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

Methylene blue (MB) is an effective antidote for methemoglobinemia. MB is a basic dye, yielding a blue solution. In the human body, hemoglobin is the oxygen-carrying protein including a ferrous atom. Hemoglobin is oxidized to methemoglobin (MetHb) with the ferric atom, which cannot bind to or carry oxygen. Equilibrium between hemoglobin and MetHb is approximately 99:1. Thus a healthy man can have about 1% of methemoglobinemia. The cytochrome b5 MetHb reductase pathway plays a major role in reducing MetHb to hemoglobin. The nicotin amide adenine dinucleotide phosphate (NADPH) MetHb reductase pathway is a minor reducing system of MetHb, and it needs NADPH as a cofactor. However, to the exceeding exogenous oxidative stress, the cytochrome b5 MetHb reductase pathway is soon exhausted, and the NADPH MetHb reductase pathway can be activated 4 to 5 times by the exogenous cofactor, MB. The decision to initiate MB therapy for methemoglobinemia depends on the MetHb level and the symptoms. The indication for MB therapy in a symptomatic patient is a MetHb level >20% and in an asymptomatic patient, a MetHb level >30%. Patients with comorbidities such as anemia, heart disease, pneumonia, chronic obstructive pulmonary disease, or liver cirrhosis can be candidates for MB therapy with an even lower MetHb level. The recommended initial dose of MB is 1 to 2 mg/kg. It can be repeated every 30 minutes to 1 hour. However, the dose should not exceed 7 mg/kg. A high dose of MB may induce methemoglobinemia paradoxically and also cause hemolytic anemia. Like other antidotes, MB has its own adverse effects.
Adenine ; Anemia ; Anemia, Hemolytic ; Antidotes ; Comorbidity ; Cytochromes b5 ; Heart Diseases ; Human Body ; