeric mice. In this study, we investigated human major cytochrome P450 (P450) in the livers of chimeric mice by mRNA, protein, and enzyme activity using real-time polymerase chain reaction, Western blot analysis, and high-performance liquid chromatography, respectively. Chimeric mice with humanized liver generated using hepatocytes from a Japanese and white donor were used. Human P450 mRNAs were expressed in the liver of chimeric mice, and major human P450 proteins such as CYP1A2, CYP2C9, and CYP3A4 were detected. The expression of P450 mRNA and protein was correlated with the hAlb concentration in the blood. The enzyme activities such as diclofenac 4'-hydroxylase activity, dexamethasone 6-hydroxylase activity, and coumarin 7-hydroxylase activity, activities that are specific to human P450 but not to murine P450, were increased in a hAlb concentration-dependent manner. The chimeric mice with nearly 90% replacement by human hepatocytes demonstrated almost the same protein contents of human P450s and drug-metabolizing enzyme activity as those of the donor. It was confirmed that genomic DNA from the livers of the chimeric mice and that from the liver of the donor exhibited the same genotype. In conclusion, the chimeric mice exhibited a similarly efficient capacity of drug metabolism as humans, suggesting that they could be a useful animal model for drug development.

Alleles↗

PubMed2004

Identification of the cytosolic carboxylesterase catalyzing the 5'-deoxy-5-fluorocytidine formation from capecitabine in human liver.

Capecitabine, a prodrug of 5-fluorouracil, is first metabolized to 5'-deoxy-5-fluorocytidine (5'-DFCR) by carboxylesterase (CES), which is mainly expressed in microsomes. Recently, we clarified that 5'-DFCR formation was catalyzed by the enzyme in cytosol as well as microsomes in human liver. In the present study, the cytosolic enzyme involved in 5'-DFCR formation from capecitabine was identified. This enzyme was purified in the cytosolic preparation by ammonium sulfate precipitation, Sephacryl S-300 gel filtration, Mono P chromatofocusing, and Superdex 200 gel filtration. The purified enzyme was identified by the amino acid sequence analysis to be CES1A1 or a CES1A1 precursor. Based on the result of the N-terminal amino acid sequence analysis, the purified enzyme has no putative signal peptide, indicating that it was CES1A1. The apparent Km and Vmax values of 5'-DFCR formation were 19.2 mM and 88.3 nmol/min/mg protein, respectively. The 5'-DFCR formation catalyzed by the purified enzyme was inhibited by both diisopropylfluorophosphate and bis(p-nitrophenyl)phosphate in a concentration-dependent manner. 7-Ethyl-10-hydroxycamptothecin (SN-38) formation from irinotecan also occurred in the purified enzyme, cytosol, and microsomes. In conclusion, the cytosolic enzyme involved in 5'-DFCR formation from capecitabine would be CES1A1. It is suggested that the cytosolic CES has significant hydrolysis activity and plays an important role as the microsomal CES in drug metabolism. It is worthy to investigate the metabolic enzyme in cytosol involved in the activation of ester-type prodrugs such as capecitabine.

Amino Acid Sequence↗

PubMed2004

Novel human CYP2A6 alleles confound gene deletion analysis.

Cytochrome P450 (CYP) 2A6 metabolizes a number of drugs and a variety of procarcinogens. CYP2A6 also catalyzes nicotine C-oxidation leading to cotinine formation, a major metabolic pathway of nicotine in humans. There are genetic polymorphisms in the human CYP2A6 gene and a relationship between the CYP2A6 genotype and smoking habits as well as the incidence of lung cancer has been indicated. CYP2A6*4 alleles are the whole deleted type and are completely deficient in the enzymatic activity. An unequal crossover junction is located in the 3'-flanking region in the CYP2A6*4A allele, whereas the junction is located in either intron 8 or exon 9 in the CYP2A6*4D allele. In the present study, a novel genotyping method to distinguish between two different whole deleted alleles of CYP2A6*4A and CYP2A6*4D was established. In the process, two novel alleles, CYP2A6*1F and CYP2A6*1G, were found. The CYP2A6*1F has a single nucleotide polymorphism (SNP) of C5717T in exon 8, and the CYP2A6*1G has two SNPs, C5717T in exon 8 and A5825G in intron 8. The SNP of C5717T corresponds to C1224T on the cDNA sequence and is a synonymous mutation. Since the CYP2A6*1F produces a recognition site of the restriction enzymes that is the same as CYP2A6*4D, the presence of the CYP2A6*1F allele could cause a mistyping as the CYP2A6*4D allele. According to an improved genotyping method, the allele frequencies of CYP2A6*4A, CYP2A6*4D, CYP2A6*1F, and CYP2A6*1G in 165 Caucasians were 3.0%, 0%, 1.8%, and 1.2%, respectively. The allele frequencies of CYP2A6*4A, CYP2A6*4D, CYP2A6*1F, and CYP2A6*1G in 94 African-Americans were 0%, 0.5%, 0%, and 13.3%, respectively. This is the first report of a method that can distinguish between CYP2A6*4A, CYP2A6*4D, and CYP2A6*1F which could otherwise cause a mistyping as CYP2A6*4D.

Aryl Hydrocarbon Hydroxylases↗