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)

Human liver P450 NF25 (CYP3A4) had been previously expressed in Saccharomyces cerevisiae using the inducible GAL10-CYC1 promoter and the phosphoglycerate kinase gene terminator [Renaud, J. P., Cullin, C., Pompon, D., Beaune, P. and Mansuy, D. (1990) Eur. J. Biochem. 194, 889-896]. The use of an improved expression vector [Urban, P., Cullin, C. and Pompon, D. (1990) Biochimie 72, 463-472] increased the amounts of P450 NF25 produced/culture medium by a factor of five, yielding up to 10 nmol/l. The availability of recently developed host cells that simultaneously overexpress yeast NADPH-P450 reductase and/or express human liver cytochrome b5, obtained through stable integration of the corresponding coding sequences into the yeast genome, led to biotechnological systems with much higher activities of yeast-expressed P450 NF25 and with much better ability to form P450 NF25-iron-metabolite complexes. 9-fold, 8-fold, and 30-fold rate increases were found respectively for nifedipine 1,4-oxidation, lidocaine N-deethylation and testosterone 6 beta-hydroxylation between P450 NF25-containing yeast microsomes from the basic strain and from the strain that both overexpresses yeast NADPH-P450 reductase and expresses human cytochrome b5. Even higher turnovers (15-fold, 20-fold and 50-fold rate increases) were obtained using P450 NF25-containing microsomes from the yeast just overexpressing yeast NADPH-P450 reductase in the presence of externally added, purified rabbit liver cytochrome b5. This is explained by the fact that the latter strain contained the highest level of NADPH-P450 reductase activity. It is noteworthy that for the three tested substrates, the presence of human or rabbit cytochrome b5 always showed a stimulating effect on the catalytic activities and this effect was saturable. Indeed, addition of rabbit cytochrome b5 to microsomes from a strain expressing human cytochrome b5 did not further enhance the catalytic rates. The yeast expression system was also used to study the formation of a P450-NF25-iron-metabolite complex. A P450 Fe(II)-(RNO) complex was obtained upon oxidation of N-hydroxyamphetamine, catalyzed by P450-NF25-containing yeast microsomes. In microsomes from the basic strain expressing P450 NF25, 10% of the starting P450 NF25 was transformed into this metabolite complex, whereas more than 80% of the starting P450 NF25 led to complex formation in microsomes from the strain overexpressing yeast NADPH-P450 reductase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Optimization of yeast-expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH-cytochrome P450 reductase and cytochrome b5. 162 42

CYP3A4 is the major human cytochrome P-450 in a superfamily of heme-thiolate proteins that catalyze the oxidation of numerous lipophilic compounds. In this investigation, we report that CYP3A4 requires a phenolic function for ortho hydroxylation of estradiol and mono-O-demethylated methoxychlor and that CYP3A4 aromatic hydroxylation in general may be dependent on the presence of a free phenolic group. Indeed, when methoxyls were present instead of phenolic hydroxyls, CYP3A4 essentially failed to catalyze ortho hydroxylation. By contrast, of eight additional cDNA-expressed P-450s (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2D6, and 2E1) examined, only CYP1A2 and CYP2B6 could catalyze ortho hydroxylation of [o-3H]methoxychlor (7.2 and 14.6 pmol/90 min/pmol P-450, respectively), indicating that these isoforms do not require a phenolic hydroxyl for aromatic hydroxylation and that methoxyls do not sterically hinder catalysis by these CYPs. However, with [o-3H]mono-O-demethylated methoxychlor, containing a phenolic group, five isoforms (CYP1A2, 2B6, 2D6, 2E1, and 3A4) supported ortho hydroxylation. Of these, CYP3A4 exhibited by far the highest rate of hydroxylation at 87.8 pmol/90 min/pmol P-450. Further studies with [2-(3)H]estradiol 3-methyl ether and with [2-(3)H]estradiol revealed a similar and dramatic augmentation of CYP3A4-mediated C2 hydroxylase activity of approximately 75-fold by the presence of the phenolic group in the 3-position. The mechanism of augmentation by the phenolic hydroxyl does not appear to involve the acidic proton of estradiol, since CYP3A4-catalyzed estradiol 2-hydroxylation and testosterone 6-beta-hydroxylation were diminished to an equal extent when incubations were performed at increasing buffer pH values from 7 to 9. Both estradiol and its 3-methoxy derivative bound with similar affinity to cDNA-expressed, microsomal CYP3A4: spectral dissociation constants were 270 and 370 microM, respectively, and both compounds exhibited type I spectra. Thus, the disparities in aromatic hydroxylation rates between compounds containing phenolic hydroxyls and those with methoxyls cannot be explained by differences in their binding affinities. To explain the mode via which the phenolic hydroxyl facilitates ortho hydroxylation, a mechanism in which the phenolic moiety attacks the iron-oxo double bond of CYP3A4, resulting in oxygen transfer to the ortho position, is proposed. It is anticipated that these findings will assist in forecasting the CYP-mediated metabolic fate of phenolic compounds.
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PMID:Catalytic characteristics of CYP3A4: requirement for a phenolic function in ortho hydroxylation of estradiol and mono-O-demethylated methoxychlor. 904 21

The effects of an immunosuppressive agent, tacrolimus (FK-506), on the activities of cytochrome P-450-linked monooxygenase systems with respect to three cytochrome P-450 isozymes in rat liver microsomes were investigated. FK-506 non-competitively inhibited the aniline p-hydroxylase, p-nitroanisole O-demethylase and lidocaine N-deethylase activities of cytochrome P-450-linked monooxygenase systems, these activities being mainly catalyzed by cytochromes P-450 CYP2E1, CYP2C11 and CYP3A4, respectively, and the Ki values of the activities for FK-506 were determined to be 605, 491 and 97 microM, respectively. The inhibition of cytochrome P-450-linked monooxygenase systems by FK-506 seemed to involve the direct inhibition of cytochromes P-450 because the NADPH-cytochrome c reductase and NADPH-ferricyanide reductase activities of NADPH-cytochrome P-450 reductase were not affected by the presence of 1 mM FK-506 at all. A spectrophotometric study showed that a reverse type I spectral change was induced on the addition of FK-506 to rat liver microsomes, and the Ks value was apparently 125 microM. On the other hand, the EPR spectra of cytochromes P-450 in rat liver microsomes were not affected by 1 mM FK-506. These results suggest direct interaction between FK-506 and cytochrome P-450 apoproteins, except for the heme iron regions of cytochromes P-450, resulting in inhibition of the drug-metabolism activities catalyzed by cytochromes P-450.
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PMID:Effects of an immunosuppressive agent, tacrolimus (FK-506), on the activities of cytochrome P-450-linked monooxygenase systems in rat liver microsomes. 930 7

Few studies have evaluated the production of reactive oxygen intermediates by human microsomes, especially the influence of the specific form of cytochrome P450. Experiments were carried out to evaluate the ability of CYP1A1, 1A2, 2B6, and 3A4 to consume NADPH, reduce iron, and catalyze production of reactive oxygen species. Microsomes enriched in each of these CYPs were obtained from commercial +/- lymphoblast cells that had been transfected with cDNA encoding the specific human CYP. On a per nanomole cytochrome P450 basis, CYP3A4 was the most active P450 evaluated in catalyzing NADPH oxidation, production of superoxide anion radical, NADPH-dependent chemiluminescence, oxidation of dichlorofluorescein diacetate, and reduction of either ferric-EDTA or ferric-citrate. CYP1A1 was the next most reactive CYP, whereas CYP1A2 and 2B6 displayed a comparable, lower activity. Nitric oxide, which reacts with and inactivates hemoproteins, inhibited superoxide production by all the CYPs to a similar extent. Because CYP3A4 is present in high amounts in human liver microsomes and is active in catalyzing the formation of reactive oxygen species, this CYP may make an important contribution in the overall ability of human liver microsomes to generate active oxygen species.
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PMID:Production of reactive oxygen species by microsomes enriched in specific human cytochrome P450 enzymes. 962 90

(R)-(+)-Menthofuran is a potent, mechanism-based inactivator of human liver cytochrome P450 (CYP or P450) 2A6. Menthofuran caused a time- and concentration-dependent loss of CYP2A6 activity. The inactivation of CYP2A6 was characterized by a Ki of 2.5 microM and a kinact of 0.22 min-1 for human liver microsomes and a Ki of 0.84 microM and a kinact of 0.25 min-1 for purified expressed CYP2A6. Addition of various nucleophiles, a chelator of iron, or scavengers of reactive oxygen species or extensive dialysis failed to protect CYP2A6 from inactivation. An antibody to metallothionein conjugates of a suspected reactive metabolite of menthofuran was used to detect reactive menthofuran metabolite adducts with CYP2A6. These adducts were formed only in the presence of NADPH-P450 reductase and NADPH. Glutathione, methoxylamine, and semicarbazide did not prevent adduction of reactive menthofuran metabolites to CYP2A6, however. The menthofuran metabolite formation/CYP2A6 inactivation partition ratio was determined to be 3.5 +/- 0.6 nmol/nmol of P450. Menthofuran was unable to inactivate CYP1A2, CYP2D6, CYP2E1, or CYP3A4 in a time- and concentration-dependent manner.
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PMID:(R)-(+)-Menthofuran is a potent, mechanism-based inactivator of human liver cytochrome P450 2A6. 966 Aug 53

1. A plasmid containing 1 kb of the CYP3A4 regulatory (promoter) region coupled to a reporter gene for secretary placental alkaline phosphatase (SPAP) was transfected into HepG2 cells. Transfected cells were dosed with several known inducers of CYP3A4 and the levels of SPAP were measured. The effect of co-transfecting a plasmid encoding the human glucocorticoid receptor on reporter gene activity was also examined. 2. Dexamethasone induced CYP3A4-dependent reporter gene expression in a concentration-dependent manner and induction was approximately doubled in the presence of the glucocorticoid receptor. Dexamethasone-dependent induction was blocked by RU-486 (a glucocorticoid receptor antagonist), in the presence of the co-transfected glucocorticoid receptor. 3. Induction of CYP3A4-dependent reporter gene expression and enhancement of the induction by the glucocorticoid receptor was also observed with pregnenolone-16alpha-carbonitrile (PCN), rifampicin, phenytoin, carbamazepine, phenylbutazone and phenobarbitone, all known in vivo inducers of CYP3A4 in man. 4. Metyrapone and sulfinpyrazone induced CYP3A4-dependent reporter gene expression, but induction was not enhanced by the glucocorticoid receptor. 5. Clotrimazole, erythromycin and triacetyloleandomycin (TAO) did not induce CYP3A4-dependent reporter gene expression, consistent with the observation that these inducers act through post-transcriptional mechanisms. 6. These results highlight differences in the molecular mechanisms of induction of CYP3A4 by the xenobiotics studied and indicate that the glucocorticoid receptor is involved in the induction of the CYP3A4 gene by some, but not all, CYP3A4 inducers. 7. We propose that the approach described here provides a useful in vitro approach for the identification of transcriptional regulators of the CYP3A4 gene.
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PMID:A reporter gene assay to assess the molecular mechanisms of xenobiotic-dependent induction of the human CYP3A4 gene in vitro. 1021 67

The drug metyrapone in the presence of glucocorticoid has been shown to induce the expression of rat hepatic cytochrome P-450 (CYP) 1A1 mRNA in vivo and in vitro through disruption of endogenous CYP1A1 regulator homeostasis and without either compound's binding to the aryl hydrocarbon receptor. Addition of metyrapone to human liver cancer cell cultures, with or without dexamethasone, did not induce CYP1A1 mRNA, in contrast to the aryl hydrocarbon receptor ligand beta-naphthoflavone. Addition of metyrapone to primary cultures of human hepatocytes also failed to induce detectable levels of CYP1A1 mRNA or CYP1A protein in two separate preparations, whereas the treatment with 2,3,7,8-tetrachlorodibenzo-rho-dioxin or omeprazole induced detectable levels of CYP1A1 mRNA in one preparation and CYP1A protein in both preparations. Addition of metyrapone to human hepatocyte cultures resulted in the induction of CYP3A4 expression. The pregnane X receptor (PXR), which has recently been shown to mediate the transcriptional induction of CYP3A4 expression in response to rifampicin, was activated by metyrapone in CV-1 cells transiently cotransfected with an expression vector encoding the human PXR and a reporter construct containing the everted repeat sequence that confers CYP3A4 induction responsiveness to inducers within its promoter. Metyrapone activated the human PXR at concentrations that also resulted in the induction of CYP3A4 in human cultured hepatocytes. Metyrapone treatment is therefore unlikely to result in the induction of CYP1A1 but may induce the expression of CYP3A4 in humans.
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PMID:Effect of the adrenal 11-beta-hydroxylase inhibitor metyrapone on human hepatic cytochrome P-450 expression: induction of cytochrome P-450 3A4. 1061 Nov 46

