Cyclophosphamide (CPA) is the most common alkylating antineoplastic agent, as well as a strong immunosuppressant that is frequently applied to autoimmune diseases and organ transplantation. It is metabolized by cytochrome P450 oxidases (CYPs) to its active metabolite which played a critical role in therapy. CPA has serious and even fatal side effects, and its efficacy and adverse reactions are significantly varied among individuals. In this review, the association of the genetic polymorphisms in the metabolic enzymes and transporters involved in the disposition of CPA with the efficacy and adverse effects of CPA were summarized, thereby providing fundamental reference for further pharmacogenomic study of CPA.
Antineoplastic Agents, Alkylating ; pharmacology ; Cyclophosphamide ; pharmacology ; Humans ; NADPH-Ferrihemoprotein Reductase ; metabolism ; Pharmacogenetics

Triterpenoids are a class of natural products of great commercial value that are widely used in pharmaceutical, health care and cosmetic industries. The biosynthesis of triterpenoids relies on the efficient synthesis of squalene epoxide, which is synthesized from the NADPH dependent oxidation of squalene catalyzed by squalene epoxidase. We screened squalene epoxidases derived from different species, and found the truncated squalene epoxidase from Rattus norvegicus (RnSETC) showed the highest activity in engineered Escherichia coli. Further examination of the effect of endogenous cytochrome P450 reductase like (CPRL) proteins showed that overexpression of NADH: quinone oxidoreductase (WrbA) under Lac promoter in a medium-copy number plasmid increased the production of squalene epoxide by nearly 2.5 folds. These results demonstrated that the constructed pathway led to the production of squalene epoxide, an important precursor for the biosynthesis of triterpenoids.
Animals ; Escherichia coli/genetics* ; NADPH-Ferrihemoprotein Reductase ; Protein Engineering ; Rats ; Repressor Proteins ; Squalene ; Squalene Monooxygenase/genetics*

OBJECTIVETo investigate the optimal conditions of tri-expression of CYP3A4, POR and cyt b5 in Sf 9 cells.

METHODSThe Sf 9 cells expressing CYP3A4, POR and cyt b5 were cultured in shaker flasks. The optimized conditions, including the temperature and rotation speed, the culture volume, the amount of surfactant and the culture time were studied. The expressed products in microsomes were used to metabolize the testosterone and their metabolic activity was determined.

RESULTSWhen the temperature and rotation speed of the shaker were 27 degree and 90 r/min, the cell density and culture volume were 5X105 cells/ml and 80-120 ml per 250 ml shaker flasks, respectively. When Pluronic F-68 was 0.1% and the culture time was 72 h, the condition was most suitable for culture of Sf 9 cells and expression of targeted proteins. When the ratio of the volume of three added viruses was 1:1:1, the expression condition was optimal, under which the Km, Vmax, and CLint for testosterone metabolism were 119.6 μmol/L,0.52 μmol/(min*g protein) and 4.34 ml/(min*g protein), respectively.

CONCLUSIONThe conditions of tri-expressing of CYP3A4, POR and cyt b5 have been optimized in the study and the product CYP3A4 is obtained with higher metabolic activity.

Animals ; Cytochrome P-450 CYP3A ; biosynthesis ; Cytochromes b5 ; biosynthesis ; Humans ; Insecta ; NADPH-Ferrihemoprotein Reductase ; biosynthesis ; Sf9 Cells

