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T Kamataki

Publications and source records attributed to T Kamataki.

At least 73 records · Page 4Linked to original sources
PubMed1998

Silencing of CYP1A1 expression in rabbits by DNA methylation.

Unlike most experimental animals, treatment of adult rabbits with 3-methylcholanthrene (MC) does not induce the expression of the CYP1A1 gene. In this study, we show that DNA methylation plays one of the key roles in the suppression of CYP1A1 gene expression. S1 nuclease protection assay showed that the induction of CYP1A1 mRNA by MC occurred in rabbit kidney RK13 cells but not in rabbit lung R9ab cells, while aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (Arnt) mRNAs were expressed in both cells at similar levels. Interestingly, the treatment of R9ab cells with a DNA demethylating agent, 5-aza-2'-deoxycitidine, resulted in the induction of the expression of the CYP1A1 gene by MC. The results indicate that DNA methylation is one of the factors involved in the loss of the MC-induced expression of the CYP1A1 gene. Thus, it seemed that the binding of the AhR/Arnt complex to the xenobiotic-responsive element (XRE) was inhibited by the hypermethylation of CpG dinucleotides within an XRE core sequence (5'-CGTG-3'). To explore this possibility, we compared the methylation status of XRE in R9ab cells with that in RK13 cells. A bisulfite sequence analysis using genomic DNAs from R9ab cells showed that the CpG site within XRE was highly methylated on both coding and non-coding strands. In contrast to this result, the hypomethylation of XRE was seen in RK13 cells. To examine whether or not the binding of the AhR/Arnt heterodimer to XRE is affected by the methylation status of XRE, a gel shift assay using a methylated XRE as a probe was carried out. As expected, the AhR/Arnt complex could not bind to the methylated XRE. From these results, we conclude that the cell type-specific transcription of the rabbit CYP1A1 gene is caused by DNA methylation.

Animals↗
PubMed1998

Establishment of a Salmonella tester strain highly sensitive to mutagenic heterocyclic amines.

Heterocyclic amines (HCAs) that are present in cooked foods require metabolic activation to exert their genotoxicity. They undergo activation via N-hydroxylation by cytochrome P450 1A2 (CYP1A2), followed by O-esterification by O-acetyltransferase (OAT). To develop a Salmonella tester strain that is highly sensitive to mutagenic HCAs, we introduced a coexpression plasmid (p1A2OR) carrying human CYP1A2 and NADPH-CYP reductase cDNAs and an expression plasmid (pOAT) carrying Salmonella OAT to Salmonella typhimurium TA1538 to yield a TA1538/ARO strain. The TA1538/ARO strain was proven to express the enzymes, as indicated by high activities of 7-ethoxyresorufin O-deethylase and isoniazid N-acetylase. The TA1538/ARO strain exhibited very high sensitivity to mutagenic HCAs 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and a somewhat higher sensitivity to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine compared with the parent Ames tester strain TA1538. The minimum concentrations of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, IQ, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine giving positive results were defined by evidence that the number of colonies increased in a dose-dependent manner and reached a number two times higher than that obtained by vehicle alone as a control in the TA1538/ARO strain at concentrations of 0.3, 3, 30, and 1000 pM, respectively. When the membrane and cytosol fractions prepared from TA1538/ARO were added to a mixture containing the parental TA1538, the sensitivity of TA1538 to IQ was much lower than that seen with TA1538/ARO. These results indicate that the intracellular expression of drug-metabolizing enzymes makes the established strain of Salmonella highly sensitive to mutagenic HCAs.

Acetyltransferases↗
PubMed1998

Separation of microsomal cytochrome b5 via phase separation in a mixed solution of Triton X-114 and charged dextran.

The successful introduction of a charged dextran into the Triton X-114 phase separation system for the selective extraction of cytochrome b5 (cyt. b5) in liver microsomes is described. In the absence of charged dextran, 55% of total microsomal proteins and 84% of cyt. b5 were extracted into the surfactant-rich phase. In the presence of anionic dextran sulfate, the extractability of total microsomal proteins was greatly reduced while that of cyt. b5 was increased. After triplicate extraction, cyt. b5 was purified more than 10-fold from microsomes with a recovery of 91% in the surfactant-rich phase. In view of its operational simplicity, this method provides a good means for the partial purification of cyt. b5 prior to chromatographic separations.

