Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q8NEX9 (reductase)
 26,410 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cyclophosphamide (CPA) and ifosfamide (IFA) are widely used anticancer prodrugs that are bioactivated in the liver by specific cytochrome P450 enzymes (CYPs). The therapeutic activity of these antitumor agents can be compromised by a low therapeutic index that is, in part, due to the systemic distribution of activated drug metabolites. Here, recombinant retroviruses were used to deliver six different CPA- or IFA-metabolizing human CYP genes to 9L gliosarcoma cells: 2B6, 2C8, 2C9, 2C18 (Met385 and Thr385 alleles), 2C19, and 3A4. Intratumoral cytochrome P450 expression conferred substantial sensitivity to CPA cytotoxicity, with the most dramatic effects seen with CYP2B6. Strong CPA chemosensitivity was also seen following transduction of CYP2C18-Met, despite a very low level of CYP protein expression (>60-fold lower than that of 2B6). In contrast to CPA, the cytotoxicity of IFA was greatest toward tumor cells transduced with CYP3A4, followed by CYPs 2B6 and 2C18-Met. A substantial further increase in chemosensitivity was achieved upon transduction of 2B6 or 2C18-Met-expressing tumor cells with P450 reductase, which provided for more efficient intratumoral prodrug activation and cytotoxicity at lower drug concentrations. With 2B6- plus P450 reductase-transduced tumor cells, CPA but not IFA conferred a strong cell contact-independent bystander cytotoxic effect on non-P450-expressing 9L cells. CPA treatment of tumors that were transduced with 2B6 or 2C18-Met together with P450 reductase and were grown s.c. in immunodeficient mice resulted in a large enhancement of the liver P450-dependent antitumor effect seen with control 9L tumors, with no apparent increase in host toxicity (growth delay of >25-50 days in P450-expressing tumors versus approximately 5-6 days without P450). CYP2B6 plus P450 reductase and CYP2C18-Met plus P450 reductase thus appear to be excellent gene combinations for use with CPA in P450/prodrug activation-based cancer gene therapy.
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PMID:Retroviral transfer of human cytochrome P450 genes for oxazaphosphorine-based cancer gene therapy. 976 69

The in vitro metabolism of [14C]-nonylphenols (NPs) by rat hepatic microsomes in vitro was examined. Product formation was NADPH dependent and inhibited by the cytochrome P450 inhibitors, piperonyl butoxide and SKF525. Hepatic microsomes isolated from various inducer-treated rats (including beta naphthoflavone, phenobarbital, ethanol, dexamethasone, and clofibrate which selectively induce CYP1A, 2B, 2E, 3A and 4A, respectively) all metabolized NPs. Only microsomes from phenobarbital-treated rats exhibited a significantly higher activity towards NPs and showed a different profile of NP metabolites compared to control, untreated rats. Microsomes from human CYP2B6 transfected cells with endogenous NADPH-P450 reductase activity but not microsomes from the non-transfected parent cells metabolized NPs. The metabolism of NPs using microsomes from phenobarbital-treated rats was inhibited by 4-amino-2, 6-dinitro-1-t-butylxylene, a specific CYP2B enzyme inhibitor. Addition of a general anti-CYP2B sera to the reaction mixture attenuated the enzyme activity of microsomes from phenobarbital-treated rats to metabolize NPs. This metabolic reaction was, however, insensitive to a specific anti-CYP2B1 sera that had been shown to inhibit enzyme activities attributed only to CYP2BI suggesting that the CYP2B2 pathway is predominant in NP metabolism. The results indicate that hepatic cytochrome P450 enzyme(s) can metabolize NPs and that CYP2B isozymes are probably involved.
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PMID:Metabolism of nonylphenol by rat and human microsomes. 981 83