Trichloroethylene (TRI) is one of the most widely used organic solvent in many industries. It is used a degreasing agent, extract and dillution solvents. TRI is metabolized by liver microsomal cytochrome P4SO to reactive intermediates, which are considered responsible for its hepatotoxicity. Cytochrome P450 is a key enzyme in the first step of TRI metabolism resulting in chloral hydrate (CH) formation, which is a rate-limiting step in TRI metabolism. This study was perfomed to find out the change of hepatic cytochrome P450 levels and of electron transport system for P-450 dependent reaction. Intraperitoneal injections of TRI were done to the male Sprague Dawley rats(mean body wights,170+/-10g) in corn oil at the dosage of 150,300,600 mg/kg for 2 days. The results of experiments are following : 1. The contents of microsomal protein, cytochrome P450 and b(5) are tended to be decreased with increasing amount of TRI, but not significantly (p>0.05) . 2. The activity of NADPH-cytochrome P450 reductase is slowly decreased by increasing amount of TRI, but not slgnificantly (p>0.05) . On the other hand, the activity of NADH-b(5) reductase is increased with increasing amount of TRI (p<0.05) 3. The activity of 7-ethoxyresolufin 0-deethylase did not show any critical changes (P>0.05) . This result suggests that the inductive pattern of TRI may not be related to P 450IAl and P450IIB.
Animals ; Chloral Hydrate ; Corn Oil ; Cytochrome P-450 Enzyme System ; Cytochromes ; Electron Transport ; Hand ; Humans ; Injections, Intraperitoneal ; Liver ; Male ; Metabolism ; NADPH-Ferrihemoprotein Reductase ; Oxidoreductases ; Rats* ; Solvents ; Trichloroethylene*

The eye is a relatively isolated organ and tends to retain or accumulate compounds within itself. Unless the eye possesses a mechanism for detoxifying drugs and chemicals that are carried to ocular tissues via the circulating blood, the compounds may cause visual impairment. The purposes of this experiment was judged by drug metabolizing action by cytochrome P-450 system, distribution and effect of metabolism of arachidonic acid and distribution of lipid peroxide formation and phospholipids in the lens, cornea and choroid-retina of pig ocular tissues. The results were as follows: 1. The activities of mixed-function oxidase, cytochrome P-450 and NADPH-cytochrome C reductase were the highest in the choroid-retina compared to the lens and cornea. 2. The activities of drug metabolizing enzymes, aryl hydrocarbon hydroxylase, 7-ethoxycoumarin-o-deethy lase and benzphetamine-N-demethy lase, heme oxygenase activity, and the activity of arachidonic acid metabolizing enzyme, cyclooxygenase were the highest in the choroid-retina compared to the lens and cornea. 3. Lipid peroxide formation was the highest in the cornea compared to the lens and choroid retina. 4. In distribution of phospholipids, sphingomyelin and phosphatidylcholine were the highest in the choroid-retina compared to the lens and cornea and phosphatidylethanolamine was the highest in the cornea campa red to the lens and choroid-retina. 5. Cytochrome P-450 isoenzyme of pig ocular tissues was confirmed in the same form of MAb(Monoclonal Antibody) PCN(pregenolone-16alpha-carbonitrile) 213-1.
Arachidonic Acid* ; Choroid ; Cornea ; Cytochrome P-450 Enzyme System* ; Cytochromes* ; Heme Oxygenase (Decyclizing) ; Metabolism* ; NADPH-Ferrihemoprotein Reductase ; Oxidoreductases ; Phosphatidylcholines ; Phospholipids ; Prostaglandin-Endoperoxide Synthases ; Retina ; Vision Disorders

4-Chloromercuribenzenesulfonate ; pharmacology ; Adult ; Case-Control Studies ; Female ; Graves Disease ; metabolism ; physiopathology ; Humans ; Hydrogen Peroxide ; metabolism ; Iodine ; metabolism ; Male ; NADPH-Ferrihemoprotein Reductase ; metabolism ; Thyroid Function Tests

