Biomedical subjects

T Kamataki

Publications and source records attributed to T Kamataki.

At least 127 records · Page 7Linked to original sources

PubMed1995

Simultaneous expression of human CYP3A7 and N-acetyltransferase in Chinese hamster CHL cells results in high cytotoxicity for carcinogenic heterocyclic amines.

To investigate whether several food-derived heterocyclic amines are activated to genotoxic products in human fetal livers, cell lines stably expressing CYP3A7, a human fetus-specific form of cytochrome P450, NADPH-cytochrome P450 reductase, and human monomorphic or polymorphic N-acetyltransferase (NAT1 or NAT2) were established. The expression of CYP3A7 mRNAs and proteins was determined by RNA blot and immunoblot analyses, respectively. The introduction of CYP3A7 cDNA to CR-68 cells which had been transfected with guinea pig NADPH-cytochrome P450 reductase, NAT1, or NAT2 cDNA resulted in increased sensitivity of the cells to aflatoxin B1 compared to parental cells. The cytotoxicity assay for 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) showed that 7P-145 cells, which expressed the reductase, CYP3A7, and NAT2, were approximately 4-, 30-, and 14-fold more sensitive to respective IQ, MeIQ, and MeIQx than parental CR-68 cells. There were no clear differences in sensitivity to these compounds among CHL, CR-68, and the cells which expressed the reductase and CYP3A7 (7R-54), the reductase and NAT1 (CNM-4), the reductase and NAT2 (CNP-40), and the reductase, NAT1, and CYP3A7 (7M-124). From these results, it was suggested that both CYP3A7 and polymorphic NAT2 are required for mutagenic activation of several heterocyclic amines in human fetal livers.

Amines↗

PubMed1995

N-oxygenation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine by the rat liver flavin-containing monooxygenase expressed in yeast cells.

N-oxygenation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a dopaminergic neurotoxin, was studied using recombinant rat liver flavin-containing monooxygenase (FMO), FMO1A1, expressed in yeast cells. The mean (+/- S.D.) kinetic parameters of MPTP N-oxygenation were: Km = 1.8 +/- 0.5 microM, Vmax = 9.5 +/- 1.6 nmol/mg per min, and Vmax/Km = 4.6 +/- 0.5 ml/mg per min. n-Octylamine, an activator of FMO, enhanced the MPTP N-oxygenation activity by 51%, while methimazole, thiobenzamide and alpha-naphthylthiourea, alternate substrates of FMO, inhibited it by 27.4, 68.0 and 59.2%, respectively. The results indicate that MPTP is efficiently N-oxygenated by the recombinant rat liver FMO1A1, and that the responses to the modulators of FMO activity found in the recombinant rat liver FMO1A1 resemble those of mouse and rat liver microsomes as reported previously. The findings suggest that the recombinant FMO expressed in yeast cells is considered as a useful tool to study an involvement of FMO in the metabolism of environmental toxins or chemicals. In addition, FMO1A1 appears to be one of the predominant enzymes responsible for the N-oxygenation of MPTP at least in rat liver.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed1995

Isolation and characterization of a new cDNA clone belonging to the cytochrome P450 2C gene subfamily in hamsters.

Cytochrome P450s are heme-containing proteins which evolved from an ancestral gene to form a large superfamily of enzymes. We previously isolated three distinct CYP2C cDNAs, namely CYP2C25, 2C26, and 2C27, from liver cDNA libraries of male and female Syrian hamsters. In the present study, we isolated another cDNA clone, assigned as CYP2C28, from the same male cDNA library. This cDNA consisted of 1556 nucleotides and encoded a polypeptide of 490 amino acids. CYP2C28 showed relatively low identities to other hamster CYP2C forms (71.4-72.6% in amino acid sequences) and showed the highest similarity to rat CYP2C24 (88.7%), while the other hamster CYP2C forms showed high similarities among themselves (> 90.2%). CYP2C28 protein expressed in yeast catalyzed the N-demethylation of aminopyrine and benzphetamine, but did not catalyze the hydroxylation of tolbutamide, testosterone, and progesterone. Northern blot analysis demonstrated that CYP2C28 mRNA was expressed in hamster livers 1 week after birth, when the other hamster CYP2C forms were not detectable. The level of CYP2C28 mRNA increased to 3 weeks and then decreased with time in males. The level in females was lower than that in males. In adult hamsters, CYP2C28 was induced almost threefold by administration of phenobarbital but not affected by 3-methycholanthrene. On the other hand, administration of pregnenolone 16 alpha-carbonitrile induced CYP2C28 in females but not in males. These results indicate that CYP2C28 is a unique form in the hamster CYP2C subfamily.

