Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

4-Ipomeanol is a pulmonary toxin in cattle and rodents that is metabolically activated by cytochromes P-450 (P-450s). P-450-mediated activation of 4-ipomeanol to DNA binding metabolites was evaluated using a vaccinia virus complementary DNA expression system and an in situ DNA-binding assay. Twelve human P-450s and two rodent P-450s were expressed in human hepatoma Hep G2 cells and examined for their abilities to metabolically activate this toxin. Three forms, designated CYP1A2, CYP3A3, and CYP3A4, were able to catalyze significant production of DNA-bound metabolites of 20-, 8-, and 5-fold, respectively, above binding catalyzed by Hep G2 cells infected with wild-type vaccinia virus. These enzymes, with highest activities, are not known to be expressed in human or rodent lung. CYP2F1 and CYP4B1, two enzymes that are expressed in lung, display only modest 3- and 2-fold respective increased abilities to metabolically activate 4-ipomeanol. Two human forms were inactive and seven other human forms showed activities ranging from 0.5- to 2-fold above control level. Surprisingly, rabbit complementary DNA-expressed CYP4B1 was the most active enzyme (180-fold above control) among all P-450s tested in producing DNA-binding metabolites from this mycotoxin. These studies demonstrate a species difference in 4-ipomeanol metabolism and suggest caution when attempting to extrapolate rodent data to humans.
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PMID:Metabolic activation of 4-ipomeanol by complementary DNA-expressed human cytochromes P-450: evidence for species-specific metabolism. 165 9

A human B-lymphoblastoid cell line, designated MCL-5, constitutively expressing human cytochrome P-450 CYP1A1 and also expressing five transfected human cDNAs encoding drug-metabolizing enzymes, has been developed. cDNAs encoding CYP1A2, CYP2A6, and microsomal epoxide hydrolase (mEH) were introduced by using a vector conferring hygromycin B resistance, and cDNAs encoding CYP2E1 and CYP3A4 were introduced by using a vector conferring resistance to 1-histidinol. MCL-5 cells stably expressed all five cDNAs and the native CYP1A1 as determined by measurement of form-specific enzyme activity levels. The mutagenicity of seven model procarcinogens to MCL-5 cells was examined at the hypoxanthine guanine phosphoribosyltransferase (hprt) and thymidine kinase (tk) loci. Exposure to benzo[a]pyrene (BP), 3-methylcholanthrene (3MC), N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), aflatoxin B1, (AFB1), 2-(acetylamino)fluorene (AAF), or benzidine (BZD) induced a statistically significant increase in mutant frequency. Linear interpolation of the concentration of procarcinogen necessary to produce a doubling of the mutant fraction at the hprt locus in MCL-5 cells and the parent AHH-1 cell line revealed that, for each of the chemicals examined, except BZD, MCL-5 cells were significantly more sensitive than the parent AHH-1 cells. The increase in sensitivity to mutagenicity ranged from 3-fold for AAF to greater than 40,000-fold for NDMA. MCL-5 cells have great potential as a screening system for the analysis of human procarcinogen/promutagen activation.
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PMID:A metabolically competent human cell line expressing five cDNAs encoding procarcinogen-activating enzymes: application to mutagenicity testing. 179 7

We have developed a human lymphoblastoid cell line, designated 3A4/Hol, which stably expresses human CYP3A4 cDNA. This cell line exhibited testosterone 6 beta-hydroxylase activity, produced immunologically detectable CYP3A4 protein and was more sensitive to the cytotoxicity and mutagenicity of the carcinogenic mycotoxin aflatoxin B1 (AFB1) than was the parent cell line. The concentration-response for AFB1 cytotoxicity and mutagenicity in 3A4/Hol cells was compared to the responses of isogenic cell lines expressing comparable levels of human CYP1A2 (1A2/Hyg cells) and human CYP2A3 (2A3/Hyg cells). 1A2/Hyg cells were 3- to 6-fold more sensitive than 3A4/Hol cells to AFB1-induced mutation. 3A4/Hol cells were 10- to 15-fold more sensitive to AFB1-induced mutation than 2A3/Hyg cells. The differences in mutagenicity were supported by the relative binding of [3H]AFB1 to cellular DNA.
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PMID:The development of a human cell line stably expressing human CYP3A4: role in the metabolic activation of aflatoxin B1 and comparison to CYP1A2 and CYP2A3. 189 12

The oxidation of O6-benzylguanine, an inactivator of O6-alkylguanine-DNA alkyltransferase, was examined using human liver cytosol, microsomes, and several P450 isoforms. Incubation of O6-benzylguanine with human liver cytosol resulted in the formation of O6-benzyl-8-oxoguanine, which was inhibited by menadione, a potent inhibitor of aldehyde oxidase. Inhibition by allopurinol, a xanthine oxidase inhibitor, was less dramatic. Oxidation of O6-benzylguanine also occurred with pooled human liver microsomes and was inhibited by both furafylline and troleandomycin, selective inhibitors of CYP1A2 and CYP3A4, respectively. Human P450s CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2E1, and CYP3A4 expressed in Hep G2 hepatoma cells using vaccinia virus vectors were incubated with 10 or 200 microM O6-benzylguanine. At 10 microM, O6-benzylguanine was oxidized primarily by CYP1A2 and to a lesser extent by CYP3A4. However, an appreciable increase in CYP3A4 contribution was noted at 200 microM. CYP1A2 exhibited a more than 200-fold higher relative catalytic activity (Vmax/Km) compared with CYP3A4. Therefore, at therapeutically relevant concentrations of O6-benzylguanine, CYP1A2 could be primarily involved in its oxidation since it shows a much lower Km value (1.3 microM) than CYP3A4 (52.2 microM) and cytosol (81.5 microM). However, one would expect interindividual variation in the extent of oxidation of O6-benzylguanine depending on the levels of aldehyde oxidase, CYP1A2, and CYP3A4.
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PMID:Human liver oxidative metabolism of O6-benzylguanine. 750 88

Nitropyrenes are ubiquitous environmental pollutants that may pose a human health hazard because some are highly potent mutagens and carcinogens. The mutagenicity (trifluorothymidine resistance at the thymidine kinase locus) of 1-, 2-, and 4-nitropyrene (1-, 2-, and 4-NP), 1,3-, 1,6-, and 1,8-dinitropyrene (1,3-, 1,6-, and 1,8-DNP), and pyrene was assessed in a quantitative forward mutation assay using a metabolically competent line (MCL-5) of human B-lymphoblastoid cells. These cells contain endogenous cytochrome P450 activity (CYP1A1) and two plasmids that express cDNAs for four additional P450s (CYP1A2, CYP2A6, CYP2E1, CYP3A4) and microsomal epoxide hydrolase found in human liver. The major finding is that 2-NP and 1,3-DNP, both potent bacterial mutagens, were nonmutagenic in this assay. The following mutagenic potency series, expressed as the minimum detectable mutagen concentration (MDMC) in nmol/ml, was obtained: 1,6-DNP (0.8), 1,8-DNP (1.5), 4-NP (3.1), 1-NP (9.1), 2-NP (> 81), 1,3-DNP (> 86), pyrene (> 494). There was over an 11-fold difference between the most potent (1.6-DNP) and the least potent (1-NP) mutagen. 1,6-DNP was approximately twice as mutagenic as 1,8-DNP, which was almost twice as mutagenic as 4-NP, which, in turn was nearly three times as potent as 1-NP. This is the first report on the testing of 2-NP and 4-NP for mutagenicity in mammalian cell cultures. The human cell mutagenicity of these compounds was discussed in terms of potency series of nitropyrenes obtained from animal carcinogenicity experiments and other mammalian cell mutagenicity assays.
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PMID:Human cell mutagenicity of mono- and dinitropyrenes in metabolically competent MCL-5 cells. 752 17

We have investigated the effect of cytokines, including interleukin-6 (Il-6), interleukin-1 alpha (Il-1 alpha), and tumor necrosis factor-alpha (TNF-alpha), on the inducible expression of cytochrome P450s (CYP) CYP1A1, CYP1A2, and CYP3A4 in human hepatocytes in primary culture. The ability of these cultures to mimic the acute phase response when stimulated with cytokines was evaluated using immunoblotting to measure the production of albumin, ferritin, fibrinogen, and ceruloplasmin. The cytokines exhibited specific patterns of action on the production of these proteins. Albumin was depressed by all the cytokines. In contrast to Il-6 and Il-1 alpha, TNF-alpha reduced the production of fibrinogen and ceruloplasmin but stimulated the production of ferritin. When cells were treated with the CYP inducer alone, large increases in the expression of CYP1A1 and CYP1A2 by beta-naphthoflavone and of CYP3A4 by rifampicin were observed at messenger RNA (mRNA) and protein levels, by ribonuclease protection and immunoblotting, respectively. When the cells were treated with the inducer plus cytokines, the induction of mRNA was greatly reduced. Again, specific patterns of action were revealed: Il-6 had the most potent effect on CYP3A4, whereas TNF-alpha was the most potent with CYP1A genes. In all cases, changes at the protein levels paralleled changes at the mRNA levels. In cells preinduced with beta-naphthoflavone or rifampicin, the decay with time of the levels of the CYP1A2 or CYP3A4 proteins, after the removal of the inducer, was not affected by cytokines. We conclude that cytokines strongly repress the inducibility of CYP1As and CYP3A4 genes at a transcriptional or a posttranscriptional level, but affect neither the rate of translation of CYP mRNAs nor the rate of degradation of the CYP proteins in these cultures.
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PMID:Differential effects of cytokines on the inducible expression of CYP1A1, CYP1A2, and CYP3A4 in human hepatocytes in primary culture. 755 64

Dithiolethiones are thought to act as potent chemoprotective agents against aflatoxin B1 (AFB1)-induced hepatocarcinogenesis in the rat by inducing glutathione S-transferases (GSTs). To determine whether these antioxidants can be similarly effective in human beings, we have investigated metabolism of AFB1, in primary human hepatocytes with or without pretreatment by oltipraz (OPZ), a synthetic derivative of the natural 1,2-dithiole-3-thione. Aflatoxin M1 (AFM1), glutathione conjugates of AFB1 oxides (AFBSGs), and unchanged AFB1 were quantitated in cultures derived from eight human liver donors. Parenchymal cells obtained from the three GST M1-positive livers metabolized AFB1 to AFM1 and to AFBSGs derived from the isomeric exo-and endo-8,9-oxides, whereas no AFBSGs were formed in the GST M1-null cells. Pretreatment of the cells with 3-methylcholanthrene or rifampicin, inducers of CYP1A2 and CYP3A4, respectively, caused a significant increase in AFB1 metabolism. Although OPZ induced GST A2, and to a lesser extent GST A1 and GST M1, it decreased formation of AFM1 and AFBSG, which involves CYP1A2 and CYP3A4. Inhibition by OPZ of AFB1 metabolism by reducing CYP1A2 and CYP3A4 was also demonstrated by decreased activity of their monooxygenase activities toward ethoxyresorufin and nifedipine, respectively. The significant inhibition by OPZ of human recombinant yeast CYP1A2 and CYP3A4 was also shown. These results demonstrate that AFBSG can be formed by GST M1-positive human hepatocytes only, and suggest that chemoprotection with OPZ is due to an inhibition of activation of AFB1, in addition to a GST-dependent inactivation of the carcinogenic exo-epoxide.
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PMID:Inhibition of CYP1A2 and CYP3A4 by oltipraz results in reduction of aflatoxin B1 metabolism in human hepatocytes in primary culture. 758 37

Two peptides that correspond to sequences within the major 33-amino acid sequence recognized by human liver-kidney microsomal-1 autoantibodies were used to elicit antibodies in rabbits (four per peptide) against CYP2D6. Peptide 1(DPAQPPRDLTEAFLA) corresponded to amino acids 263-277, and peptide 2 (LLTEHRMTWDPAQPPRDLTE) corresponded to amino acids 254-273 of CYP2D6. The peptide-keyhole limpet hemocyanin conjugates elicited good immune responses against their respective peptides as judged by enzyme-linked immunosorbent assay (titers of 1/10,000 to 1/30,000). The antisera recognized CYP2D6 on Western blots and, to varying extents, inhibited recombinant CYP2D6 and liver microsomal CYP2D6 activity. Immunization with peptide 2 produced antisera with the greatest inhibitory potency. Antiserum from a rabbit (#236) immunized with peptide 2 inhibited up to 95% of dextromethorphan O-demethylase activity in human liver microsomes at the highest concentration tested (40% v/v) but did not significantly inhibit CYP1A2, CYP2C9, CYP2E1, or CYP3A4 marker activities. On Western blot, only a single immunoreactive protein comigrating with recombinant CYP2D6 was recognized. In liver microsomes from a CYP2D6-deficient individual, no proteins were recognized, and the antisera did not cross-react with recombinant CYP1A2, CYP2C9, CYP2E1, or CYP3A4. There was a significant correlation between the quantity of immunoreactive CYP2D6 as determined by immunoblotting with anti-peptide 2 antiserum and dextromethorphan O-demethylation in a panel of 10 human liver microsomes (r = 0.95). These data identify a peptide sequence (peptide 2) that can be used to raise antisera that specifically recognize and inhibit CYP2D6.
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PMID:Antipeptide antibodies against overlapping sequences differentially inhibit human CYP2D6. 758 52

