Quantitative RT-PCR for CYP3A4 mRNA in human peripheral lymphocytes: induction of CYP3A4 in lymphocytes and in liver by rifampicin.
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Animals↗
Biomedical subjects
Y Funae
Publications and source records attributed to Y Funae.
Chemopreventive effects of coumaperine from pepper on the initiation stage of chemical hepatocarcinogenesis in the rat.
This study was designed to investigate the chemopreventive action of three natural products, coumaperine, aurapten and an extract from rosemary, against the initiation stage of rat hepato-carcinogenesis. Coumaperine has been isolated from white pepper as a naturally occurring antioxidative agent, but its potential modifying effects on carcinogenesis remain unclear. In experiment 1, a modification of the model developed by Tsuda et al. was applied, with assessment of numbers and areas of induced glutathione S-transferase placental form (GST-P)-positive hepatocellular foci in male F344 rats. Coumaperine, aurapten and the extract from rosemary were administered i.g. at 100 mg / kg / day once daily for 5 days with initiation by diethylnitrosamine (DEN) on day 4 (20 mg / kg, i.p.). Numbers and areas of GST-P-positive foci in each group given test chemicals tended to be decreased as compared to the vehicle control group values, significance being achieved for number with coumaperine. Experiment 2 was planned to investigate the mechanism of the inhibitory effects of coumaperine. Livers at 8 h after initiation by DEN were examined with coumaperine administered at 100 mg / kg / day once daily for 3 days. Proliferating cell nuclear antigen (PCNA)-positive cells tended to be decreased as compared to the vehicle control, but no effects on apoptosis or cytochrome P-450 (CYP) 2E1 expression were apparent. Our results suggest that coumaperine provides protection against initiation of hepatocarcinogenesis, and that this is related to inhibition of cell proliferation.
Animals↗
Induction of hepatic CYP2B1/2 by a teratogenic compound cis-1-[-4-(p-menthane-8-yloxy)phenyl]piperadine (YM9429) in rats.
A teratogenic compound cis-1-[-4-(p-menthane-8-yloxy)phenyl]piperadine (YM9429) selectively induces skeletal malformations characterized by cleft palate in rat fetuses. In the present study, we investigated the effect of YM9429 on hepatic cytochrome P-450s and their activities in rats. Oral administrations of YM9429 at a dose of 250, 500 or 750 mg/kg daily for 3 days induced cytochrome P-450 contents in a dose-dependent manner. Concomitant induction of enzyme activities of benzphetamine N-demethylase, erythromycin N-demethylase and, to a lesser extent, aminopyrine N-demethylase was observed. Immunoblot analysis revealed that YM9429 up-regulated hepatic levels of CYP2B1/2 and CYP3A1/2 proteins. A single dose of YM9429 at 250 mg/kg induced CYP2B1/2 protein levels significantly. These results suggest that YM9429 is a strong inducer of cytochrome P-450 with characteristics resembling those of phenobarbital.
Animals↗
Prediction of in vivo drug-drug interactions between tolbutamide and various sulfonamides in humans based on in vitro experiments.
Drug-drug interactions between tolbutamide and sulfonamides have extensively been reported. We attempted to predict the in vivo interaction between tolbutamide and sulfonamides from the in vitro metabolic inhibition studies. The inhibition constant (K(i)) was derived from the inhibitory effects of eight sulfonamides (sulfaphenazole, sulfadiazine, sulfamethizole, sulfisoxazole, sulfamethoxazole, sulfapyridine, sulfadimethoxine, and sulfamonomethoxine) on tolbutamide metabolism. We found that the inhibitory effect of sulfaphenazole was greatest among the eight sulfonamides examined. Furthermore, the contribution of each P450 enzyme to tolbutamide metabolism was investigated by using recombinant P450 enzymes. Although cytochrome P450 (CYP) 2C8, 2C9, and 2C19 metabolized tolbutamide, the main enzyme involved was CYP2C9. The K(i) values of several sulfonamides were comparable between human liver microsomes and recombinant CYP2C9. The maximum unbound plasma concentration of sulfonamides in the portal vein was calculated from literature data on the pharmacokinetics of sulfonamides. Using the K(i) values obtained from in vitro inhibition studies, the degree of increase in tolbutamide area under the plasma concentration-time curve (AUC) was predicted. About 4.8- and 1.6-fold increases in tolbutamide AUC were predicted by coadministration of sulfaphenazole and sulfamethizole, respectively, which agreed well with the reported increases in humans. Furthermore, the increase in tolbutamide AUC by coadministration of sulfadiazine, sulfisoxazole, and sulfamethizole was predicted to be 1.5- to 2. 6-fold, although the corresponding in vivo effects have not been reported. It is concluded that some of these sulfonamides have to be carefully coadministered with CYP2C9 substrates such as tolbutamide although coadministration of sulfaphenazole needs the greatest care.