Amino Acid Sequence↗

PubMed1995

Fetus-specific CYP3A7 and adult-specific CYP3A4 expressed in Chinese hamster CHL cells have similar capacity to activate carcinogenic mycotoxins.

To assess whether CYP3A4 and CYP3A7 have a similar capacity to activate carcinogenic mycotoxins, we established cell lines stably expressing human CYP3A4 and CYP3A7, which are adult- and fetal-specific forms of cytochrome P450 in human livers, respectively. Each cDNA was introduced into CR-119 cells which had been established by introducing guinea pig NADPH-cytochrome P450 reductase cDNA into Chinese hamster lung cells. The cell lines (4-line and 7-line) stably expressed the mRNA and the protein corresponding to CYP3A4 and CYP3A7, respectively. The concentration-response for aflatoxin B1 (AFB1) cytotoxicity in 4-line and 7-line, respectively, was compared. 4-10 and 7-40 cells were approximately 17- and 20 times more sensitive to AFB1 than the parental CR-119 cells, respectively. In addition, the sensitivities to AFB1 of both 4-10 and 7-40 cells were enhanced approximately seven times by the addition of 10 microM alpha-naphthoflavone, a known activator of CYP3A enzyme, while the sensitivities were suppressed approximately four times by the addition of 100 microM troleandomycin, which forms a metabolite intermediate complex with CYP3A enzyme. Moreover, both cell lines showed approximately 10 and 2 times higher sensitivity to sterigmatocystin and aflatoxin G1 than CR-119 cells, respectively. These results indicate that CYP3A4 and CYP3A7 have essentially similar capacities to activate AFB1, sterigmatocystin, and aflatoxin G1 to produce toxic metabolites.

Adult↗

PubMed1995

Occurrence of autoimmune antibodies to liver microsomal proteins associated with lethal hepatitis in LEC rats: effects of TJN-101 ((+)-(6S,7S,R-biar)- 5,6,7,8-tetrahydro-1,2,3,12-tetramethoxy-6,7-dimethyl-10,11- methylenedioxy-6-dibenzo[a,c]cyclooctenol) on the development of hepatitis and the autoantibodies.

Long Evans Cinnamon (LEC) rats, that spontaneously develop hepatitis, were found to possess autoantibodies to liver microsomal proteins (anti-LM) before the development of hepatitis. Anti-LM antibody was assumed to appear in association with the lethal hepatitis in the LEC rats. Thus, the purpose of this study was to investigate the effects of an anti-hepatitis drug on the development of hepatitis and the occurrence of the antibody in LEC rats. Mortality, blood biochemical parameters and the titer of serum anti-LM antibody were measured. In control LEC rats, 4 of 8 rats died before 20 weeks of age. In rats treated with TJN-101 ((+)-(6S,7S,R-biar)-5,6,7,8-tetrahydro-1,2,3,12-tetramethoxy -6,7-dimethyl-10,11 - methylenedioxy-6-dibenzo[a,c]cyclooctenol), 4 of 7 rats died of hepatitis, but the time of death was delayed by 7-10 weeks compared to the control rats. The titer of the anti-LM antibody increased 3-7 weeks before death in the non-survivors in control and TJN-101-treated rats, supporting the idea that anti-LM antibody occurs in association with acute lethal hepatitis.

Animals↗

PubMed1995

Expression of CYP3A7, a human fetus-specific cytochrome P450, in cultured cells and in the hepatocytes of p53-knockout mice.

CYP3A7 is a major form of cytochrome P450 in human fetal livers. To elucidate toxicological significance of CYP3A7 in fetal livers, CYP3A7 cDNA was introduced into Chinese hamster lung (CHL) cells. Transformants carrying the CYP3A7 gene were more sensitive to mycotoxins than parental CHL cells. In additional studies, we established a hepatocyte cell line from CYP3A7-transgenic/p53-knockout mice. In hepatocyte cells from CYP3A7-transgenic/p53-knockout mice, CYP3A7 mRNA was expressed and the catalytic activity of CYP3A7 protein was detected. The cells are expected to show cytotoxicity to mycotoxins and teratogens. These cell lines provide a valuable panel for studying the fetal toxicities of chemicals in humans.

Animals↗

PubMed1995

Hormonal regulation of the mRNA level of male-dominant cytochrome P450 (CYP2C27) in hamster livers.

