Biomedical subjects

T Kamataki

Publications and source records attributed to T Kamataki.

At least 91 records · Page 5Linked to original sources

PubMed1997

Regulation of CYP1A and CYP3A mRNAs by ascorbic acid in guinea pigs.

In previous studies, we found that the ascorbic acid (AsA) deficiency caused changes in the amounts of the various forms of cytochrome P450 (P450) in liver microsomes from guinea pigs in a form-specific manner. Thus, the aim of this study was to clarify whether the changes seen in the protein contents of the various forms of P450 were associated with the levels of the expression of their mRNAs. Prior to determining the mRNA level, we isolated four cDNA clones, encoding CYP1A2, CYP3A14, CYP3A15, and CYP3A17, from guinea pig liver cDNA libraries to use them as probes in further experiments. The amino acid sequence of the guinea pig CYP1A2 showed identity ranging from 73 to 77% with those of other mammalian P450s. The amino acid sequences among guinea pig CYP3As had about 94% identities with each other. The AsA deficiency apparently decreased the expression of mRNA for CYP1A1 and CYP1A2. These results were in agreement with the decrease in the content of CYP1A1 and CYP1A2 proteins. The amount of P450 protein(s) immunochemically cross-reactive with antibodies to human CYP3A4 was likely unaffected while that of human CYP3A7 tended to be decreased by the AsA deficiency. It suggested that the expression of each CYP3A isozyme was regulated differently by AsA. In fact, the level of mRNA for CYP3A14 was unaffected by the AsA deficiency, while those for CYP3A15 and CYP3A17 were significantly decreased by the AsA deficiency, clearly indicating that the expression of each isozyme within the CYP3A subfamily is differently regulated by AsA. These results support the idea that the transcription of P450 is regulated by AsA in guinea pigs.

Amino Acid Sequence↗

PubMed1997

Inhibition of the transcription of CYP1A1 gene by the upstream stimulatory factor 1 in rabbits. Competitive binding of USF1 with AhR.Arnt complex.

A xenobiotic-responsive element (XRE)-binding factor(s) other than the AhR.Arnt complex was found to inhibit the transcription of CYP1A1 gene in the liver from adult rabbits, known to be nonresponsive to CYP1A1 inducers. The constitutive factor(s) in liver nuclear extracts bound to the core sequence of XRE. The binding was eliminated by the presence of an excess amount of the AhR.Arnt complex synthesized in vitro. To identify the constitutive factor(s), a sequence similar to rabbit XRE was sought. It was found that the sequence of rabbit XRE overlapped with that of the upstream stimulatory factor 1 (USF1)-binding site in the mouse metallothionein I promoter. In fact, a super shift assay using a specific antibody against human USF1 indicated that USF1 was capable of binding to rabbit XRE. Additionally, the AhR.Arnt-mediated activation of XRE-TK/Luc reporter gene in RK13 cells was blocked by the transfection with a USF1 expression vector with the amounts of the expression vector transfected. These results indicate that the XRE of the rabbit CYP1A1 gene is recognized by the basic helix-loop-helix proteins to regulate the expression of CYP1A1 in both an agonistic (AhR.Arnt) and an antagonistic (USF1) manner.

Animals↗

PubMed1997

Upstream stimulatory factor 1 (USF1) suppresses induction of CYP1A1 mRNA by 3-methylcholanthrene (MC) in HepG2 cells.

In this study, an endogenous factor(s) involved in the suppression of the induction of CYP1A1 was studied. Analyzing the sequences, we found that the sequence of xenobiotic responsive element (XRE) in the upstream region of the human CYP1A1 gene was overlapped with that of the upstream stimulatory factor 1 (USF1)-binding site in mouse metallothionein I promoter. In fact, a gel shift assay using a specific competitor or mutant probes showed that the core sequence of human XRE was specifically recognized by USF1. The amount of USF1 in the nuclear extracts from HepG2 cells was smaller than that from rat and rabbit livers as assayed by the binding to XRE. To determine whether or not USF1 could inhibit the interaction of aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (Arnt) complex with XRE, we transfected USF1-SR alpha expression vector into HepG2 cells. The results showed that no interaction of AhR/Arnt complex with XRE occurred even when the cells were treated with 2,3,7,8-tetrachlorodibenzofuran (TCDF). Furthermore, the S1 nuclease protection assay showed that the induction of CYP1A1 mRNA by 3-methylcholanthrene (MC) was depressed by the transfection of USF1-SR alpha into HepG2 cells. Thus, it is highly possible that USF1 negatively regulates the induction of CYP1A1 in humans.