Adult↗
[Arachidonic acid metabolites (HETEs and EETs) produced by cytochrome P450s].
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Animals↗
Inhibition of liver glutathione S-transferase placental form-positive foci development in the rat hepatocarcinogenesis by Porphyra tenera (Asakusa-nori).
The effects of Asakusa-nori, Porphyra tenera (PT), a popular edible seaweed in Japan, on the development of putative preneoplastic lesions, glutathione S-transferase placental form (GST-P)-positive foci, in the male F344 rat liver were examined using a medium-term bioassay system. PT significantly decreased both the number and area of GST-P-positive foci in rat livers initiated with diethylnitrosamine (DEN). To investigate possible mechanisms of inhibition, effects of PT on 5-bromo-2'-deoxyuridine (BrdU) labeling in GST-P-positive foci and the surrounding area of hepatocytes were studied. The ratio of the GST-P-positive foci to surrounding tissue labeling indices was decreased in the PT-treated group as compared with the DEN alone group. Ornithine decarboxylase activity in the liver was slightly increased and spermidine/spermine N'-acetyltransferase activity was slightly decreased in the PT-treated animals. These results suggest that PT possesses chemopreventive effects against DEN-induced hepatocarcinogenesis.
Acetyltransferases↗
Isoforms of cytochrome P450 on organic nitrate-derived nitric oxide release in human heart vessels.
Glutathione S-transferases and the cytochrome P450 system have been proposed for the vascular biotransformation systems in the metabolic activation of organic nitrates. The present study was designed to elucidate the role of human cytochrome P450 isoforms on nitric oxide formation from organic nitrates using lymphoblast microsomes transfected with human CYP isoforms cDNA. CYP3A4-transfected microsomes had the most effective potential of nitric oxide formation from isosorbide dinitrate. Anti-CYP3A2 antibody (which cross-reacts with CYP3A4) or ketoconazole (an inhibitor of the CYP3A superfamily) inhibited nitric oxide formation from isosorbide dinitrate in rat heart microsomes. Immunohistochemistry of human heart also showed intense bindings of CYP3A4 antibody in the endothelium of the endocardium and coronary vessels. These results suggest that the CYP3A4-NADPH-cytochrome P450 reductase system specifically participates in nitric oxide formation from isosorbide dinitrate.
Animals↗
Hepatic cytochrome P450 is directly inactivated by nitric oxide, not by inflammatory cytokines, in the early phase of endotoxemia.
BACKGROUND/AIMS: Although the activity of the liver in metabolizing and eliminating various drugs decreases in endotoxemia, the mechanism remains to be elucidated. The generation of nitric oxide by the inducible type of nitric oxide synthase increases in endotoxemia. Nitric oxide readily reacts with heme proteins such as cytochrome P450 that metabolize various compounds, including steroids and eicosanoids. The purpose of this study was to determine the effect of nitric oxide on the function of hepatic cytochrome P450 in endotoxemic rats. METHODS: To determine the dynamic aspects of nitric oxide metabolism, hepatic levels of the inducible type of nitric oxide synthase and heme-iron nitrosyl complexes, and plasma levels of nitrite and nitrate were determined in rats before and after intravenous administration of lipopolysaccharide. Changes in the levels of P450 isoforms and testosterone hydroxylation activity in hepatic microsomes were also determined. To evaluate in vivo CYP3A2 activity, midazolam sleep time was measured. RESULTS: When lipopolysaccharide increased the hepatic inducible type of nitric oxide synthase and plasma levels of nitric oxide metabolites, the intensity of low-spin signal of electron spin resonance responsible for the ferric form of P450 decreased with a concomitant increase in heme-iron nitrosyl complexes in the liver. Lipopolysaccharide-related nitric oxide generation is followed by an early decrease in the levels of cytochrome P450 and of testosterone hydroxylation activity in liver microsomes. Midazolam sleep time was prolonged by lipopolysaccharide. All these early changes were prevented by the inhibitor of nitric oxide synthase, N(G)-iminoethyl-L-ornithine. Moreover, lipopolysaccharide suppressed the gene expression of CYP2C11 and CYP3A2. Decreases in levels of cytochrome P450 and their mRNAs were more pronounced at 24 h after LPS administration, but apparently they are NO-independent. CONCLUSIONS: These results suggest that lipopolysaccharide-induced modulation of cytochrome P450 may occur via the interplay of two different mechanisms and that, especially in the early phase, nitric oxide-dependent inhibition is more important.