Hormonal regulation in the expression of CYP2C27, a male-predominant form of cytochrome P450 in the hamster, was investigated. The mRNA level of CYP2C27 was five-fold higher in male than in female livers. The mRNA level was suppressed by both gonadectomy and hypophysectomy in male, while the mRNA level was increased by the same treatment in female, resulting in the disappearance of sex difference. In both sexes, treatment of the gonadectomized animals with testosterone induced the cytochrome over the level of sham- operated males. Treatment of the hypophysectomized animals with testosterone elevated the level in females. On the other hand, oestradiol did not show any effects on either the gonadectomized or the hypophysectomized animals of both sexes. Twice-daily injection of growth hormone to hypophysectomized animals restored the level in males. Continuous infusion of growth hormone to hypophysectomized animals suppressed the level in both sexes. These results indicate that the expression of CYP2C27 in hamsters is under the control of endocrine factors and suggest that the gonadal-pituitary axis pathway is a control mechanism as proposed in rats and mice.

Androgens↗

PubMed1995

Polymorphic drug metabolism: studies with recombinant Chinese hamster cells and analyses in human populations.

Most promutagens and procarcinogens exert their genotoxicity after undergoing metabolic activation. Metabolism of chemicals is an important factor in limiting the extent of the action of a chemical. In this study, we established cell lines which carried cDNAs coding for human CYP1A2 and N-acetyltransferase (NAT); the latter functions as O-acetyltransferase for N-hydroxyarylamines formed by CYP1A2. A cell line which expresses CYP1A2 together with P450 reductase activated aflatoxin B1, but not heterocyclic amines. A cell line which carries CYP1A2 and polymorphic NAT (NAT2) in addition to P450 reductase efficiently activated IQ and some other heterocyclic amines. However, a cell line which carries CYP1A2 and monomorphic NAT (NAT1) had only low activity toward the same heterocyclic amines. In order to determine the presence of and frequency of genetic polymorphisms of CYP1A2 and NAT2 in humans, we performed in caffeine phenotyping test on 205 Japanese volunteers. Analyses of metabolic ratios of urinary metabolites showed a bimodal distribution, indicating that about 86% and 91% of Japanese were extensive metabolizers (EM) of CYP1A2 and NAT2, respectively. The genotype NAT2 determined by the PCR-RFLP method agreed completely with the phenotype. To determine the mechanism of the differences in CYP1A2 activity, genomic DNA from peripheral lymphocytes of poor metabolizers (PM) and EM was subjected to DNA sequencing. No differences in nucleotide sequence were observed between PMs and EMs in the exons, exon-intron junctions and 5'-flanking region of the CYP1A2 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Inhibition of UDP-glucuronosyltransferase by aglycons of natural glucuronides in kampo medicines using SN-38 as a substrate.

7-Ethyl-10-[4-(piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11), a potent anticancer agent for lung and gynecological cancers, is metabolized in vivo to the active compound, 7-ethyl-10-hydroxycamptothecin (SN-38), which is subsequently conjugated to SN-38-glucuronide by UDP-glucuronosyltransferase (UDP-GT). Three purified aglycons of natural glucuronides, baicalein, luteolin and glycyrrhetic acid, inhibited UDP-GT activity towards SN-38 as a substrate. The inhibitory potencies of these aglycons toward UDP-GT were similar to that of 1-naphthol. Based on these results, together with our previous finding that the corresponding glucuronides used in the present study strongly inhibited beta-glucuronidase in gut flora, we propose that materials in Kampo (Japanese herbal) medicines containing these aglycons of natural glucuronides could be used in vivo to decrease the enterohepatic circulation of SN-38 and other drugs.

Animals↗

PubMed1995

[Molecular toxicology of cytochrome P450: focusing on interspecies homology].

It is theoretically impossible to extraporate the toxicity of drugs in humans from data obtained with experimental animals. In fact, accumulated data on species differences in the drug metabolism can provide only a little information for the estimation of the metabolism and the related toxicity of drugs under development in humans. The recent development in the molecular biology has realized the quantitative estimation of genetic distance between humans and experimental animals. Thus, this study was undertaken to estimate the distance between humans and experimental animals to propose the use of monkeys in an initial phase of the development of a new drug. Cell lines carrying human drug metabolizing enzymes are also expected to be a good tool for the estimation of the drug metabolism and the toxicity of drugs in humans. To show an example of the usefulness of such cell lines, we established new cell lines expressing CYP3A7, a form of cytochrome P450 specifically present in human fetal livers.

Amino Acid Sequence↗

PubMed1995

S-oxidation of (+)-cis-3,5-dimethyl-2-(3-pyridyl)-thiazolidin-4-one hydrochloride by rat hepatic flavin-containing monooxygenase 1 expressed in yeast.