Animals↗
PubMed1998

High catalytic activity of human cytochrome P450 co-expressed with human NADPH-cytochrome P450 reductase in Escherichia coli.

Forms of human cytochrome P450 (P450 or CYP), such as CYP1A1, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4, were expressed or co-expressed together with human NADPH-P450 reductase in Escherichia coli. When P450 was expressed alone in E. coli, the expression level of holo-P450 ranged from 310 to 1620 nmol/L of culture. The expression level of holo-P450 decreased by co-expression with the reductase, and the level ranged from 66 to 381 nmol/L of culture. The expression level of the reductase varied depending on the forms of P450 co-expressed, and ranged from 204 to 937 U/L of culture. We assayed the catalytic activity of P450 using E. coli cells disrupted by freeze-thaw. When co-expressed with the reductase, human P450 catalyzed the oxidation of representative substrates at efficient rates. The rates appeared comparable to the reported activities of P450 in a reconstituted system containing purified preparations of P450 and the reductase.

Amino Acid Sequence↗
PubMed1998

Inhibition of intestinal microflora beta-glucuronidase modifies the distribution of the active metabolite of the antitumor agent, irinotecan hydrochloride (CPT-11) in rats.

PURPOSE: SN-38, a metabolite of irinotecan hydrochloride (CPT-11), is considered to play a key role in the development of diarrhea as well as in the antitumor activity of CPT-11. We have previously found that the inhibition of beta-glucuronidase, which hydrolyzes detoxified SN-38 (SN-38 glucuronide) to reform SN-38, in the lumen by eliminating the intestinal microflora with antibiotics, markedly ameliorates the intestinal toxicity of CPT-11 in rats. In this study we compared the disposition of CPT-11 and its metabolites in rats treated with and without antibiotics. METHODS: Rats were given drinking water containing 1 mg/ml penicillin and 2 mg/ml streptomycin from 5 days before the administration of CPT-11 (60 mg/kg i.v.) and throughout the experiment. CPT-11, SN-38 glucuronide and SN-38 concentrations in the blood, intestinal tissues and intestinal luminal contents were determined by HPLC. RESULTS: Antibiotics had little or no effect on the pharmacokinetics of CPT-11, SN-38 glucuronide or SN-38 in the blood, or in the tissues or contents of the small intestine, which has less beta-glucuronidase activity in its luminal contents. In contrast, antibiotics markedly reduced the AUC1-24 h of SN-38 (by about 85%) in the large intestine tissue without changing that of CPT-11, and this was accompanied by a complete inhibition of the deconjugation of SN-38 glucuronide in the luminal contents. CONCLUSIONS: These results suggest that SN-38, which results from the hydrolysis of SN-38 glucuronide by beta-glucuronidase in the intestinal microflora, contributes considerably to the distribution of SN-38 in the large intestine tissue, and that inhibition of the beta-glucuronidase activity by antibiotics results in decreased accumulation of SN-38 in the large intestine.

Administration, Oral↗
PubMed1998

Preclinical approach for identifying drug interactions.

Pharmacokinetic drug interactions may be divided into two categories: induction and inhibition of enzymes involved in the metabolism of a drug. The induction and inhibition of such enzymes result in decreases or increases in the blood concentrations of the drug, causing drug effects to be altered. Cytochrome P450 (P450 or CYP) is an enzyme responsible for the metabolism of a wide variety of drugs, including some anticancer agents. If a drug with a high affinity to bind to a specific form of P450 is given to a patient in combination with other drugs mainly metabolized by this enzyme, the former may potentiate the pharmacological actions of the latter by preventing their metabolism and thus increasing their serum concentration. Alternatively, if a drug inhibits or inactivates essentially all forms of P450 nonspecifically, it may be possible that the pharmacological effects of other drugs used in combination with it will be enhanced. CYP3A4 is one of the major forms of P450 in human liver microsomes. In previous studies using human liver microsomes, docetaxel was determined to be metabolized mainly by this isozyme. Thus it was assumed that inducers and inhibitors of CYP3A4 might affect the pharmacokinetics of docetaxel. In our studies, administration of dexamethasone, a known inducer of CYP3A, to mice resulted in decreases in serum docetaxel concentrations. In contrast, ketoconazole, an inhibitor of CYP3A, is assumed to increase the serum and hepatic concentrations of docetaxel. As an example of a drug which might inhibit the metabolism of other drugs, we found that bis-aceto-ammine-dichloro-cyclohexylamine platinum(IV) (JM216), which is currently being developed as a potential anticancer agent, inhibits essentially all major forms of P450 present in human liver microsomes. Since its inhibition potency is relatively high, careful assessment of the effects of this drug on the metabolism of other drugs appears to be necessary.

