Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.13.97 (CYP3A4)
 6,365 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The stability of cytochrome P450 enzymes, cytochrome b5, and NADPH-cytochrome c reductase was examined in (A) human liver samples frozen in liquid nitrogen and stored at -80 degrees C, (B) human liver microsomes suspended in 250 mM sucrose and stored at -80 degrees C, and (C) human liver microsomes subjected to as many as 10 cycles of thawing and freezing. In study A, microsomes from five human livers were prepared from fresh (unfrozen) tissue and from tissue that was stored frozen at -80 degrees C for 1, 2, 4, or 6 months. The apparent concentration of cytochromes P450 and b5 and the activity of NADPH-cytochrome c reductase decreased 20-40% as a result of freezing the liver, regardless of whether the liver was stored for 1 or 6 months. Similar decreases were observed in the activities of cytochrome P450 enzymes belonging to several gene families, namely CYP1A2 (7-ethoxyresorufin O-dealkylation and caffeine N3-demethylation), CYP2A6 (coumarin 7-hydroxylation), CYP2C9 (tolbutamide methylhydroxylation), CYP2C19 (S-mephenytoin 4'- hydroxylation), CYP2D6 (dextromethorphan O-de-methylation), CYP2E1 (chlorzoxazone 6-hydroxylation), CYP3A4solidus5 (testosterone 6beta-hydroxylation), and CYP4A9solidus11 (lauric acid 12-hydroxylation). Freezing human liver did not convert cytochrome P450 to its inactive form, cytochrome P420, but it increased the contamination of liver microsomes with hemoglobin or other heme-containing proteins, which resulted in a uniform decrease in the specific activity of cytochromes P450 and b5 and in the specific activity of all P450 enzymes. In study B, the concentration of cytochromes P450 and b5, the activity of NADPH-cytochrome c reductase, and the activity of individual cytochrome P450 enzymes were determined in 10 samples of human liver microsomes stored at -80 degrees C for approximately 0, 1, or 2 years. The sample-to-sample variation in the concentration and activity of cytochrome P450, cytochrome b5, and NADPH-cytochrome c reductase was nominally affected by long-term storage of human liver microsomes at -80 degrees C, indicating there was no differential loss of cytochrome P450 activity, cytochrome b5 concentration, or NADPH-cytochrome c reductase activity. In study C, microsomes from a pool of human livers were subjected to 1, 2, 3, 5, 7, or 10 cycles of freezing at -80 degrees C followed by thawing at room temperature. Freezing/thawing liver microsomes for up to 10 cycles did not convert cytochrome P450 to P420, nor did it cause significant loss of CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4/5, or CYP4A9/11 activity. Overall, these results suggest that our current methods for storing and processing human liver are well suited to preserving microsomal P450 enzyme activity.
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PMID:Effects of freezing, thawing, and storing human liver microsomes on cytochrome P450 activity. 866 Jun 94

Interindividual variation in the spontaneous and in the glucocorticoid-or rifampicin-inducible expression of the CYP3A cytochromes P450, the dominant froms of this supergene family that catalyze the oxidation of numerous drugs and environmental chemicals in human liver, remains largely unexplained, due in part to the lack of a validated animal model. We analyzed the 5'-flanking sequences of CYP3A genes from the rat (CYP3A23, CYP3A2), rabbit (CYP3A6), and human (CYP3A4, CYP3A5, CYP3A7) and found variable regions separated by three areas (consensus I, II, and III) of sequence homology immediately upstream of their respective promoters. We used trans-species gene transfer in cellulo as a new approach for determining the basis for qualitative differences among species in liver expression of different forms of CYP3A. When we transfected into cultured rat hepatocytes vectors containing 5'-flanking DNA from CYP3A23, CYP3A4, or CYP3A6 genes, we found that CAT activity was induced on treatment with dexamethasone or pregnenolone-16 alpha-carbonitrile only if consensus II sequences were included. Rifampicin treatment had no effect. When the same constructions containing consensus II were transfected into rabbit hepatocytes, increased activity was observed on treatment of the cells with dexamethasone or with rifampicin but not with pregnenolone-16 alpha-carbonitrile. These results suggest that the host cellular environment rather than the structure of the gene dictates the pattern of CYP3A inducibility. The application of this new model system will provide a unique technique for identifying mechanisms of induction and advancing the development of appropriate toxicological models for human safety assessment.
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PMID:Trans-species gene transfer for analysis of glucocorticoid-inducible transcriptional activation of transiently expressed human CYP3A4 and rabbit CYP3A6 in primary cultures of adult rat and rabbit hepatocytes. 870 Jan 1

Cytochromes P450 comprise a remarkably diverse superfamily of heme-thiolate proteins critical in the metabolism of numerous endogenous ligands and xenobiotics. Among the myriad of P450 substrates are many compounds of toxicological and pharmacological significance. The precise complement of cytochrome P450 isoforms in any given tissue may therefore be an important determinant of susceptibility to chemical-mediated toxicity. We have used a histological approach to study the distribution of individual P450s in human and rabbit gastro-intestinal tissues. We have focused primarily on P450 enzymes of importance in the metabolism of carcinogens, namely CYP1A1, CYP1A2, CYP2E1, CYP3A4/3A5 and CYP4B1. Here we give an overview of the distribution of these enzymes in human and rabbit tissues and discuss the possible toxicological implications of the results. In addition we will discuss the value of archival human tissue specimens for histological analysis of P450 distribution.
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PMID:Localization of cytochromes P450 in human tissues: implications for chemical toxicity. 874 22

Recent findings about individual isoforms of the cytochromes P450 involved in the metabolism of phenytoin (PHT) and carbamazepine (CBZ) make prediction of inhibition-based interactions possible. PHT is eliminated principally by hydroxylation to p-HPPH, a reaction catalyzed primarily by CYP2C9 and secondarily by CYP2C19 (S-mephenytoin hydroxylase). The principle of isoform specificity (drugs metabolized by the same isoform should exhibit interactions with the same inhibitors) was applied to the interactions of PHT with 17 inhibitors using two probes for CYP2C9, S-warfarin and tolbutamide. Eleven of 17 interactions (sulfaphenazole, phenylbutazone, fluconazole, azapropazone, cotrimoxazole, propoxyphene, miconazole, amiodarone, disulfiram, metronidazole, and stiripentol) could be explained by inhibition of CYP2C9. The remaining interactions (felbamate, omeprazole, cimetidine, fluoxetine, imipramine, and diazepam) were attributed to inhibition of CYP2C19. For CBZ, studies utilizing chemical inhibitors, immunoinhibition, liver bank correlations, and expressed enzymes established that CYP3A4 is the main enzyme catalyzing formation of CBZ-10, 11-epoxide. This explains the pronounced interactions of CBZ with erythromycin, troleandomycin, and other macrolide antibiotics (clarithromycin, josamycin, flurythromycin, and ponsinomycin). Work is in progress to explain the interactions of CBZ with other inhibitors. The literature contains no other information on isoforms involved in the metabolism of other major antiepileptic drugs.
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PMID:Cytochrome P450 isozymes and antiepileptic drug interactions. 880 99

To investigate the role of newly synthesized apocytochrome P450 (P450) in the regulation of 5-aminolevulinate synthase (ALAS), we overexpressed P450 in primary cultures of chick embryo hepatocytes and measured the subsequent effects on ALAS mRNA by semiquantitative RT-PCR. Hepatocytes were co-transfected with a vector for expression of P450 cDNAs (CYP3A4 or CYP2H1) and a vector directing the synthesis of a cell surface antibody. Transfected hepatocytes were isolated with hapten-coated magnetic beads at different times after electroporation (4, 8 and 20 h). Overexpression of human CYP3A4 was demonstrated by high levels of the corresponding mRNA and apoprotein as analyzed by RT-PCR and western-blot analysis. Similarly, chicken CYP2H1 was expressed to levels even higher than those induced with phenobarbital. However the level of ALAS mRNA did not change in these cells. Our results demonstrate that the induction of ALAS by drugs is not a direct consequence of increased P450 apoprotein synthesis and heme utilization.
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PMID:Induction of 5-aminolevulinate synthase by drugs is independent of increased apocytochrome P450 synthesis. 880 6