Animals↗
Non-carcinogenicity, but dose-related increase in preneoplastic hepatocellular lesions, in a two-year feeding study of phenobarbital sodium in male F344 rats.
Phenobarbital sodium (PB) was administered at dietary levels of 0 (control), 8, 30, 125 and 500 ppm to groups of 20 male F344/DuCrj rats for 104 weeks. There were no treatment-related clinical signs or adverse effects on survival rate, body weights, food consumption, and haematology or blood biochemistry data. Statistically significant increases of relative liver weights were found in the 500 and 125 ppm, but not the 30 and 8 ppm groups. Quantitative analysis of glutathione S-transferase placental form positive (GST-P+) hepatocyte foci/areas revealed clear increases limited to the 500 and 125 ppm groups. Western blotting revealed CYP2B1, 2C6 and 3A2 proteins to be also increased only with these high doses. In addition, significant increase of regenerative hepatocellular hyperplasias was noted in the 500 ppm group. No hepatocellular adenomas were observed, but a hepatocellular carcinoma arose in single rats of the 8 ppm and 125 ppm groups. No treatment-related changes were found in any other organs or tissues. Thus, under the experimental conditions used, the highest dose of PB (500 ppm) was not carcinogenic in male F344 rats. Furthermore, increase in putative preneoplastic proliferative hepatocytic lesions was only noted with 500 and 125 ppm.
Administration, Oral↗
Involvement of CYP3A-derived arachidonic acid metabolite(s) in responses to endothelium-derived K+ channel opening substance in monkey lingual artery.
1. In monkey lingual artery strips partially contracted with prostaglandin F2alpha, acetylcholine-induced, concentration-related relaxations were abolished by removal of the endothelium. The response was not significantly influenced by indomethacin but attenuated by NG-nitro-L-arginine (L-NOARG); the effect of the nitric oxide (NO) synthase inhibitor was reversed by L-arginine. 2. The response to acetylcholine resistant to L-NOARG was suppressed in the strips exposed to high K+ media. Charybdotoxin partially inhibited the relaxation, and the remaining relaxation was abolished by additional treatment with apamin, whereas glibenclamide, iberiotoxin or apamin alone was without effect. Relaxations induced by sodium nitroprusside were not influenced by charybdotoxin. 3. The L-NOARG-resistant acetylcholine-induced relaxation was inhibited by metyrapone, proadifen and 17-octadecynoic acid, non-selective cytochrome P450 mono-oxygenase (CYP) inhibitors, and progesterone and ketoconazole, inhibitors selective to CYP3A. The inhibitors did not affect the nitroprusside-induced relaxation. Selective inhibitors of other CYP isoforms, such as debrisoquine and lauric acid, did not reduce the response to acetylcholine. 4. Reaction mixture containing human liver microsome rich in CYPs, arachidonic acid and NADPH incubated at 37 degrees C and filtrated relaxed endothelium-denuded monkey lingual artery strips, used as bioassay tissues. This response was abolished in the strips exposed to high K+ media. The response was also suppressed by combined treatment of the assay tissue with charybdotoxin plus apamin, but was not affected by treatment with iberiotoxin. The reaction mixture co-incubated with ketoconazole failed to relax the strips. 5. It is concluded that the monkey lingual arterial relaxation dependent on the endothelium is mediated by NO and also by a charybdotoxin plus apamin-sensitive but iberiotoxin-insensitive Ca2+-activated K+ channel opening substance(s) that may be a CYP3A-derived arachidonic acid metabolite(s).