Animals↗

PubMed1997

Expression of cytochrome P450 3A7 in Escherichia coli: effects of 5' modification and catalytic characterization of recombinant enzyme expressed in bicistronic format with NADPH-cytochrome P450 reductase.

Cytochrome P450 3A7 is the major P450 form present in fetal liver tissue and may be responsible for the detoxification of many drugs that reach the fetal circulation. We report the development of bacterial expression systems for P450 3A7. Maximal yields (up to 50 nmol P450/liter culture) were obtained with a construct in which the 5'-terminus of the 3A7 cDNA was modified to include the MALLLAVFL N-terminal sequence of recombinant bovine P450 17A (H. J. Barnes, M. P. Arlotto, and M. R. Waterman, Proc. Natl. Acad. Sci. USA 88, 5597-5601, 1991) and to incorporate several downstream amino acid substitutions derived from the P450 3A5 sequence. This sequence also appeared optimal for expression of P450 3A4 and 3A5. Recombinant P450 3A7 was partially purified using ion-exchange and hydroxylapatite chromatography and reconstituted with NADPH-cytochrome P450 reductase, cytochrome b5, and lipids. Activity comparable to that of P450 3A4 was demonstrated toward a number of procarcinogens. An alternative approach was used to further characterize recombinant 3A7 due to low yields of recombinant protein in the expression and poor recovery in the purification. P450 3A7 was subcloned into a bicistronic vector containing human NADPH-cytochrome P450 reductase and expressed in bacteria. Recombinant P450 3A7 coexpressed in bacterial membranes with NADPH-cytochrome P450 reductase showed similar levels of activity toward erythromycin (N-demethylation) and ethylmorphine (N-demethylation) to P450 3A4 and 3A5 expressed in the same system, whereas 3A7 was less active toward midazolam (1'- and 4-hydroxylation) and nifedipine (oxidation).

Amino Acid Sequence↗

PubMed1997

Determination of FAD-binding domain in flavin-containing monooxygenase 1 (FMO1).

The flavin-containing monooxygenases (FMOs) are a family of flavoenzymes and contain one molecule of FAD per monomer. In order to demonstrate where FMO interacts with FAD, four mutants for the rat liver FMO1 protein were expressed in yeast and characterized. All four mutants were immunochemically similar to the unmodified form, although the contents of FAD in all four mutants were much lower than that in the unmodified form. Interestingly, the mutant generated by changing the first glycine of the proposed FAD-binding domain (GxGxxG) to alanine revealed catalytic activities, but was lower than those seen with the unmodified form. The conversion of the first glycine to alanine markedly increased and decreased the Km and Vmax values for imipramine N-oxidation, respectively. The other three mutants (RFMOm2, RFMOm3, and RFMOm4) were catalytically inactive. Our results suggest that three glycines, especially the second and third glycines, in the proposed FAD-binding domain were necessary for FMO to show catalytic activities. Using RFMOm1 and the unmodified form, the effects of n-octylamine on the activity of FMO1 were investigated. The activities of both wild-type and RFMOm1 enzymes for all of the compounds examined were enhanced by n-octylamine. The Km and Vmax values of both RFMOm1 and the unmodified form for imipramine N-oxidation were lowered and raised by n-octylamine, respectively.

Animals↗

PubMed1997

Establishment of a novel host, high-red yeast that stably expresses hamster NADPH-cytochrome P450 oxidoreductase: usefulness for examination of the function of mammalian cytochrome P450.