Codeine is metabolized by glucuronidation, by O-demethylation to morphine, and by N-demethylation to norcodeine. The enzyme responsible for the O-demethylation to morphine has been identified as cytochrome P4502D6 (CYP2D6). The purpose of the present study was to identify the specific P450 enzyme responsible for codeine N-demethylation. Microsomal preparations (250 pmol of P450) obtained from 12 human liver donors were incubated with 20 microM codeine and analyzed for norcodeine formation. Codeine N-demethylation activity was linearly correlated with nifedipine oxidation activity (r = 0.90, p < 0.001), a marker of CYP3A4, but not with codeine O-demethylation, a marker of CYP2D6. Preincubation with troleandomycin (50 microM), or gestodene (50 microM) inhibitors of CYP3A4, decreased the rate of production of norcodeine by 60 and 45% compared to control values, respectively. Similarly, ketoconazole (10 microM) and erythromycin (10 microM) inhibited codeine N-demethylation by 75 and 35%, respectively. In contrast, the presence of quinidine, sulfaphenazole, or diethyldithiocarbamate in the incubation mixture had no effect on norcodeine formation. Preincubation with antibodies raised to CYP3A4 (5 mg lgG/nmol P450) caused 96% inhibition of norcodeine production, whereas preimmune IgG or antibodies raised to CYP2A6 and CYP2C had no effect. Additionally, significant norcodeine production was observed with purified CYP3A4 derived from human liver microsomes. In conclusion, codeine N-demethylation activity cosegregates with CYP3A4 activity. Coadministration of codeine with selective inhibitors of CYP3A4 may result in increased morphine production and enhanced pharmacodynamic effects due to shunting down the CYP2D6 pathway.
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PMID:Microsomal codeine N-demethylation: cosegregation with cytochrome P4503A4 activity. 881 73

Cyclobenzaprine (Flexeril) is a muscle relaxant, possessing a tricyclic structure. Numerous therapeutic agents containing this structure are known to be metabolized by polymorphic cytochrome P4502D6. The aim of this study was to determine if cytochrome P4502D6 and other isoforms are involved in the metabolism of cyclobenzaprine in human liver microsomes. Selective cytochrome P450 inhibitors for CYP1A1/2 (furafylline and 7,8-benzoflavone) and CYP3A4 (troleandomycin, gestodene, and ketoconazole) inhibited the formation of desmethylcyclobenzaprine, a major metabolite of cyclobenzaprine, in human liver microsomes. Antibodies directed against CYP1A1/2 and CYP3A4 inhibited the demethylation reaction whereas anti-human CYP2C9/10, CYP2C19, and CYP2E1 antibodies did not show any inhibitory effects. When a panel of microsomes prepared from human B-lymphoblastoid cells that expressed specific human cytochrome P450 isoforms were used, only microsomes containing cytochromes P4501A2, 2D6, and 3A4 catalyzed N-demethylation. In addition, demethylation catalyzed by these recombinant cytochromes P450 can be completely inhibited with selective inhibitors at concentrations as low as 1 to 20 microM. Interestingly, cyclobenzaprine N-demethylation was significantly correlated with caffeine 3-demethylation (1A2) and testosterone 6 beta-hydroxylation (3A4) but not with dextromethorphan O-demethylation (2D6) in human liver microsomes. To further determine the involvement of cytochrome P4502D6 in cyclobenzaprine metabolism, liver microsomes from a human that lacked CYP2D6 enzyme activities was included in this study. The data showed that cyclobenzaprine N-demethylation still occurred in the incubation with this microsome. These results suggested that cytochrome P4502D6 plays only a minor role in cyclobenzaprine N-demethylation whereas 3A4 and 1A2 are primarily responsible for cyclobenzaprine metabolism in human liver microsomes. Due to the minimum involvement of CYP2D6 in the vitro metabolism of cyclobenzaprine, the polymorphism of cytochrome P4502D6 in man should not be of muci concern in the clinical use of cyclobenzaprine.
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PMID:Identification of human liver cytochrome P450 isoforms involved in the in vitro metabolism of cyclobenzaprine. 881 77