Animals↗
PubMed1998

Genetically engineered cells stably expressing cytochrome P450 and their application to mutagen assays.

Genetically engineered cells transiently and stably expressing cytochrome P450 (P450), a key enzyme for biotransformation of a wide variety of compounds, have provided new tools for investigation of P450 functions such as P450-mediated metabolic activation of chemicals. This review will focus on the development of mammalian cell lines stably expressing P450s and application to toxicology testings. Stable expression systems have an advantage over transient ones in that a series of the process from metabolic activation of test compounds to the appearance of toxicological consequences occurs entirely in the same intact cells. Indeed, many cell lines stably expressing a single form of mammalian P450 have been established so far and applied to cytotoxic or genotoxic assays, the endpoints of which contained mutations at hprt and other gene loci, chromosomal aberrations, sister chromatid exchanges, micronuclei, morphological transformation, and 32P-postlabeling. Analyses of metabolites of toxic substances have also been carried out, using the intact cells or microsomal fractions prepared from the cells. The stable expression systems clearly indicate the form of P450 enzyme capable of activating a certain chemical. More recently, coexpression of P450 together with other components of microsomal electron transfer systems such as NADPH-cytochrome P450 reductase has been successfully performed to increase the metabolic capacity of the heterologously expressed P450. In addition, to reconstruct the entire metabolic activation system for certain heterocyclic amines, cell lines which simultaneously express a form of human P450 and a phase II enzyme, N-acetyltransferase, were established. These cells were highly sensitive to some carcinogenic heterocyclic amines. In genetic toxicology, such a coexpression system for two or more enzymes will provide useful materials which mimic in vivo activation systems.

Animals↗
PubMed1998

UGT1A1 genotypes and glucuronidation of SN-38, the active metabolite of irinotecan.

BACKGROUND: Irinotecan (CPT-11) is metabolized by esterase to form a SN-38, which is further conjugated by UGT1A1. Genetic polymorphism has been shown in a promoter region of UGT1A1 and is related to its activity. We investigated whether there might be an inter-individual difference in pharmacokinetics of SN-38 and its glucuronide, depending on the genotypes of UGT1A1. PATIENTS AND METHODS: Nine male patients with lung cancer were treated with irinotecan (50 mg/m2) and carboplatin. Pharmacokinetic parameters were calculated with full sampling plasma data. Genotypes were determined by analyzing the sequence of TATA box of UGT1A1 of genomic DNA from the patients. RESULTS: The genotyping analysis revealed one heterozygote (6/7) and one homozygote (7/7) for (TA)7TAA allele (UGT1A1*28). The remaining seven patients were homozygote for (TA)6TAA allele (6/6, wild type). The metabolic ratios (SN-38/SN-38 glucuronide) in the patient with 7/7 genotype were uncharacteristically higher than those in the patients with other genotypes (6/6 and 6/7). Biliary index was 6980 versus 2180 +/- 1110 (range 840-3730) in patients with 7/7 versus 6/6 genotypes, respectively. CONCLUSION: These results support the idea that the patient with 7/7 genotype has an impaired capacity for glucuronidation of SN-38.

Aged↗
PubMed1998

Genetic polymorphism of drug metabolizing enzymes: new mutations in CYP2D6 and CYP2A6 genes in Japanese.

Genetic polymorphism of drug metabolizing enzymes, particularly cytochrome P450(CYP), is an important cause of adverse drug reactions. Multiple gene mutations in CYP have been shown to be phenotype. The occurrence of genetic polymorphism has been seen in genes for CYP1A1, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A5. This review discusses the molecular mechanism of two genetic polymorphisms, debrisoquine/sparteine (CYP2D6) coumarin (CYP2A6) polymorphisms. In addition, elucidation of gene mutations of CYP2D6 and CYP2A6 in Japanese will be discussed.

Aryl Hydrocarbon Hydroxylases↗
PubMed1998

Potent and non-specific inhibition of cytochrome P450 by JM216, a new oral platinum agent.

Bis-acetato-ammine-dichloro-cyclohexylamine-platinum (IV), JM216, is the first antineoplastic platinum compound that can be given to patients orally. Several phase II clinical trials of JM216 monotherapy have already been reported. However, no information on the potential drug interactions caused by JM216 is available. In this study, the capacity of JM216 to inhibit cytochrome P450 (CYP) in human liver microsomes was investigated by measuring the inhibition potential (IC50 and Ki) on prototype reactions. Specific substrates of CYP included testosterone (catalysed by CYP3A4), paclitaxel (CYP2C8), 7-ethoxyresorufin (CYP1A1, CYP1A2), coumarin (CYP2A6), aniline (CYP2E1) and (+/-)-bufuralol (CYP2D6). JM216 inhibited the catalytic activities of CYP isozymes. The IC50 values were between 0.3 microM and 10 microM, indicating strong and non-specific inhibitory effects of JM216. The inhibition occurred in a non-competitive manner, and the Ki value was 1.0 and 0.9 microM for metabolite formation of testosterone and paclitaxel respectively. Therefore, some in vivo studies should be conducted to determine whether or not there is a correlation between in vivo and in vitro results.

Antineoplastic Agents↗
PubMed1998

Molecular cloning and regulation of a novel guinea pig cytochrome P450 (CYP3A20) which differs from guinea pig CYP3A14 in only two amino acid residues.

A full length cDNA clone of cytochrome P450, encoding 503 amino acid residues, was isolated from a male guinea pig liver cDNA library. The sequence was highly homologous to other members in the CYP3A subfamily and was designated CYP3A20. CYP3A20 and CYP3A14, another guinea pig CYP3A, shared 99.4% nucleotide and 99.6% deduced amino acid sequence homology. There were only two amino acid differences between CYP3A20 and CYP3A14. No significant induction of CYP3A20 mRNA in the livers from male guinea pigs treated with dexamethasone was observed by S1 mapping analysis although CYP3A14 mRNA was induced. The expression of CYP3A20 mRNA in the livers did not change between 5 and 10 weeks after birth while that of CYP3A14 mRNA in livers significantly increased at 10 weeks. This is the first report that the two highly resembling forms of cytochrome P450 display differently regulated expression from each other.

Amino Acid Sequence↗
PubMed1998

Expression and characterization of dog CYP2D15 using baculovirus expression system.

Dog CYP2D15 was expressed in Sf9 cells with a recombinant baculovirus. Infection of Sf9 insect cells with a recombinant dog CYP2D15-virus resulted in the expression of a protein which cross-reacted with a polyclonal antibody against a dog CYP2D15-specific peptide. The difference spectrum of CO-complex of reduced P450 of the infected cell microsomes had a maximal absorbance at 449 nm. The specific content of P450 was calculated to be 0.56 nmol/mg of Sf9 cell microsomal protein. Although the expressed dog CYP2D15 showed high catalytic activity for the hydroxylations of bunitrolol and imipramine at low substrate concentration (10 microM), the catalytic activity for that of debrisoquine (50 microM) was extremely low as compared with that of CYP2D from other species. Dog liver microsomes also showed bunitrolol and imipramine hydroxylase activities, but not debrisoquine hydroxylase activity at the same substrate concentrations. In addition, the expressed CYP2D showed high catalytic activity for imipramine N-demethylation. Thus, our study reveals that the expressed dog CYP2D15 engages in high catalytic activity and has a unique substrate specificity from other CYP2D subfamilies. Western blot analysis suggested that the dog CYP2D15 contents were less than 4% of the total liver P450 content, assuming that 100% of expressed CYP2D15 incorporated heme.

Animals↗
PubMed1998

A new deleted allele in the human cytochrome P450 2A6 (CYP2A6) gene found in individuals showing poor metabolic capacity to coumarin and (+)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride (SM-12502).