Andrographolide is a main active ingredient in traditional Chinese medicine Andrographis paniculata，with a variety of pharmacological activity，widely used in clinical practice. However its biosynthetic pathway has not been resolved. Cytochrome P450 reductase provides electrons for CYP450 and plays an important role in the CYP450 catalytic process. In this study，the coding sequence of A. paniculata CPR was screened and cloned by homologous alignment，named ApCPR4. The ApCPR4 protein was obtained by prokaryotic expression. After isolation and purification，the enzyme activity was identified . The results showed that ApCPR4 could reduce the cytochrome c and ferricyanide in NADPH-dependent manner. In order to verify its function，ApCPR4 and CYP76AH1 were co-transformed into yeast engineering bacteria. The results showed that ApCPR4 could help CYP76AH1 catalyze the formation of rustols in yeast. Real-time quantitative PCR results showed that the expression of ApCPR4 increased gradually in leaves treated with methyl jasmonate (MeJA). The expression pattern was consistent with the trend of induction and accumulation of andrographolide by MeJA，suggesting that ApCPR4 was associated with biosynthesis of andrographolide.
Acetates ; Andrographis ; enzymology ; genetics ; Biosynthetic Pathways ; Cloning, Molecular ; Cyclopentanes ; Diterpenes ; metabolism ; NADPH-Ferrihemoprotein Reductase ; genetics ; Oxylipins ; Plant Leaves ; enzymology ; Plant Proteins ; genetics

PURPOSE: To date, paraquat poisoning has almost universally resulted in unfavorable outcomes, and it has become a big issue in clinical toxicology. Current efforts to overcome its toxicity have focused on drugs with anti-oxidant capacity such as ascorbic acid in order to combat over-production of reactive oxygen species (ROS) by paraquat radicals, which are mainly induced by NADPH-cytochrome P450 reductase. Unfortunately, this strategy of treatment has not yielded satisfactory results. In search of a new approach to cope with PQ toxicity, we developed an in vitro culture model of cells resistant to lethal doses of PQ, and we then investigated resistance mechanisms using DNA microarray technology, a tool for simultaneously measuring a number of gene expression changes. METHODS: This experiment was conducted in vitro using the hepatocelluar carcinoma cell line (HepG2) to assay xenobitotics metabolism. We induced resistant of these cells to up to 100 uM PQ by treating with escalating doses of PQ for about 5 months. Cytotoxicity was studied using the MTT method, and optical density was measured at 540 nm using an ELISA reader. We examined morphological changes in cells after drug treatment using an inverted microscope, and we investigated gene expression profiles in control and resistant cells by use of DNA microarray. RESULTS: Results of MTT assays indicated that resistant cells showed relatively high survivals against a 100 mM dose, but that the control group had zero percents of survival at a 1 mM dose. In the comparing gene expression levels between the control group and the resistant group, 6,717 genes found to be differentially expressed. In the analysis of anti-apoptosis genes in particular, the resistant group showed more expression of genes with anti-apoptotic functions than did the control group. In examining the expression of cytochrome P450 genes related to xenobiotic metabolism and PQ radical induction, expression of the cytochrome P450 1B1 gene was significantly higher in the resistant group than in the control group. CONCLUSION: Although cytochrome P450 is known to be responsible for redox cycling of PQ as an electron transferor, this study suggest that up-regulation of the cytochrome P450 1B1 gene can corelate with PQ resistance. Therefore, induction of cytochrome P450 1B1 can be a new therapeutic approach to reduce PQ toxicity through actual PQ degradation, rather than simply through neutralization of ROS.
Ascorbic Acid ; Cell Line ; Cytochrome P-450 Enzyme System ; DNA ; Electrons ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Hep G2 Cells ; NADPH-Ferrihemoprotein Reductase ; Oligonucleotide Array Sequence Analysis ; Oxidation-Reduction ; Paraquat ; Phosphatidylethanolamines ; Reactive Oxygen Species ; Toxicology ; Transcriptome ; Up-Regulation

