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)

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

Betel quid chewing is known to cause cheek cancer in a wide area covering Africa to Asia. Areca nut contained in the betel quid is believed to give rise to carcinogenic N-nitrosamines. In the present study, the roles of human cytochromes P450 (P450 or CYP) in the mutagenic activation of betel quid-specific N-nitrosamines such as 3-(N-nitrosomethylamino)propionitrile (NMPN), 3-(N-nitrosomethylamino)propionaldehyde (NMPA) and N-nitrosoguvacoline (NG) were examined by using genetically engineered Salmonella typhimurium YG7108 expressing each form of human P450 together with NADPH-P450 reductase, which had been established in our laboratory. Among typical P450s (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2A13, CYP2D6 or CYP3A4) examined, CYP2A6 was the most efficient activator of NMPN, followed by CYP1A1 and CYP1B1. The mutagenic activation of NMPN by CYP2A6 was seen at the substrate concentrations of microM levels (approximately 100 microM). The activation of NMPA was catalyzed predominantly by CYP2A13 and to lesser extents by CYP2A6, CYP1A1, CYP1A2 and CYP1B1. The activation of NMPA by CYP2A13 was detectable at the substrate concentrations of microM levels (approximately 1 microM). NG was activated by CYP2A13 and CYP2A6, the genotoxicity of NG being much lower than that of NMPA or NMPN. Based on these data, we conclude that human CYP2A subfamily members play important roles in the mutagenic activation of essentially all betel quid-related N-nitrosamines tested in the present study.
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PMID:Mutagenic activation of betel quid-specific N-nitrosamines catalyzed by human cytochrome P450 coexpressed with NADPH-cytochrome P450 reductase in Salmonella typhimurium YG7108. 1572 15

The metabolism of the bicyclic monoterpene Delta(3)-carene was investigated in vitro using human liver microsomes as well as human smoker/non-smoker lung microsomes and 12 different recombinant cytochrome P450 enzymes coexpressed with human CYP-reductase in Escherichia coli cells. We detected two metabolites using GC-MS analysis. The mass fragmentation indicated for one metabolite hydroxylation in the allyl position and for the other metabolite epoxidation at the double bond. For clear identification the suggested metabolites were synthesized in a four-step reaction. Comparison of GC retention times and mass spectra lead to the identification of the metabolites as Delta(3)-carene-10-ol ((1S, 6R)-7,7-Dimethylbicyclo[4.1.0]hept-3-en-3-yl-methanol) and Delta(3)-carene-epoxide ((1S, 3S, 5R, 7R)-3,8,8-Trimethyl-4-oxa-tricyclo[5.1.0.0(3,5)]octane). Delta(3)-carene-10-ol was formed by human liver microsomes and recombinant human CYP2B6, CYP2C19 and CYP2D6. Delta(3)-Carene-epoxide was obviously catalyzed only by CYP1A2. In both cases there was a clear correlation between the metabolite formation, incubation time and enzyme concentration, respectively. Further kinetic analysis revealed that CYP2B6 exhibited the highest activity for Delta(3)-carene 10-hydroxylation. Michaelis-Menten K(m) and V(max) for oxidation of Delta(3)-carene were 0.6 mM and 28.4 nmol/min/nmol P450 using human CYP2B6. For the formation of Delta(3)-carene-epoxide 98.2 mM and 3.9 nmol/min/nmol P450 were determined as K(m) and V(max) by using human CYP1A2. To our knowledge, this is the first time that Delta(3)-carene-10-ol and Delta(3)-carene-epoxide are described as human metabolites of Delta(3)-carene.
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PMID:Metabolism of Delta(3)-carene by human cytochrome p450 enzymes: identification and characterization of two new metabolites. 1637 71

Genetic polymorphisms can lead to drug adverse effects because certain allelic variants of genes that encode enzymes, targets or carriers involved in drug metabolism, are associated with an increase or a loss of function. Drug metabolism takes place essentially in the liver and is regulated by phase I enzymes (including several cytochrome P450 isoenzymes), the role of which is to make drug metabolites more polar by hydroxylation, and by phase II enzymes that catalyse conjugation reactions. Cytochromes P450 isoenzymes control 80% of oxidative reactions, owing to their low substrate specificity. The most extensively studied polymorphisms are those of CYP2D6 and CYP2C9, which are frequent and affect the metabolism of many drugs. For example, several CYP2C9 gene variants are associated with lower activity of the corresponding enzyme, potentially leading to drug overdose. Thiopurine methyl transferase and UDP-glucuronyl transferase are phase II enzymes that conjugate respectively 6-mercaptopurine metabolites with a methyl radical and metabolites of irinotecan (an anti-tumour drug) with a glucuronyl radical. Mutations in the corresponding genes can, through a loss of function, lead to excessively high levels of active metabolites, with a risk of bone marrow aplasia. The action of vitamin K antagonists is influenced by polymorphisms of vitamine K epoxyde reductase, the target molecule of vitamin K. A mutation in the methylene tetrahydrofolate reductase gene diminishes the folate pool and thereby increases the effects of methotrexate, a folic acid antagonist. Resistance to the anti-platelet effect of aspirin can be due to polymorphisms that affect other platelet aggregation pathways. Genotyping results must be confirmed by phenotyping, which examines the rate of transformation of a drug into its metabolites and shows whether the increase or decrease in this rate is linked to a specific polymorphism. Somatic mutations in malignant tumours can modify the response to anticancer drugs, and should therefore be taken into account. The National Academy of Medicine recommends:--development of genomic analyses for frequent polymorphisms in patients warranting treatment with drugs that have severe adverse effects;--collection of DNA samples from patients participating in drug trials in order to examine the possible relation between adverse effects and gene polymorphisms;--creation of biological resource centers in hospitals for the storage and analysis of tissue specimens.
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PMID:[Pharmacogenetics and pharmacogenomics]. 1687 43

This work provides functional data showing that the bacterial CYP102A1 recognises compounds metabolised by human CYP3A4, CYP2E1 and CYP1A2 and is able to catalyse different reactions. Wild-type cytochrome CYP102A1 from Bacillus megaterium is a catalytically self-sufficient enzyme, containing an NADPH-dependent reductase and a P450 haem domain fused in a single polypeptidie chain. An NADPH-dependent method (Tsotsou et al. in Biosens. Bioelectron. 17:119-131, 2002) together with spectroscopic assays were applied to investigate the catalytic activity of CYP102A1 towards 19 xenobiotics, including 17 commercial drugs. These molecules were chosen to represent typical substrates of the five main families of drug-metabolising human cytochromes P450. Liquid chromatography-mass spectrometry analysis showed that CYP102A1 catalyses the hydroxylation of chlorzoxazone, aniline and p-nitrophenol, as well as the N-dealkylation of propranolol and the dehydrogenation of nifedipine. These drugs are typical substrates of human CYP2E1 and CYP3A4. The KM values calculated for these compounds were in the millimolar range: 1.21+/-0.07 mM for chlorzoxazone, 2.52 +/- 0.08 mM for aniline, 0.81+/-0.04 mM for propranolol. The values of vmax for chlorzoxazone and propranolol were 46.0+/-9.0 and 7.6+/-3.4 nmol min-1 nmol-1, respectively. These values are higher then those measured for the human enzymes. The vmax value for aniline was 9.4+/-1.3 nmol min-1 nmol-1, comparable to that calculated for human cytochromes P450. The functional data were found to be in line with the sequence alignments, showing that the identity percentage of CYP102A1 with CYP3A4 and CYP2E1 is higher than that found for CYP1A2, CYP2C9 and CYP2D6 families.
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PMID:Wild-type CYP102A1 as a biocatalyst: turnover of drugs usually metabolised by human liver enzymes. 1723 82

The hemoprotein cytochrome b(5) (cyt b5) has been demonstrated to affect the kinetics of drug oxidation by the microsomal cytochromes P450 (P450s). However, the mechanisms through which cyt b5 exerts these effects are variable and P450 isoform-dependent. Whereas the effects of cyt b5 on the major drug-metabolizing enzymes CYP2D6, CYP2E1, and CYP3A4 are well studied, fewer studies conducted over limited ranges of cyt b5 concentrations have been performed on CYP2C9. In the present study with CYP2C9, cyt b5 exerted complex actions upon P450 oxidative reactions by affecting the rate of metabolite formation, the consumption of NADPH by cytochrome P450 reductase, and uncoupling of the reaction cycle to hydrogen peroxide and water. Cytochrome b(5) devoid of the heme moiety (apo-b5) exhibited effects similar to those of native cyt b5. All rates were highly dependent on the cyt b5 to CYP2C9 enzyme ratio, suggesting that the amount of cyt b5 present in an in vitro incubation is an important factor that can have an impact on the reliability of extrapolating in vitro generated data to predict the in vivo condition. The major effects of cyt b5 are hypothesized to result from a cyt b5-induced conformational change in CYP2C9 that results in an increased collision frequency between the iron-oxygen species (Cpd I) and the substrate, and a decrease in the oxidase activity. Together, these findings suggest that cyt b5 can alter multiple steps in the P450 catalytic cycle via complex interactions with P450 and P450 reductase.
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PMID:CYP2C9 protein interactions with cytochrome b(5): effects on the coupling of catalysis. 1744 62

Members of the CYP2D family constitute only about 2-4% of total hepatic CYP450s, however, they are responsible for the metabolism of 20-25% of commonly prescribed therapeutic compounds. CYP2D enzymes have been identified in a number of different species. However, vast differences in the metabolic activity of these enzymes have been well documented. In the horse, the presence of a member of the CYP2D family has been suggested from studies with equine liver microsomes, however its presence has not been definitively proven. In this study a cDNA encoding a novel CYP2D enzyme (CYP2D50) was cloned from equine liver and expressed in a baculovirus expression system. The nucleotide sequence of CYP2D50 was highly homologous to that of human CYP2D6 and therefore the activity of the enzyme was characterized using dextromethorphan and debrisoquine, two isoform selective substrates for the human orthologue. CYP2D50 displayed optimal catalytic activity with dextromethorphan using molar ratios of CYP2D50 to NADPH CYP450 reductase of 1:15. Although CYP2D50 and CYP2D6 shared significant sequence homology, there were striking differences in the catalytic activity between the two enzymes. CYP2D50 dextromethorphan-O-demethylase activity was nearly 180-fold slower than the human counterpart, CYP2D6. Similarly, rates of formation of 4-hydroxydebrisoquine activity were 50-fold slower for CYP2D50 compared to CYP2D6. The results of this study demonstrate substantial interspecies variability in metabolism of substrates by CYP2D orthologues in the horse and human and support the need to fully characterize this enzyme system in equids.
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PMID:Complementary DNA cloning, functional expression and characterization of a novel cytochrome P450, CYP2D50, from equine liver. 1869 86

Three neurotransmitters, namely adrenaline, serotonin and tryptamine inhibit the in vitro activity of several cytochrome P450 (CYP) isozymes (CYP1A2, CYP2C9, CYP2D6 and CYP3A). In order to test whether this effect is related to inhibition of the CYP-coupled NADPH reductase activity, we assayed the potential inhibitory effect of these neurotransmitters and their main metabolites on the NADPH reductase activity. Of the five compounds analyzed: tryptamine, tryptophol, serotonin, 5-hydroxytryptamine and adrenaline, only adrenaline significantly decreased NADPH reductase activity at the fixed concentration of 500 microM. However, the effect became negligible when adrenaline concentration was decreased to 100 microM: whereas a high inhibitory effect was observed in CYP2D6, CYP2C9 and CYP3A4 enzyme activities, the NADPH reductase activity remains unchanged. This study indicates that the effect of these endogenous neurotransmitters on CYP enzymes is not related to changes in the reductase activity. In the light of these findings further studies on the inhibitory effect of these neurotransmitters on CYP enzymes can be designed ruling out the modulation of the coupled NADPH reductase activity as a confounding factor.
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PMID:Effect of neurotransmitters on NADPH-cytochrome P450 reductase in vitro activity. 1935 39