The S-oxidation of (4)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride (SM-12502) and the 7-hydroxylation of coumarin are primarily catalyzed by cytochrome P450 2A6 (CYP2A6). The activities of SM-12502 S-oxidase and coumarin 7-hydroxylase were investigated with liver microsomes from 20 human individuals. Liver microsomes from individual H16 showed the lowest activities of both enzymes. The expression of CYP2A6 protein was not detectable in liver microsomes from individuals H4, H5, H7, H8, H12 and H16. CYP2A6 mRNA was hardly detectable in the liver of the individual H16. A new SacI-restriction fragment length polymorphism showing the lack of a 2.6 kb fragment was found in two of forty genomic DNA preparations from individuals H16 and No. 594, using CYP2A6 cDNA as a probe. This deletional 2.6 kb fragment was isolated from a genomic library prepared from one individuals showing normal coumarin 7-hydroxylase activity and was sequenced. This fragment contained a CYP2A6 gene region from 319 bp upstream of a putative exon 6 to a SacI site in exon 9, indicating that this region was deleted in the two individuals in this study. We also demonstrated by polymerase chain reaction analysis that the exon 8 of CYP2A6 gene was deleted in individuals H16 and No. 594. These results indicate that the reduced activity of SM-12502 S-oxidase and no activity of coumarin 7-hydroxylase are caused by the lack of CYP2A6 mRNA and CYP2A6 protein caused by the CYP2A6 gene deletion.

Aryl Hydrocarbon Hydroxylases↗
PubMed1998

[Genetic polymorphism of drug metabolizing enzymes: new mutations in CYP2D6 and CYP2A6 genes in Japanese].

Interindividual variation of drug effects in humans can be attributed to many factors. Among the factors, the rate of drug metabolism has been regarded as the most important one. A genetic defect of enzymes involved in drug metabolism, particularly cytochrome P450 (CYP), has been believed to be one of the important causal factors of adverse drug reactions. There are multiple gene mutations for CYP causing the poor metabolizer phenotype. The occurrence of genetic polymorphism has been seen in genes for CYP1A1, CYP2A6, CYP2C9, CYP2D19, CYP2D6, CYP2E1 and CYP3A5. Among them, the molecular mechanisms of genetic polymorphisms of CYP2D6 (debrisoquine/sparteine type) and CYP2A6 (coumarin type) in Japanese have been the focus of investigations. Only 20-30% of the Japanese population that shows the CYP2D6 poor metabolizer phenotype can be diagnosed by gene analysis. By other means, we found two new mutations, CYP2D6/J9 and CYP2D6/C8, in Japanese. With regard to CYP2A6, we discovered SM-12502, a new probe drug in addition to coumarin, that is currently under development; this drug is mainly metabolized by CYP2A6. Using this drug as a probe, we found poor metabolizers and analyzed the genes for CYP2A6. We found a new mutation (CYP2A6 whole deletion) responsible for the poor metabolizer phenotype. These results should contribute to the selection of an effective drug prescription and a reduced incidence of adverse effects.

Alleles↗
PubMed1998

Oxidation of histamine H1 antagonist mequitazine is catalyzed by cytochrome P450 2D6 in human liver microsomes.

Mequitazine [10-(3-quinuclidinylmethyl) phenothiazine] is a long-acting and selective histamine H1-receptor antagonist that is mainly biotransformed by human liver microsomes to yield hydroxylated and S-oxidized metabolites. Mequitazine hydroxylase was inhibited by propranolol and quinidine. Lineweaver-Burk plots for the hydroxylation and the S-oxidation indicated that the hydroxylation occurred with a low Km (0.72 +/- .26 microM) in human liver microsomes. Microsomes from genetically engineered human B-lymphoblastoid cells expressing cytochrome P450 2D6 (CYP2D6) efficiently metabolized mequitazine to the hydroxylated and S-oxidized metabolites. The results indicate that CYP2D6 isozyme is a major form of CYP responsible for the metabolism of mequitazine in human liver microsomes. Inhibition of CYP3A-catalyzed midazolam 1'-hydroxylase by various histamine H1 antagonists, including mequitazine, suggested that mequitazine and some other histamine H1 antagonists could also be inhibitors of CYP3A in human liver microsomes.

1-Propanol↗