A novel strain of Saccharomyces cerevisiae useful for expression studies of mammalian microsomal cytochrome P450s was established and named High-red yeast. Hamster NADPH-cytochrome P450 oxidoreductase (P450 reductase) cDNA to be introduced into yeast was isolated from a hamster liver cDNA library. The cDNA was 2421 bp long and contained an entire coding region for 667 amino acids. The NH2-terminal amino acid sequence deduced from the hamster P450 reductase cDNA was identical with that of the enzyme purified from hamster livers except for deletion of the initial methionine. A delta-sequence derived from yeast retrotransposon Ty was cloned and used as a sequence for homologous recombination in a yeast genome. S. cerevisiae YPH500 was transformed with a multi-integration cassette containing the expression unit of the hamster P450 reductase and the delta-sequence. The transformant showing the highest activity of the P450 reductase was named High-red yeast. High-red yeast carried more than six copies of the multi-integration cassettes in a single chromosome and retained the multi-integration cassettes over a period of 100 generations under nonselective culture conditions, indicating that this yeast was a mitotically stable transformant. The microsomes prepared from High-red yeast had 20 times the P450 reductase activity of the microsomes prepared from the parental yeast. Due to the high activity of the hamster P450 reductase, the 7-ethoxycoumarin deethylase activity of mouse CYP1A1 expressed in High-red yeast was 250 times higher than the activity of mouse CYP1A1 expressed in the parental yeast.

Amino Acid Sequence↗

PubMed1997

Mouse cytochrome P450 (Cyp3a11): predominant expression in liver and capacity to activate aflatoxin B1.

S1 mapping analysis for the expression of Cyp3a11 and Cyp3a13 indicated that Cyp3a11 mRNA is predominantly expressed in mouse liver, compared with that of Cyp3a13. In addition, all of six inducers, such as dexamethasone, 3-methylcholanthrene, phenobarbital, polychlorinated biphenyl, pregnenolone 16 alpha-carbonitrile, and rifampicin, increased the expression of the Cyp3a11 mRNA more extensively than that of Cyp3a13. The level of mRNAs corresponding to Cyp3a11 and Cyp3a13 reached the maximum level between 4 and 8 weeks after birth. Cyp3a11 enzyme was expressed into CR119 cells which had been established as a cell line stably expressing NADPH-cytochrome P450 reductase cDNA of guinea pigs. These transformants showed aflatoxin B1-dependent cytotoxicity in proportion to the amounts of Cyp3a11 mRNA. This cytotoxicity was enhanced by 7,8-benzoflavone, a known activator of CYP3A protein. Based on these results, we confirm that CYP3A in the mouse, which is an animal species known to be relatively insensitive to aflatoxin B1 genotoxicity, can activate this mycotoxin efficiently.

Aflatoxin B1↗

PubMed1997

Marmoset liver cytochrome P450s: study for expression and molecular cloning of their cDNAs.

Northern blot and immunoblot analyses indicated that considerable levels of CYP2B, CYP2C, CYP2D, CYP2E, and CYP3A were expressed in the liver of untreated marmosets. CYP1A was also expressed but to lesser extents. CYP3A mRNA was also detectable in the small intestine of untreated marmoset; the amount was increased by treatment with polychlorinated biphenyl. From a liver cDNA library, two cDNA clones coding for CYP2D19 and CYP3A21 (clones CM2D-1 and CM3A-10, respectively) were isolated. CM2D-1 and CM3A-10 contained an entire coding region for polypeptide 497 and 503 amino acid residues, respectively. The deduced amino acid sequences of CYP2D19 and CYP3A21 showed 90% identities to human CYP2D6 and CYP3A4, respectively. The value of CYP3A21 was 3% lower than that of cynomolgus monkey CYP3A8. On the other hand, these values were 11 to 23% higher than those of the other experimental animals, including dogs, rabbits, guinea pigs, rats, mice, and hamsters. These results indicate that the marmoset stands at a midpoint between human and nonprimate experimental animals.