The inhibitory effects of six commonly used calcium channel blockers on three major cytochrome P-450 activities were examined and characterized in human liver microsomes. All six compounds reversibly inhibited CYP2D6 (bufuralol 1'-hydroxylation) and CYP2C9 (tolbutamide methyl hydroxylation) activities. The IC(50) values for the inhibition of CYP2D6 and CYP2C9 for nicardipine were 3 to 9 microM, whereas those for all others ranged from 14 to >150 microM. Except for nifedipine, all calcium channel blockers showed increased inhibitory potency toward CYP3A activities (testosterone 6beta-hydroxylation and midazolam 1'-hydroxylation) after 30-min preincubation with NADPH. IC(50) values for the inhibition of testosterone 6beta-hydroxylase obtained in the NADPH-preincubation experiment for nicardipine (1 microM), verapamil (2 microM), and diltiazem (5 microM) were within 10-fold, whereas those for amlodipine (5 microM) and felodipine (13 microM) were >200-fold of their respective plasma concentrations reported after therapeutic doses. Similar results also were obtained based on midazolam 1'-hydroxylase activity. Unlike the observations with mibefradil, a potent irreversible inhibitor of CYP3A, the NADPH-dependent inhibition of CYP3A activity by nicardipine and verapamil was completely reversible on dialysis, whereas that by diltiazem was partially restored (80%). Additional experiments revealed that nicardipine, verapamil, and diltiazem formed cytochrome P-450-iron (II)-metabolite complex in both human liver microsomes and recombinant CYP3A4. Nicardipine yielded a higher extent of complex formation ( approximately 30% at 100 microM), and was a much faster-acting inhibitor (maximal inhibition rate constant approximately 2 min(-1)) as compared with verapamil and diltiazem. These present findings that the CYP3A inhibition caused by nicardipine, verapamil, and diltiazem is, at least in part, quasi-irreversible provide a rational basis for pharmacokinetically significant interactions reported when they were coadministered with agents that are cleared primarily by CYP3A-mediated pathways.
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PMID:Drug interactions with calcium channel blockers: possible involvement of metabolite-intermediate complexation with CYP3A. 1064 May 8

Phenyldiazene reacted with lymphoblast-expressed CYP3A4 to give a stable phenyl-iron complex that could be induced to rearrange in situ producing approximately equal amounts of four N-phenyl-protoporphyrin IX isomers (N(B):N(A):N(C):N(D), 01:01:02:02). In the presence of 10 mM MgCl(2), the formation profile of the protoporphyrin isomers was markedly altered compared with control, favoring the N(A) isomer (N(B):N(A):N(C):N(D), 01:34:01:02). In addition, an investigation of MgCl(2) effects on CYP3A4-mediated metabolism of triazolam revealed that 10 mM MgCl(2) increased the apparent K(m) of triazolam 4-hydroxylation from 83 to 173 microM and reduced the V(max) for the reaction from 3.4 to 2.4 min(-1). Moreover, when the reaction kinetics of the oxidation of pyrene by CYP3A4 was examined in the absence of MgCl(2), it was found that the substrate-velocity curve was best approximated by a sigmoidal velocity curve (Hill coefficient 1.7 +/- 0.1). However, when the reaction was conducted in the presence of 10 mM MgCl(2), the resulting pyrene kinetics was not sigmoidal but rather biphasic (Hill coefficient 0.80 +/- 0.07). Based on the current results, it appears that CYP3A4 is conformationally sensitive to its in vitro environment and parameters, such as the presence of a divalent magnesium, can have a measurable effect on active site topography and consequently catalytic activity.
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PMID:Topological alteration of the CYP3A4 active site by the divalent cation Mg(2+). 1099 40

Priming of the liver for ethanol-induced injury, by nutrients such as polyunsaturated fat and iron, plays a key role in alcoholic liver disease. The objective of this work was to evaluate the effect of the combination of Fe-nitrilotriacetic acid (Fe-NTA) and arachidonic acid (AA) on the viability of HepG2 cells (E47 cells) transfected to express human CYP2E1. Cells were plated, preloaded with arachidonic acid, washed, and exposed to Fe-NTA for variable periods. Fe-NTA (10 microM) or AA (5 microM) alone showed low toxicity to E47 cells (18 and 8%, respectively, at 24 h), whereas the combination produced synergistic injury (62% toxicity at 24 h). Exposure of cells not expressing any cytochrome P450 (P450), or HepG2-C3A4 cells (expressing CYP3A4) to 10 microM Fe-NTA plus 5 microM AA produced lower toxicity (14 and 32%, respectively), demonstrating a role for P450, and in particular CYP2E1, in the development of toxicity by exposure to Fe + AA. Lipid peroxidation was induced in the E47 cells exposed to Fe plus arachidonic acid and the synergistic toxicity was prevented by antioxidants, which also decreased lipid peroxidation. Damage to mitochondria plays a role in the CYP2E1-dependent toxicity of Fe + AA, because the mitochondrial transmembrane potential decreased early in the process, and cyclosporin A prevented the toxicity. Toxicity in E47 cells exposed to Fe + AA is mainly necrotic in nature. Hepatocytes from pyrazole-treated rats, with high levels of CYP2E1, were more sensitive to Fe + AA toxicity than were saline control hepatocytyes. The results presented suggest that low concentrations of Fe and AA can act as priming or sensitizing factors for CYP2E1-induced injury in HepG2 cells, and such interactions may play a role in alcohol-induced liver injury.
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PMID:Synergistic toxicity of iron and arachidonic acid in HepG2 cells overexpressing CYP2E1. 1156 36


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