The promutagenic and procarcinogenic heterocyclic amines (HAs) found in cooked meats are N-hydroxylated by microsomal cytochrome P450 enzymes as the first step in their metabolic activation. In cynomolgus monkeys, one of the HAs, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), has been shown to be a potent hepatocarcinogen. However, the structurally similar HA 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) lacks this potency to induce hepatocellular carcinoma in monkeys. Liver microsomes from cynomolgus monkeys show a striking substrate specificity for the metabolic activation of IQ and MeIQx, the former being a far better substrate for N-hydroxylation. Western blot analysis showed that cynomolgus monkey hepatic microsomes constitutively express P450s immunologically related to the human CYP3A, CYP2C, and low levels of CYP1A1. For comparison, Western blot analysis of rat, human and patas monkey microsomes was also carried out. Treatment of cynomolgus monkeys with rifampicin induced hepatic cytochromes P450 related to human CYP3A4 and CYP2C9/10 without inducing CYP1A1 or CYP1A2. Immunoblot analysis also showed that chronic exposure of cynomolgus monkeys to IQ induced hepatic microsomal cytochrome CYP1A1 and CYP1A2, similarly but lesser in magnitude to that observed with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCCD) induction. Using the Ames Salmonella mutagenicity assay, we examined the effect of the inducers on the mutagenic activation (i.e. N-hydroxylation) of IQ and MeIQx by cynomolgus monkey hepatic microsomes. We also examined the mutagenic activation of these HAs by rat, human and patas monkey liver microsomes. Microsomes from cynomolgus monkeys treated with rifampicin showed a 3-fold increase in the mutagenic activation of IQ but showed no increase in the mutagenic activation of MeIQx. Since cytochromes P4503A and/or P4502C are constitutively expressed in cynomolgus monkey hepatic microsomes, and upon induction with rifampicin are associated with an increased metabolic activation of IQ but not MeIQx, it appears that CYP3A and/or CYP2C are the isoform(s) showing the selective substrate specificity in the metabolic activation of IQ over MeIQx. Treatment of monkeys with TCDD significantly increased the mutagenic activation of both IQ and MeIQx, concomitant with an induction of CYP1A isozymes. Thus, it appears that TCDD-inducible CYP1A enzymes N-hydroxylate both substrates without selectivity. Together, these findings suggest that CYP3A and CYP2C are the principal isoforms in the cynomolgus monkey, associated with the metabolic activation implicated in the induction of hepatocarcinogenicity by IQ. Furthermore, the poor metabolic activation of MeIQx by CYP3A and CYP2C, coupled with low constitutive levels of CYP1A isozymes, provide a metabolic explanation for the low hepatocarcinogenic potency of MeIQx in cynomolgus monkeys.
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PMID:Cytochromes P450 in cynomolgus monkeys mutagenically activate 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) but not 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). 761 88

1. The mechanism of the interaction between ciprofloxacin and theophylline was investigated in nine healthy subjects. 2. Subjects were given a single oral dose of theophylline (3.4 mg kg-1), before and after 60 h of ciprofloxacin therapy at a dose of 500 mg twice daily. 3. Ciprofloxacin reduced the oral clearance of theophylline by 19% (-7.73 +/- 6.42 ml kg-1 h-1 (95% confidence limits -12.66, -2.79)). Some subjects (group A, n = 4) showed little decrease in clearance (mean 4.4%; -1.6 +/- 0.7 ml kg-1 h-1 (-2.6, 0.5)), whereas others (group B, n = 5) showed a marked decrease (mean 30%; -12.7 +/- 3.7 ml kg-1 h-1 (-17.2, -8.1)). 4. Comparing groups A and B, the decrease in oral clearance of theophylline in group B could not be ascribed to differences in the AUC of ciprofloxacin. Group A subjects showed only slight inhibition of 1-demethylation (-12.8 +/- 5.5% (-21.5, -4.0)), while group B subjects showed a significantly greater inhibition of 1-demethylation (-49.9 +/- 9.8% (-62.1, -37.7)), 3-demethylation (-44.8 +/- 8.6% (-55.4, -34.1)) and 8-hydroxylation (-27.0 +/- 3.7% (-31.6, -22.4)). 5. The results suggest that inter-individual variability in the inhibition of theophylline metabolism by ciprofloxacin can be attributed to inter-individual differences in the level of CYP1A2 expression and/or in the degree of inhibition of hepatic CYP1A2 and CYP3A4. 6. The interaction between ciprofloxacin and theophylline can be clinically significant.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The effect of ciprofloxacin on theophylline pharmacokinetics in healthy subjects. 761 73


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