Amino Acid Sequence↗

PubMed1997

In vivo activation of aflatoxin B1 in C57BL/6N mice carrying a human fetus-specific CYP3A7 gene.

The in vivo activation of aflatoxin B1 (AFB1) was assessed by using two transgenic mouse lines, M2 and M10, in which the human fetus-specific CYP3A7 was expressed in the kidney (M2) and the liver (M10), respectively. Male mice of 8 weeks old from these two lines were treated with a single i.p. injection of AFB1 (4 mg/kg body weight). AFB1-N7-guanine adduct was quantified by high-performance liquid chromatography. DNA damage was measured using the alkaline elution technique 2 and 6 h after AFB1 treatment. Administration of AFB1 resulted in a significantly higher level of AFB1-N7-guanine in the livers of M10 transgenic mice compared with their nontransgenic littermates (16.5 +/- 4.2 versus 10.4 +/- 1.2 ng/mg DNA, P < 0.01). The level of this biomarker was also significantly higher in the kidney of the M2 mice compared with control mice (73.0 +/- 6.3 versus 50.2 +/- 9.5 ng/mg DNA, P < 0.01). Similar results were also observed with DNA damage expressed as normalized area above curve (NAAC) in the two transgenic lineages, e.g., NAAC values were significantly higher in the livers of M10 and the kidneys of M2 mice. A dose-response relationship of NAAC values was observed in the livers of M10 mice when treated with AFB1 at different doses ranging from 1 to 16 mg/kg body weight, whereas in nontransgenic mice, only slight but not statistically significant increases of NAAC values were observed. Both the mouse CYP3A11 and GST-Yc subunit were expressed at identical levels in these transgenic lines. The results of this study present further evidence that human fetuses are also under the carcinogenic attack of AFB1 as adults if exposed to this potent carcinogen.

Aflatoxin B1↗

PubMed1997

Isolation of a promoter region in mouse cytochrome P450 3A (Cyp3A16) gene and its transcriptional control.

An 11.5 kb fragment of the mouse Cyp3a16 gene containing the 5' flanking region was isolated from the lambda DASHII mouse genomic library. A part of the 5' flanking region and the first exon of Cyp3a16 gene were sequenced. S1 mapping analysis showed the presence of two transcriptional initiation sites. The first exon was completely identical to Cyp3a16 cDNA. The identity of 5' flanking sequences between Cyp3a16 and Cyp3a11 genes was about 69%. A typical TATA box and a basic transcription element (BTE) were found as seen with other CYP3A genes from various animal species Moreover, some putative transcriptional regulatory elements were also found in addition to the sequence motif seen for the formation of Z-type DNA. To examine the transcriptional activity of Cyp3a11 gene, DNA fragments in the 5'-flanking region of the gene were inserted front of the luciferase structural gene, and the constructs were transfected in primary hepatocytes. The analysis of the luciferase activity indicated that the region between -146 and -56 was necessary for the transcription of CYP3a16 gene.

Animals↗

PubMed1997

Gene structure of mouse Cyp3a11: evidence for an enhancer element within its 5' flanking sequences.

A mouse Cyp3a11 gene was isolated from a mouse sperm DNA library with mouse Cyp3a11 cDNA as a probe. The nucleotide sequences determined for the gene and the 5' flanking region revealed that the mouse Cyp3a11 gene was composed of 13 exons and 12 introns. The exons spun about 23 kb. The nucleotide sequence of the exons was completely identical to Cyp3a11 cDNA. Within the 5' flanking sequence, putative binding sites of several transcriptional factors were found. Transient transfection assays were carried out with HepG2 cells, a human hepatoma cell line, using constructs containing different lengths of 5' flanking sequence fused to a reporter, chloramphenicol acetyltransferase gene. The results showed that a cis-acting element(s) was located from -1609 to -907.

Animals↗

PubMed1997

A comparison of hepatic cytochrome P450 protein expression between infancy and postinfancy.

We immunochemically measured the contents of 9 different cytochrome P450 (CYP) isoenzymes expressed in the liver and compared them between two groups: one group of 6 infant and 4 perinatal patients and one group of 10 patients after infancy (over 1 year old). CYP protein expressed in human liver can be divided into three groups on the basis of expression pattern: (a) CYP2A6, 2C9, 2D6, 2E1, and 3A were present in all samples and no difference was observed between the two groups; (b) CYP1A2, 2B6, and 2C8 were expressed more after infancy than during infancy; and (c) CYP3A7, which has been considered a major CYP enzyme in fetal liver microsomes, was expressed in all infants as well as the four perinatal patients, whereas it was detected in only 2 patients after infancy. These results implied that CYP2A6, 2C9, 2D6, 2E1, and 3A are already expressed during perinatal and infant period, while CYP1A2, 2B6, and 2C8 are expressed highly in subjects over 1 year old, and CYP3A7 disappeared after infancy.

Adolescent↗

PubMed1997

Opposite effects of isoniazid and fasting on the expression of CYP2E1 protein and mRNA in house musk shrew (Suncus murinus).

The effects of isoniazid and fasting on hepatic CYP2E1 in the suncus were investigated. Aniline hydroxylation and N-nitrosodimethylamine demethylation, which are known to be catalyzed by CYP2E1, in liver microsomes were induced and suppressed by the treatment with isoniazid and the fasting, respectively. Immunoblot analysis indicated that CYP2E1 protein in liver microsomes from isoniazid-treated suncus was increased in contrast to the result with the fasting of the suncus. Northern blot analysis showed that the treatment of suncus with isoniazid increased the expression of CYP2E1 mRNA in livers although the fasting of the suncus significantly decreased CYP2E1 mRNA. These results suggest that the regulation of hepatic CYP2E1 in the suncus by treatment with isoniazid and fasting was different from that in rats.

Amino Acid Sequence↗

PubMed1997

Marmoset CYP1A2: primary structure and constitutive expression in livers.

Complementary DNA of marmoset CYP1A2 was isolated by means of screening the cDNA library and reverse-transcriptase polymerase chain reaction. The deduced amino acid sequence of marmoset CYP1A2 consisted of 516 residues and showed 88.2 and 90.0% identities to corresponding forms in human and cynomolgus monkey, respectively. S1 nuclease protection assay demonstrated that CYP1A2 mRNA was expressed constitutively in the liver, but not in the lung, kidney and small intestine. The level of CYP1A2 mRNA in the liver was increased by treatment with 3-methylcholanthrene and polychlorinated biphenyls. Marmoset CYP1A2 expressed in recombinant yeast activated 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx) efficiently, and also activated 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), but at a relatively lower rate in the umu mutagenicity test. Marmoset CYP1A2 also showed ethoxyresorufin O-de-ethylase activity. Based on these results, we demonstrate that marmosets constitutively express CYP1A2 in the liver as in humans.

Amino Acid Sequence↗

PubMed1997

Relevance of irinotecan hydrochloride-induced diarrhea to the level of prostaglandin E2 and water absorption of large intestine in rats.

For characterization of the mechanism(s) of severe diarrhea due to the anticancer agent, irinotecan hydrochloride (CPT-11), examination was made of the relation of CPT-11-related diarrhea to colonic prostaglandin E2 (PGE2) and water absorption in rats. Acute diarrheal symptoms were observed within 1 hr after the administration of CPT-11 to rats, with increased PGE2 and decreased water absorption in the colon. Treatment with atropine at 1 mg/kg, s.c. was noted to inhibit intestinal PGE2 and the CPT-11-related acute diarrheal symptoms, indicating that these diarrheal symptoms were mediated through the cholinergic nervous system accelerated functionally by CPT-11. On the other hand, daily treatment of CPT-11 at the same dose resulted in chronic diarrheal symptoms in all animals 3 days after CPT-11 treatment. Histopathological changes observed in the descending colon and ileum of the rats included degeneration and necrosis of villi and cryptal cells and a decrease in the number of the goblet cells. Significantly increased PGE2 and impaired water absorption of the descending colon were also observed during the chronic diarrheal stage. It can be considered that the chronic diarrheal symptoms appear as a consequence of the gastrointestinal injury characterized by significant increase in PGE2 accompanied by impaired water absorption.