1-(4-Methylphenyl)-2-pyrrolidin-1-ylhexan-1-one (4'-methyl-alpha-pyrrolidinohexanophenone, MPHP) is a new designer drug that appeared on the illicit drug market. It is mainly metabolized to 4'-hydroxymethyl-alpha-pyrrolidinohexanophenone (HO-MPHP) followed by oxidation to the respective carboxylic acid. For studies on the quantitative involvement of human cytochrome P450 (CYP) isoenzymes in the initial hydroxylation, a reference standard of HO-MPHP was needed. Therefore, the aim of this study was to synthesize this metabolite using a biotechnological approach. MPHP.HNO(3) (250 micromol) was incubated with 1 L culture of the fission yeast (Schizosaccharomyces pombe) strain CAD64 heterologously co-expressing human CYP reductase and CYP2D6. After centrifugation, the product was isolated from the incubation supernatants by solid-phase extraction. Further product cleanup was achieved by semi-preparative high-performance liquid chromatography (HPLC). After extraction of HO-MPHP from the respective eluent fractions, it was precipitated as its hydrochloric salt. The final product HO-MPHP.HCl was obtained in a yield of 138 micromol (43 mg, 55%). Its identity was confirmed by full scan gas chromatography-mass spectrometry (after trimethylsilylation), (1)H-NMR, and (13)C-NMR. The product purity as estimated from HPLC-ultraviolet analysis was greater than 99%. The described biotechnological approach proved to be a versatile alternative to the chemical synthesis of HO-MPHP.
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PMID:Biotechnological synthesis of the designer drug metabolite 4'-hydroxymethyl-alpha-pyrrolidinohexanophenone in fission yeast heterologously expressing human cytochrome P450 2D6--a versatile alternative to multistep chemical synthesis. 1947 Feb 20

The initial objectives of this study were to evaluate the extent of 3, 4-methylenedioxymethamphetamine (MDMA) induced loss of cell viability (cytotoxicity), induction of reactive oxygen species formation and damage to sub-cellular organelles (e.g. mitochondria/lysosomes) in freshly isolated rat hepatocytes under normothermic conditions (37 degrees C) and to compare the results with the effects obtained under hyperthermic conditions (41 degrees C). MDMA induced cytotoxicity, reactive oxygen species formation, mitochondrial membrane potential decline and lysosomal membrane leakiness in isolated rat hepatocytes at 37 degrees C. A rise in incubation temperature from 37 degrees C to 41 degrees C had an additive/synergic effect on the oxidative stress markers. We observed variations in mitochondrial membrane potential and lysosomal membrane stability that are significantly (P<0.05) higher than those under normothermic conditions. Antioxidants, reactive oxygen species scavengers, lysosomal inactivators, mitochondrial permeability transition (MPT) pore sealing agents, NADPH P450 reductase inhibitor, and inhibitors of reduced CYP2E1 and CYP2D6 prevented all MDMA induced hepatocyte oxidative stress cytotoxicity markers. It is therefore suggested that metabolic reductive activation of MDMA by reduced cytochrome P450s and glutathione could lead to generation of some biological reactive intermediates which could activate reactive oxygen species generation and cause mitochondrial and lysosomal oxidative stress membrane damages. We finally concluded that hyperthermia could potentiate MDMA induced liver toxicity probably through a mitochondrial/lysosomal toxic cross-talk in freshly isolated rat hepatocytes.
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PMID:Involvement of mitochondrial/lysosomal toxic cross-talk in ecstasy induced liver toxicity under hyperthermic condition. 2059 25


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