1. Rat hepatic flavin-containing monooxygenase 1 (FMO1) expressed in yeast catalyzed the S-oxidation of (+)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride (SM-12502) in vitro. 2. S-oxidation was inhibited by 1-(1-naphthyl)-2-thiourea and thiobenzamide, known inhibitors of FMO, but was not enhanced by n-octylamine, a known enhancer of FMO. 3. The rate of S-oxide formation from SM-12502 was about four-fold lower than that from (+/-)-trans-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride (SM-9979) and enantioselectivity and diastereoselectivity of the S-oxidation reaction were observed. 4. The ability of the recombinant yeast to produce the S-oxide from SM-12502 was maintained for long periods and exemplifies the recombinant yeast as a bioreactor to produce a large amount of the S-oxide.

Animals↗

PubMed1995

Efficient complementary DNA directed expression of human fetal liver cytochrome P450 (CYP3A7) in insect cells using baculovirus.

CYP3A7 is a form of cytochrome P450, which is expressed specifically in human fetal livers. NPVHF1, a recombinant baculovirus containing the entire coding region of CYP3A7, was constructed and infected to Spodoptera frugiperda (Sf9) cells. Upon infection with NPVHF1, the Sf9 cells expressed the CYP3A7 to the maximum content of 0.2 nmol per mg of whole cell lysates 72 hours after infection. A 5.5-fold expression level (1.1 nmol per mg of whole cell lysates) was attainable when cultured in the presence of externally added hemin. A catalytic activity of the CYP3A7 expressed in the Sf9 cells was confirmed by the umu gene expression mutation assay, in which aflatoxin B1 was activated to a mutagen by the expressed CYP3A7 in the presence of NADPH-cytochrome P450 reductase and cytochrome b5. From these results, it is concluded that the baculovirus expression system enables the high-level expression of CYP3A7 and will be a very useful tool for the characterization of CYP3A7.

Aflatoxin B1↗

PubMed1994

Molecular cloning and functional expression of a mouse cytochrome P-450 (Cyp3a-13): examination of Cyp3a-13 enzyme to activate aflatoxin B1 (AFB1).

A cDNA encoding a novel member of the cytochrome P-450 superfamily, Cyp3a-13, has been isolated from mouse liver cDNA library by hybridization screening. The Cyp3a-13 encoded 503 amino acid residues and shared 71% amino acid identity with Cyp3a-11. When Cyp3a-13 cDNA was expressed in CR119 cells which had been established as a cell line stably expressing NADPH-cytochrome P-450 reductase cDNA of guinea pigs, aflatoxin B1-dependent cytotoxicity was observed. This cytotoxicity was enhanced by alpha-naphthoflavone (7,8-benzoflavone), which is known to augment the CYP3A enzymatic activity. The results indicate that CYP3A in mice, which are relatively insensitive to aflatoxin B1, can activate aflatoxin B1 to a genotoxic product.

Aflatoxin B1↗

PubMed1994

A novel form of mouse cytochrome P450 3A (Cyp3a-16). Its cDNA cloning and expression in fetal liver.

A complementary DNA clone coding for a novel form of cytochrome P450, Cyp3a-16, in mouse fetal livers was isolated and completely sequenced. This clone encoded a polypeptide of 504 deduced amino acids and showed 87.3% and 66.6% amino acid identities with mouse Cyp3a-11 and Cyp3a-13, respectively. Cyp3a-16 transcript was detectable before birth and remarkably diminished five weeks after birth in mice. We conclude that Cyp3a-16 is a fetal- and puberty-specific cytochrome P450 in mice.

Amino Acid Sequence↗

PubMed1994

Possible occurrence of P450 related to P450 HFLb in extrahepatic tissues of human fetuses and its contribution to metabolic activation of promutagens.

P450 HFLb purified from human fetal livers has been shown to be constitutively expressed in fetal livers. In the present study, the occurrence of proteins immunochemically related to P450 HFLb in extrahepatic tissues of human fetuses and their contribution to mutagenic activation of promutagens were investigated. The mutagenic activation of aflatoxin B1 (AFB1), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and benzo[a]pyrene were observed in human fetal extrahepatic tissues, including adrenal glands, kidneys and lungs, at varying rates. Immunoblot analysis of homogenates of extrahepatic tissues with antibodies to P450 HFLb revealed the occurrence of proteins immunochemically related to P450 HFLb in adrenal glands, kidneys and lungs. Immuno-inhibition studies suggested that in fetal adrenal gland and kidney, the proteins cross-reactive with antibodies to P450 HFLb were capable of activating IQ and MeIQ to mutagens.