Animals↗
Cytochrome P450 isoforms responsible for the N-deethylation and cyclohexane-hydroxylation of NS-21.
1. Cytochrome P450 (P450) isoforms responsible for the N-deethylation and cyclohexane-hydroxylation of (+/-)-4-diethylamino-1,1-dimethylbut-2-yn-1-yl 2-cyclohexyl-2-hydroxy-2-phenylacetate monohydrochloride monohydrate (NS-21) have been identified in rat and man. 2. Anti-CYP2C11 antibody inhibited the N-deethylation of S- and R-NS-21 in rat hepatic microsomes by 84 and 66% respectively, indicating that CYP2C11 is mainly responsible for these activities in male rats. 3. Of several human recombinant P450 isoforms, CYP3A4 had the activities for the N-deethylation of S- and R-NS-21. In addition, triacetyloleandomycin (TAO), an inhibitor of the CYP3A subfamily, significantly inhibited the N-deethylation of S- and R-NS-21 in human hepatic microsomes by 67 and 69%, respectively. CYP3A4 therefore contributes to it in man. 4. Quinine, an inhibitor of the rat CYP2D subfamily, significantly inhibited the cyclohexane-4-cis-hydroxylation of S-NS-21 by 48% in rat hepatic microsomes. In contrast, this inhibitor had little effect on the cyclohexane-4-trans-hydroxylation of S-NS-21, and the cyclohexane-4-cis- and trans-hydroxylation of R-NS-21. 5. Human recombinant CYP3A4 catalysed the cyclohexane-4-trans-hydroxylation of S-NS-21, and CYP2D6 supported the cyclohexane-4-cis- and trans-hydroxylation of S-NS-21. Quinidine, an inhibitor of human CYP2D6, had little effect on these latter activities in human hepatic microsomes. TAO significantly inhibited the cyclohexane-4-trans-hydroxylation of S-NS-21 by 75%, indicating that CYP3A4 catalyses this reaction.
Animals↗
Isoform-selective metabolism of mianserin by cytochrome P-450 2D.
The involvement of cytochrome P-450 (CYP) 2D isoforms in the metabolism of mianserin and the stereoselectivity of their catalytic activities were investigated by using five CYP2D isoforms (CYP2D1, 2D2, 2D3, 2D4, and 2D6). Using RS-mianserin as a substrate, we found that five CYP2D isoforms had similar levels of 8-hydroxylation activity. However, N-demethylation activity differed among the isoforms; CYP2D3 and 2D4 efficiently demethylated RS-mianserin compared with the other three isoforms. N-Oxidation activity was specific to CYP2D1 although its level was relatively low. Another metabolite, assigned as 8-hydroxy-N-desmethylmianserin by liquid chromatography/mass spectrometry analysis, was formed by CYP2D4 and 2D6. The metabolism exhibited stereoselectivity. CYP2D1 and 2D4 selectively 8-hydroxylated the R(-)-enantiomer, and CYP2D6 predominately N-demethylated R(-)-enantiomer. N-Oxidation by CYP2D1 was specific to R(-)-enantiomer. In conclusion, CYP2D isoforms are involved in several metabolic pathways of mianserin acting in an isoform-specific manner. Stereoselectivity of the catalytic activities was clearly observed in the reactions of CYP2D1, 2D4, and 2D6.
Alcohol Oxidoreductases↗
Developmental changes in the catalytic activity and expression of CYP2D isoforms in the rat liver.