Animals↗

PubMed1997

Preventive effects of Hange-shashin-to on irinotecan hydrochloride-caused diarrhea and its relevance to the colonic prostaglandin E2 and water absorption in the rat.

The possible preventive effect of Kampo medicine Hange-shashin-to (TJ-14) on chronic diarrheal symptoms induced by the administration of the anticancer agent irinotecan hydrochloride (CPT-11) was investigated in the rat. Repeated oral administrations of TJ-14 at 125 and 500 mg/kg significantly prevented the reduction in body weight and the onset of chronic diarrheal symptoms due to CPT-11 in a dose-dependent manner, even though it failed to show a definite effect on acute diarrheal symptoms. In addition, treatment with TJ-14 accelerated the healing of the intestinal tract injured by repeated dosing of CPT-11 and inhibited significantly the increase of colonic prostaglandin E2 (PGE2) which is closely related to the onset of diarrhea. TJ-14 also improved colonic water absorption impaired by repeated dosing of CPT-11 in rats. These results demonstrate that TJ-14 is an effective medicine for the prevention and/or treatment of CPT-11-induced chronic diarrheal symptoms.

Animals↗

PubMed1997

Low activity of aldehyde dehydrogenase (ALDH) in liver of Suncus murinus: possible explanation for high sensitivity to alcohol.

Suncus murinus (suncus) is an animal species highly sensitive to ethanol (Lin, S.-C., Saito, H., Yohro, T. and Shiga, J.,J. Toxicol. Env. Health, 18: 575-587, 1986). To elucidate the mechanism involved in sensitivity to alcohol, we investigated that properties of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in the suncus in comparison with the rat. The activities of ALDH in microsomes and mitochondria of suncus livers were lower than those in rat livers, although the activity of cytosolic ADH was not different between the suncus and the rat. We propose that the high sensitivity of the suncus to alcohol can be accounted for, at least in part, by the low activity of ALDH in suncus liver.

Alcohol Dehydrogenase↗

PubMed1997

Molecular cloning of mouse liver flavin containing monooxygenase (FMO1) cDNA and characterization of the expression product: metabolism of the neurotoxin, 1,2,3,4-tetrahydroisoquinoline (TIQ).

A mouse liver cDNA clone, MFMO1, coding for a flavin-containing monooxygenase (FMO) was isolated. This cDNA clone encoded a protein of 532 amino acids. Based upon its predicted amino acid sequence, this clone was assumed to belong to the FMO1 subfamily. The deduced amino acid sequence showed 94, 84, 83, and 83% identity with FMO1s of rats, pigs, rabbits and humans, respectively, while it showed only 50-59% identity with human FMO3 and FMO4, rabbit FMO2, FMO3, FMO4 and FMO5, and guinea-pig FMO2. RNA blot analysis showed that the mouse FMO1 was also expressed in the lung and kidney and to lesser extents in the heart, spleen, testis and brain. Mouse FMO1 expressed in yeast showed activities of thiobenzamide S-oxidation, and NADPH oxidation associated with the S- or N-oxidation of chlorpromazine, N,N-dimethylaniline, N,N-dimethyl-hydrazine, imipramine, nicotine, thioacetamide, thiourea and trimethylamine. Moreover, 1,2,3,4-tetrahydroisoquinoline (TIQ), a substance known to induce a parkinsonism-like syndrome in monkeys, was also metabolized by the mouse FMO1. The K(m) values for chlorpromazine, imipramine and TIQ were determined to be 2,4, 16.0, 435 mM, respectively. This is the first report to show that an expressed FMO can metabolize a neurotoxin, TIQ.

Amines↗