4-Dimethylamino-4'-(imidazol-1-yl)chalcone (RL3142) is a newly developed antimalarial cysteine protease inhibitor. Four metabolites (M1-M4) were found in human liver microsomes and their structures were identified by LC/MS/MS. Two primary metabolites, M2 (minor) and M4 (major), were determined to be the N-demethylated product (M2) and the product (M4) resulting from 1,2-hydrogenation of the alpha, beta-unsaturated ketone moiety of the parent compound. A combined approach utilizing selective P450 inhibitors, immunoinhibition with CYP3A and NADPH P450 reductase antibodies, and cDNA expressed human CYP3A4 and NADPH P450 reductase, was used for identification of enzymes responsible for the biotransformation. For formation of M2, both a rabbit CYP3A polyclonal antibody (110 microliter/mg microsomal protein) and ketoconazole (2 micromol/l), a CYP3A inhibitor, showed about 50% inhibitory effects; other specific inhibitors of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP2E1 showed no significant effects. For formation of M4, neither CYP3A antibody nor the above mentioned CYP inhibitors exhibited inhibitory effects. Anti-rat NADPH P450 reductase serum (50 microliter/100 microgram microsomal protein) exhibited 70 and 58% inhibitory effects on M2 and M4 formation, respectively. Incubation of RL3142 with cDNA expressed human NADPH P450 reductase yielded formation of M4, but not M2. Carbon monoxide inhibited formation of M2 and M1 (the reduced product of M2), but had no effect on M4 and M3 (the reduced product of M4) formation. Collectively, NADPH P450 reductase solely catalyzed reduction of RL3142 to M4, whereas CYP3A contributed in part to formation of M2.
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PMID:In vitro biotransformation of a novel antimalarial cysteine protease inhibitor in human liver microsomes. 992 71

Nicotine C-oxidation by recombinant human cytochrome P450 (P450 or CYP) enzymes and by human liver microsomes was investigated using a convenient high-performance liquid chromatographic method. Experiments with recombinant human P450 enzymes in baculovirus systems, which co-express human nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH)-P450 reductase, revealed that CYP2A6 had the highest nicotine C-oxidation activities followed by CYP2B6 and CYP2D6; the Km values by these three P450 enzymes were determined to be 11.0, 105, and 132 microM, respectively, and the Vmax values to be 11.0, 8.2, and 8.6 nmol/min per nmol P450, respectively. CYP2E1, 2C19, 1A2, 2C8, 3A4, 2C9, and 1A1 catalysed nicotine C-oxidation only at high (500 microM) substrate concentration. CYP1B1, 2C18, 3A5, and 4A11 had no measurable activities even at 500 microM nicotine. In liver microsomes of 16 human samples, nicotine C-oxidation activities were correlated with CYP2A6 contents at 10 microM substrate concentration, whereas such correlation coefficients were decreased when the substrate concentration was increased to 500 microM. Contribution of CYP2B6 (as well as CYP2A6) was demonstrated by experiments with the effects of orphenadrine (and also coumarin and anti-CYP2A6) on the nicotine C-oxidation activities by human liver microsomes at 500 microM nicotine. CYP2D6 was found to have minor roles since quinidine did not inhibit microsomal nicotine C-oxidation at both 10 and 500 microM substrate concentrations. These results support the view that CYP2A6 has major roles for nicotine C-oxidation at lower substrate concentration and both CYP2A6 and 2B6 play roles at higher substrate concentrations in human liver microsomes.
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PMID:Roles of CYP2A6 and CYP2B6 in nicotine C-oxidation by human liver microsomes. 1035 Jan 85

High levels of cytochrome P450 are present in the olfactory mucosa (OM) in mammalian animals and contribute to the known tissue-selective toxicity of numerous chemical compounds. Olfactory toxicity in the perinatal period may have a greater impact on behavior, growth, and development than in adults. To establish a molecular basis for determining the risk of developmental toxicity in OM, the expression of several cytochrome P450 enzymes, as well as NADPH-cytochrome P450 reductase and microsomal epoxide hydrolase, was examined in hepatic and nasal microsomes prepared from human fetal tissues at gestational day 91-125. The relative microsomal concentrations of these biotransformation enzymes were determined on immunoblots. Expression of CYP2A, CYP2J2, the reductase, and epoxide hydrolase was detected in both OM and liver. The microsomal levels of these enzymes were generally lower in OM than in liver of the same fetuses, except for the CYP2A-related proteins, which were expressed in OM at much higher levels. OM expression of CYP2A6, CYP2A13, CYP2B6, and CYP2J2 mRNAs was detected using RNA-PCR. These results document, for the first time, prenatal expression of xenobiotic-bioactivating cytochrome P450 enzymes in human OM and suggest that the human fetal OM may be a preferred target tissue for the toxicity of maternally derived chemical compounds that are activated by the CYP2A enzymes.
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PMID:Expression of biotransformation enzymes in human fetal olfactory mucosa: potential roles in developmental toxicity. 1082 11