Developmental changes in bufuralol 1'-hydroxylation activity, which is known as a typical activity of cytochrome P-450 (CYP)2D isoforms, in the liver of rats were investigated. The catalytic activities of hepatic microsomes increased with development especially from 3 to 7 weeks. Eadie-Hofstee plots of bufuralol 1'-hydroxylation were obtained for monophasic kinetics (Km: 0.037 microM) at 1 week and for biphasic kinetics (Km: 0.051 and 6.4 microM) at 7 weeks of age. Quinine completely inhibited bufuralol 1'-hydroxylation activity of hepatic microsomes of 1- and 7-week-old rats. These results indicated that at least two kinds of CYP2D isoforms, which differ markedly in their affinity for bufuralol, were present at 7 weeks of age and that the CYP2D isoform that had low affinity for bufuralol was expressed with development. To assess the affinity of CYP2D isoforms for bufuralol, the kinetic properties of CYP2D1, 2D2, 2D3, and 2D4 expressed in yeast cells were investigated. The Km value of CYP2D2, 0.044 microM, was extremely small compared with that of the other rat CYP2D isoforms. We further investigated developmental changes of CYP2D isoform mRNA by reverse transcription-polymerase chain reaction. CYP2D3 mRNA increased with development although CYP2D1 and 2D2 mRNA were not changed. The CYP2D4 mRNA was not detected. These findings indicated that CYP2D2, which had high affinity for bufuralol, was expressed in immature and mature rats, but CYP2D3, which had low affinity for bufuralol, was expressed only in mature rats.
Animals↗
Effects of ACTH administration on zonation of the guinea pig adrenal cortex.
Experiments were done to determine the actions of ACTH on the morphologic and functional characteristics of the zona fasciculata (ZF) and zona reticularis (ZR) in the guinea pig adrenal cortex. In control guinea pigs, a number of morphologic differences distinguished the ZF from the ZR, including the presence of far more lipid in the ZF than in the ZR. Treatment with ACTH decreased the lipid droplet content of the ZF cells, equalizing the amount of lipid in the two zones. Other morphologic differences between the ZF and ZR were also diminished by ACTH treatment. Immunohistochemical analyses indicated that CYP17 protein was found in both the ZF and ZR in control animals, but with greater immunostaining intensity in the ZF. The enzyme protein distribution corresponded with higher 17alpha-hydroxylase activity in the ZF than in the ZR. After ACTH treatment, the intensity of staining and enzyme activities in the two zones were similar, attributable largely to increases in the ZR. In situ hybridization-and immunohistochemistry showed that in control animals CYPD216 was highly expressed in the ZR but not in the ZF. ACTH treatment dramatically reduced the intensity of CYP2D16 mRNA and protein staining in the ZR. Bufuralol 1'-hydroxylase activity, a marker for CYP2D subfamily members, was also decreased significantly in the ZR by ACTH treatment. The data indicate that administration of ACTH to guinea pigs has opposite effects on the expression of CYP17 and CYP2D16 in the ZR, and diminishes or eliminates some of the structural and functional differences between the ZF and ZR. The results suggest a role for ACTH in establishing and maintaining adrenocortical zonation.
Adrenal Cortex↗
Dopamine formation from tyramine by CYP2D6.
Dopamine is formed form L-tyrosine by tyrosine hydroxylase and aromatic L-amino acid decarboxylase. In addition to this pathway, however, the formation of catecholamines, including dopamine, from trace amines such as tyramine by hepatic microsomes has been demonstrated. In this study, we investigated the formation of dopamine from trace amines, using human hepatic microsomes and human cytochrome P450 (CYP) isoforms expressed in yeast. Among the 11 isoforms of human CYP expressed in yeast, CYP2D6 was the only isoform exhibiting strong ability to convert p-tyramine and m-tyramine to dopamine. In studies with human hepatic microsomes, the hydroxylation of tyramine to dopamine was inhibited by bufuralol, a typical substrate for CYP2D isoforms, and anti-CYP2D1 antiserum. This is the first report showing that CYP2D is capable of converting tyramine to dopamine. The Km values of CYP2D6, expressed in yeast, for p-tyramine and m-tyramine were 190.1 +/- 19.5 microM and 58.2 +/- 13.8 microM, respectively. Tyramine is an endogenous compound which exists in the brain as a trace amine but is also an exogenous compound which is found in foods such as cheese and wine. Our results suggest that dopamine is formed from endogenous and/or exogenous tyramine by this CYP2D isoform.
Animals↗
P450 isoforms in a murine macrophage cell line, RAW264.7, and changes in the levels of P450 isoforms by treatment of cells with lipopolysaccharide and interferon-gamma.