Adrenal Glands↗

PubMed1994

Chiral inversion of drug: role of intestinal bacteria in the stereoselective sulphoxide reduction of flosequinan.

Chiral inversion at a sulphoxide position of flosequinan enantiomers [(+/-)-7-fluoro-1-methyl-3-methylsulphinyl-4-quinolone] occurred in conventional rats but not in either germ-free rats or rats treated with antibiotics after an oral administration of each enantiomer. Thus, it was postulated that the chiral inversion occurred by mechanisms mediated by intestinal bacteria. The intestinal content isolated from conventional rats reduced R(+)- and S(-)-flosequinan to produce the sulphide, while intestinal content from rats treated with antibiotics did not reduce the drug. Several strains of facultative anaerobes possessed a high flosequinan reducing activity. Escherichia coli, Klebsiella oxytoca and Klebsiella pneumoniae reduced R(+)-flosequinan to the sulphide stereoselectively. On the other hand, Enterobacter aerogenes and Micrococcus agilis exclusively reduced S(-)-flosequinan. The sulphide, which could be produced by intestinal bacteria from R(+)- and S(-)-flosequinan, was readily absorbed upon an oral administration to rats, and was oxidized fairly rapidly to R(+)- and S(-)-flosequinan and further to the sulphone form. Based on these data, it has been confirmed that chiral inversion at the sulphoxide position of flosequinan enantiomers occur via stereoselective reduction of sulphoxide by intestinal bacteria to form the sulphide, followed by oxidation of the sulphide in the body to produce R(+)- and S(-)-flosequinan.

Animals↗

PubMed1994

Stable expression of human CYP1A2 and N-acetyltransferases in Chinese hamster CHL cells: mutagenic activation of 2-amino-3-methylimidazo[4,5-f]quinoline and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline.

In order to investigate the metabolic activation pathway of food-derived heterocyclic amines, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), cultured cell lines which stably expressed human cytochrome P4501A2 (CYP1A2) and N-acetyltransferases (NATs) were developed by the method of complementary DNA (cDNA) transfection. First, a cell line expressing CYP1A2, designated A2R-5, was established from the cell line CR-68, which was previously established by introducing NADPH-cytochrome P-450 reductase cDNA into Chinese hamster CHL cells. The expression of CYP1A2 in the transfected cells was confirmed by determining sensitivity to aflatoxin B1. As the next step, the A2R-5 as well as CR-68 cells were further transfected with human monomorphic NAT (NAT1) or polymorphic NAT (NAT2) cDNAs. The expression of NAT in the transfected cells was confirmed using p-aminobenzoic acid and sulfamethazine as substrates, while no activity was seen in parental CR-68 and A2R-5 cells. The cell line, ANP-25, which expressed both CYP1A2 and NAT2, was approximately 370- and 100-fold more sensitive to IQ and MeIQx, respectively, than parental CR-68 cells in cytotoxicity assays. There were no clear differences in sensitivity to both compounds among CR-68, A2R-5, and the cell lines which expressed NAT1 alone, NAT2 alone, and CYP1A2 plus NAT1. Mutagenicity of IQ and MeIQx at the hypoxanthine-guanine phosphoribosyltransferase locus was also detectable only in ANP-25 cells but not in A2R-5 or the cell line expressing CYP1A2 plus NAT1. From these results, it is proposed that both CYP1A2 and NAT2 (but not NAT1) are required for mutagenic activation of these compounds, implying that acetylator polymorphism may be an important risk factor in the carcinogenicity of these compounds.

Acetyltransferases↗

PubMed1994

Mouse NADPH-cytochrome P-450 oxidoreductase: molecular cloning and functional expression in yeast.

We published isolation of a mouse NADPH-cytochrome P-450 oxidoreductase cDNA and afterward ascribed the cDNA to the guinea-pig instead of the mouse (Ohgiya, S. et al. (1992) Biochim. Biophys. Acta 1171, 103-105 and Corrigendum (1993) Biochim. Biophys. Acta 1174, 313). We report here nucleotide and deduced amino acid sequences of an NADPH-cytochrome P-450 oxidoreductase cDNA isolated from the ddY mouse. The mouse cytochrome P-450 oxidoreductase shares 98.4% identity with its rat counterpart. In particular, clusters of acidic residues that presumably participate in interaction with cytochrome P-450 are highly conserved in primary structures of mammalian cytochrome P-450 oxidoreductases. The mouse cytochrome P-450 oxidoreductase was functionally expressed in yeast using a modified cDNA clone lacking whole noncoding regions.

Amino Acid Sequence↗