S-Methyl N,N-diethyldithiocarbamate (MeDDC), a metabolite of the alcohol deterrent disulfiram, is converted to MeDDC sulfine and then S-methyl N,N-diethylthiocarbamate sulfoxide, the proposed active metabolite in vivo. Several isoforms of CYP450 and to a lesser extent flavin monooxygenase (FMO) metabolize MeDDC in the liver. The human kidney contains FMO1 and several isoforms of CYP450, including members of the CYP3A, CYP4A, CYP2B, and CYP4F subfamilies. In this study the metabolism of MeDDC by the human kidney was examined, and the enzymes responsible for this metabolism were determined. MeDDC was incubated with human renal microsomes from five donors or with insect microsomes containing human FMO1, CYP4A11, CYP3A4, CYP3A5, or CYP2B6. MeDDC sulfine was formed at 5 microM MeDDC by renal microsomes at a rate of 210 +/- 50 pmol/min/mg of microsomal protein (mean +/- S.D., n = 5) and by FMO1 at 7.6 +/- 0.2 nmol/min/nmol (n = 3). Oxidation of 5 microM MeDDC was negligible by all CYP450 tested (< or =0.03 nmol/min/nmol). Inhibition of FMO by methimazole or heat diminished MeDDC sulfine formation 75 to 89% in renal microsomes. Inhibition of CYP450 in renal microsomes by N-benzylimidazole or antibody to the CYP450 NADPH reductase had no effect on MeDDC sulfine production. Benzydamine N-oxidation, a probe for FMO activity, correlated with MeDDC sulfine formation in renal microsomes (r = 0.951, p = 0.013). The K(M) values for MeDDC sulfine formation by renal microsomes and recombinant human FMO1 were 11 and 15 microM, respectively. These results demonstrate a role for the kidney and FMO1 in the metabolism of MeDDC in humans.
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PMID:Metabolism of a disulfiram metabolite, S-methyl N,N-diethyldithiocarbamate, by flavin monooxygenase in human renal microsomes. 1115 1

Drug oxidation activities of 12 recombinant human cytochrome P450s (P450) coexpressed with human NADPH-P450 reductase (NPR) in bacterial membranes (P450/NPR membranes) were determined and compared with those of other recombinant systems and those of human liver microsomes. Addition of exogenous membrane-bound NPR to the P450/NPR membranes enhanced the catalytic activities of CYP2C8, CYP2C9, CYP2C19, CYP3A4, and CYP3A5. Enhancement of activities of CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2D6, and CYP2E1 in membranes was not observed after the addition of NPR (4 molar excess to each P450). Exogenous purified human cytochrome b5 (b5) further enhanced catalytic activities of CYP2A6, CYP2B6, CYP2C8, CYP2E1, CYP3A4, and CYP3A5/NPR membranes. Catalytic activities of CYP2C9 and CYP2C19 were enhanced by addition of b5 in reconstituted systems but not in the P450/NPR membranes. Apo b5 (devoid of heme) enhanced catalytic activities when added to both membrane and reconstituted systems, except for CYP2E1/NPR membranes and the reconstituted system containing purified CYP2E1 and NPR. Catalytic activities in P450/NPR membranes fortified with b5 were roughly similar to those measured with microsomes of insect cells coexpressing P450 with NPR (and b5) and/or human liver microsomes, based on equivalent P450 contents. These results suggest that interactions of P450 and NPR coexpressed in membranes or mixed in reconstituted systems appear to be different in some human CYP2 family enzymes, possibly due to a conformational role of b5. P450/NPR membrane systems containing b5 are useful models for prediction of the rates for liver microsomal P450-dependent drug oxidations.
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PMID:Roles of NADPH-P450 reductase and apo- and holo-cytochrome b5 on xenobiotic oxidations catalyzed by 12 recombinant human cytochrome P450s expressed in membranes of Escherichia coli. 1192 48

Tamoxifen is primarily used in the treatment of breast cancer. It has been approved as a chemopreventive agent for individuals at high risk for this disease. Tamoxifen is metabolized to a number of different products by cytochrome P450 enzymes. The effect of tamoxifen on the enzymatic activity of bacterially expressed human cytochrome CYP2B6 in a reconstituted system has been investigated. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of purified CYP2B6 was inactivated by tamoxifen in a time- and concentration-dependent manner. Enzymatic activity was lost only in samples that were incubated with both tamoxifen and NADPH. The inactivation was characterized by a K(I) of 0.9 microM, a k(inact) of 0.02 min(-1), and a t(1/2) of 34 min. The loss in the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity did not result in a similar percentage loss in the reduced carbon monoxide spectrum, suggesting that the heme moiety was not the major site of modification. The activity of CYP2B6 was not recovered after removal of free tamoxifen using spin column gel filtration. The loss in activity seemed to be due to a modification of the CYP2B6 and not reductase because adding fresh reductase back to the inactivated samples did not restore enzymatic activity. A reconstituted system containing purified CYP2B6, NADPH-reductase, and NADPH-generating system was found to catalyze tamoxifen metabolism to 4-OH-tamoxifen, 4'-OH-tamoxifen, and N-desmethyl-tamoxifen as analyzed by high-performance liquid chromatography analysis. Preliminary studies showed that tamoxifen had no effect on the activities of CYP1B1 and CYP3A4, whereas CYP2D6 and CYP2C9 exhibited a 25% loss in enzymatic activity.
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PMID:Effect of tamoxifen on the enzymatic activity of human cytochrome CYP2B6. 1202 23