Tanshinones are the bioactive components of the Chinese medicinal herb Salvia miltiorrhiza, while its biosynthetic pathway remains to be characterized. Rapid identification and characterization of the genes correlated to tanshinones biosynthesis is very important. As one of the intermediates of tanshinones biosynthesis, the ferruginol content is relative low in both root and engineered bacteria. It is urgent to construct an efficient system for conversion of miltiradiene to ferruginol to obtain large amount of ferruginol as the substrates for further identifying other downstream genes involved in tanshinones biosynthesis. In this study, we constructed the whole-cell yeast biocatalysts co-expressing miltiradiene oxidase CYP76AH1 and cytochrome P450 reductases (SmCPR1) from Salvia miltiorrhiza, and then characterized it with RT-PCR. After permeabilization, the yeast whole-cell could catalyze turnover of miltiradiene to ferruginol efficiently through single-step biotransformation with a conversion efficiency up to 69.9%. The yeast whole-cell biocatalyst described here not only provide an efficient platform for producing ferruginol in recombinant yeast but also an alternative strategy for identifying other CYP genes involved in tanshinones biosynthesis.
Biosynthetic Pathways ; Biotransformation ; Cytochrome P-450 Enzyme System ; genetics ; metabolism ; Diterpenes ; metabolism ; Diterpenes, Abietane ; biosynthesis ; chemistry ; Electrophoresis, Agar Gel ; Gene Amplification ; NADPH-Ferrihemoprotein Reductase ; genetics ; metabolism ; Open Reading Frames ; Plasmids ; Saccharomyces cerevisiae ; genetics ; metabolism ; Salvia miltiorrhiza ; chemistry

CYP2D6 is an important drug-metabolizing enzyme. The polymorphism of CYP2D6 leads to metabolism difference and the different reactions of drugs in the individuals and different races are normal phenomenon in clinical medication. CYP2D6*10 is an important subtype in Asian people and 51.3% Chinese are classified with this subtype. To obtain recombinant active CYP2D6*1/CYP2D6*10 in baculovirus system by optimizing coexpression with CYPOR, and detect their activity to catalyze dextromethorphan, three recombinants pFastBac-CYP2D6*1, pFastBac-CYP2D6*10 and pFastBac-CYPOR were constructed and transformed into DH10Bac cell to obtain the recombinant Bacmid-CYPOR, Bacmid-CYP2D6*1 and Bacmid-CYP2D6*10. And then the recombinant CYP2D6*1 and CYP2D6*10 virus were obtained by transfecting Sf9. Then homogenate protein activity was determined with dextromethorphan as substrate. The multiple of infection (MOI) and its ratio of recombinant CYP2D6 virus to CYPOR virus were adjusted by detecting the activity of the homogenate protein. The Km and Vmax are 26.67 +/- 2.71 micromol x L(-1) (n=3) and 666.7 +/- 56.78 pmol x nmol(-1) (CYP2D6) x min(-1) (n=3) for CYP2D6*1 to catalyze dextromethaphan. The Km and Vmax are 111.36 +/- 10.89 micromol x L(-1) (n=3) and 222.2 +/- 20.12 pmol x nmol(-1) (CYP2D6) x min(-1) (n=3) for CYP2D6*10 to catalyze dextromethorphan. There is significant difference between CYP2D6*1 and CYP2D6*10 for Vmax and Km (P < 0.01). The clearance ratio of CYP2D6*1 is 25.0 and the clearance ratio of CYP2D6*10 is 2.0. The expressed CYP2D6*1 and CYP2D6*10 are useful tools to screen the metabolism profile of many xenobiotics and endobiotics in vitro, which are benefit to understand individual metabolism difference.
Animals ; Baculoviridae ; enzymology ; genetics ; Catalysis ; Cells, Cultured ; Chromatography, High Pressure Liquid ; methods ; Cytochrome P-450 CYP2D6 ; genetics ; metabolism ; Dextromethorphan ; metabolism ; Isoenzymes ; metabolism ; NADPH-Ferrihemoprotein Reductase ; genetics ; metabolism ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; Spectrometry, Mass, Electrospray Ionization ; Spodoptera ; cytology ; virology ; Transfection