The metabolic dealkylation of nine nitrosodialkylamines, including five symmetrical (nitrosodimethylamine, nitrosodiethylamine, nitrosodipropylamine, nitrosodibutylamine and nitrosodiamylamine) and four asymmetrical nitrosodialkylamines (nitrosomethylethylamine, nitrosomethylpropylamine, nitrosomethylbutylamine and nitrosomethylamylamine), was investigated in 14 samples of human liver microsomes. All these nitrosodialkylamines were dealkytated to aldehydes that were separated by reversed phase HPLC and UV detected as dinitrophenylhydrazones. As the length of the alkyl chain increased from methyl to pentyl, dealkylation of symmetrical nitrosodialkylamines became less efficiently catalyzed by cytochrome P450. Conversely, oxidation of the methyl moiety of asymmetrical nitrosomethylalkylamines increased with the size of the alkyl moiety, while dealkylation of the longer alkyl group decreased. N-Dealkylase activities were significantly correlated with P450 activities measured in human liver microsomes. These catalytic activities involve CYP2A6 (coumarin 7-hydroxylation), CYP2C (mephenytoin 4-hydroxylation and tolbutamide hydroxylation), CYP2D6 (dextromethorphan O-demethylation), CYP2E1 (chlorzoxazone and p-nitrophenol hydroxylation) and CYP3A4 (nifedipine oxidation). By using 10 heterologously expressed P450s, it was shown that nitrosodimethylamine was mainly demethylated by CYP2E1. However, such enzyme specificity was lost with increasing size of the alkyl group. Therefore, the chain length of the alkyl group of nitrosodialkylamines determined the P450 involved in its oxidation. All these results emphasize that the catalytic site of P450 2EI has a geometric configuration such that only small molecules like nitrosodimethylamine fit favorably within the putative active site of the enzyme. Furthermore, there is good evidence that P450s other than P450 2E1, such as P450 2A6, 2C8/2C9/2C19 and 3A4, are involved in the metabolism of nitrosodialkylamines bearing bulky alkyl chains.
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PMID:Cytochrome P450 metabolic dealkylation of nine N-nitrosodialkylamines by human liver microsomes. 882 31

Human bronchial epithelial cells (BEC), a primary defense against inhaled materials, are the progenitor cells for bronchogenic carcinomas and have important metabolic capabilities. We used reverse transcriptase-polymerase chain reaction (RT-PCR) to identify xenobiotic metabolism enzymes expressed in primary BEC and alveolar macrophages (AM) of non-smoking volunteers. Cytochromes P450 (CYP) 1A1, 1B1, 2B7, 2E1, and 4B1 and microsomal epoxide hydrolase (mEH) were expressed in BEC but not AM. CYP2F1 was expressed in BEC, but it was expressed at barely detectable levels or not at all in AM. NADPH oxidoreductase (NADPH OR), microsomal glutathione transferase (GST 12), glutathione transferase mu, phenol sulfotransferase (PST), thermolabile phenol sulfotransferase (TL PST), and the clara cell-specific gene, CC10 were expressed in both BEC and AM. CYP3A4 and glucuronosyl transferases-1 and 2 were not expressed in either BEC or AM. In contrast to primary BEC, of the genes evaluated, the immortalized human bronchial epithelial cell line BEP2D constitutively expressed only CYP1A1, CYP2E1, NADPH OR, glucuronosyl transferase 1, GST 12, GST mu, PST, TL PST, and CC10. The loss of xenobiotic metabolism enzyme gene expression in the BEP2D cell line may result from either reduced exposure to inducing agents, or loss of differentiative characteristics in culture. It is clear from the data comparing BEC and AM that there are important intertissue differences in expression of xenobiotic metabolism enzymes.
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PMID:Xenobiotic metabolism enzyme gene expression in human bronchial epithelial and alveolar macrophage cells. 884 77

The multiplicity of the drug-metabolizing cytochrome P450 system was discovered 20 years ago. During the past 10 years the complementary DNAs of the most important P450 enzymes have been cloned and sequenced, and much has been learned about their substrate specificities, selective inhibitors, and functional characteristics. Cytochrome P4501A2 (CYP1A2), CYP2C19, CYP2D6, and CYP3A4 are the most important P450s catalyzing the biotransformation of psychotropic drugs. Assessment of the activity of individual P450 enzymes makes it possible to forecast an appropriate initial dose in a patient. At present, this strategy can be recommended only for CYP2D6 before treatment with tricyclic antidepressants and certain neuroleptics. Important drug-drug interactions can be predicted if two substrates or a substrate and an inhibitor of a particular P450 are co-administered. Therapeutic drug monitoring is of invaluable help in discovering and handling this type of interaction.
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PMID:Drug-metabolizing enzymes and therapeutic drug monitoring in psychiatry. 885 57


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