The in vitro metabolism of tolperisone, 1-(4-methyl-phenyl)-2-methyl-3-(1-piperidino)-1-propanone-hydrochloride, a centrally acting muscle relaxant, was examined in human liver microsomes (HLM) and recombinant enzymes. Liquid chromatography-mass spectrometry measurements revealed methyl-hydroxylation (metabolite at m/z 261; M1) as the main metabolic route in HLM, however, metabolites of two mass units greater than the parent compound and the hydroxy-metabolite were also detected (m/z 247 and m/z 263, respectively). The latter was identified as carbonyl-reduced M1, the former was assumed to be the carbonyl-reduced parent compound. Isoform-specific cytochrome P450 (P450) inhibitors, inhibitory antibodies, and experiments with recombinant P450s pointed to CYP2D6 as the prominent enzyme in tolperisone metabolism. CYP2C19, CYP2B6, and CYP1A2 are also involved to a smaller extent. Hydroxymethyl-tolperisone formation was mediated by CYP2D6, CYP2C19, CYP1A2, but not by CYP2B6. Tolperisone competitively inhibited dextromethorphan O-demethylation and bufuralol hydroxylation (K(i) = 17 and 30 microM, respectively). Tolperisone inhibited methyl p-tolyl sulfide oxidation (K(i) = 1200 microM) in recombinant flavin-containing monooxygenase 3 (FMO3) and resulted in a 3-fold (p < 0.01) higher turnover number using rFMO3 than that of control microsomes. Experiments using nonspecific P450 inhibitors-SKF-525A, 1-aminobenzotriazole, 1-benzylimidazole, and anti-NADPH-P450-reductase antibodies-resulted in 61, 47, 49, and 43% inhibition of intrinsic clearance in HLM, respectively, whereas hydroxymethyl-metabolite formation was inhibited completely by nonspecific chemical inhibitors and by 80% with antibodies. Therefore, it was concluded that tolperisone undergoes P450-dependent and P450-independent microsomal biotransformations to the same extent. On the basis of metabolites formed and indirect evidences of inhibition studies, a considerable involvement of a microsomal reductase is assumed.
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PMID:Identification of metabolic pathways involved in the biotransformation of tolperisone by human microsomal enzymes. 1269 52

epsilon-Viniferin, a dimer of resveratrol, was isolated in wine at concentration between 0.5 and 5 microM. As resveratrol and polyphenols from red wine were reported to inhibit cytochrome P450 (CYP) activities, this led us to investigate the inhibitory effects of epsilon-viniferin on human CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2E1, CYP3A4 and CYP4A activities. These effects were compared to those of resveratrol and non volatiles compounds from red wine or various Cognac(R) beverages (enriched with oak-polyphenols). Assays were carried out on human liver microsomes and heterologously expressed CYPs. Ethoxyresorufin, coumarin, benzoxyresorufin, chlorzoxazone, testosterone and lauric acid were used as selective substrates for CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2E1, CYP3A4 and CYP4A, respectively. epsilon-viniferin displayed a more potent inhibitory effect than resveratrol for all the CYP activities tested (Ki 0.5 to 20 microM vs. 10 to 100 microM, respectively). This effect was not due to an inhibition of the NADPH reductase. A particularly potent inhibitory effect was shown for CYP1A1, CYP1B1 and CYP2B6 which are involved in bioactivation of numerous carcinogens. epsilon-viniferin was not a mechanism-based inhibitor of human CYPs. It displayed, like resveratrol, mixed-type inhibitions for all the CYP tested, except for CYP2E1 (non-competitive). Comparison of the inhibitory effects exerted on CYP activities by epsilon-viniferin, resveratrol and non volatile components from red wine or various Cognac beverages showed that neither resveratrol, nor epsilon-viniferin is the main CYP inhibitor present in red wine solids.
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PMID:Differential inhibition of human cytochrome P450 enzymes by epsilon-viniferin, the dimer of resveratrol: comparison with resveratrol and polyphenols from alcoholized beverages. 1281 27


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