The presence of P450 in a murine macrophage cell line, RAW264.7, was investigated to clarify the biological role and regulation of P450. Microsomes of RAW264.7 cells were isolated and subjected to immunoblotting with anti-rat CYP2A1, 2B1, and 4A2 antibodies. The microsomes gave staining bands with all these antibodies, suggesting the presence of mouse Cyp2a, 2b, and 4a isoforms in RAW264.7. RAW264. 7 cells were treated with typical inducers of P450 (phenobarbital, clofibrate, beta-naphthoflavone and 3-methylcholanthrene). None of these chemicals induced these P450s. Stimulation of RAW264.7 cells with lipopolysaccharide (LPS) and interferon-gamma (INF-gamma) which increase inducible nitric oxide synthase (iNOS) and cytokines in cells decreased Cyp4a protein but not Cyp2a and 2b proteins. To identify P450 isoforms in RAW264.7, we used polymerase chain reaction (PCR) primers for mouse Cyp2a4, 2a12, 2b9/10, 4a10, and 4a12. Total RNA was isolated from these cells and converted to cDNA by reverse transcriptase. PCR was done with these primers and the amplified nucleotides were analyzed by a DNA sequencer. Only Cyp2b9/10 and 4a12 primers gave clear bands, although all primers gave clear bands from liver total RNA. Nucleotide sequences of these products amplified by PCR were identical with Cyp2b9 and 4a12. These findings indicate that Cyp2b9 and 4a12 were present in a macrophage cell line, RAW264.7, and the regulation of P450 by inducers and cytokine differed from that in liver.
Animals↗
Cloning and characterization of the CYP2D1-binding protein, retinol dehydrogenase.
A CYP2D1-binding protein, 29 k-protein (p29), has been isolated and its N-terminal amino acid sequence has been reported (Ohishi et al. (1993) Biochim. Biophys. Acta 1158, 227-236). In this study, p29 cDNA was isolated by PCR with oligonucleotide probes designed from the N-terminal amino acid sequence and p29 was found to be a microsomal retinol dehydrogenase, a member of the short-chain alcohol dehydrogenase family which metabolize hydroxysteroids and prostaglandins. CYP2D1 and p29 were expressed in Saccharomyces cerevisiae to characterize these proteins. CYP2D1 had an absorption maximum at 448 nm in a CO-reduced form. Expressed p29 in yeast cells was detected with anti-p29 antibody. Solubilized CYP2D1 and p29 from yeast microsomes were mixed and applied to an anti-CYP2D1 antibody-binding column. Both proteins were retained in the column and eluted with glycine buffer (pH 2.8). However, when applied alone, p29 was not retained in the column. The findings indicated that CYP2D1 bound tightly with p29. Catalytic activities of p29 expressed in yeast were investigated. p29 had retinal reductase activity in the presence of NADPH. Addition of CYP2D1 and NADPH-P450 reductase increased the retinal reductase activity of p29. These findings suggest that the complex of CYP2D1, p29, and NADPH-P450 reductase has an important role in the metabolism of retinoids.
Alcohol Oxidoreductases↗
Tissue distributions of CYP2D1, 2D2, 2D3 and 2D4 mRNA in rats detected by RT-PCR.
The tissue distributions of four isoforms (CYP2D1/5, 2D2, 2D3 and 2D4/18) in rat CYP2D subfamily were investigated. Twelve kinds of tissue (liver, kidney, brain, lung, heart, spleen, adrenal gland, small intestine mucosa, bladder, testis, ovary and gonecystis) were removed from Sprague-Dawley male and female rats. The expression of CYP2D mRNA in these tissues was detected by RT-PCR. Specific primers were designed to recognize the four isoforms individually. In liver, kidney and small intestine mucosa, the mRNA expression of all four CYP2D isoforms was detected as high-intensity PCR products. mRNA of CYP2D1/5 was expressed in all tissues used in this study except the brain, although the intensity of PCR products varied among tissues. mRNAs of CYP2D2 and CYP2D3 were mainly expressed in liver, kidney and small intestine mucosa, which were exposed to xenobiotics such as drugs, food components and environmental contaminations. mRNA of CYP2D4/18 was expressed in liver, kidney, small intestine mucosa and brain. In brain, only mRNA of CYP2D4/18 was expressed. CYP2D4/18 mRNA was also expressed in ovary, testis and gonecystis. The tissue distributions help to clarify the differences in physiological and pharmacological functions between CYP2D isoforms.
Animals↗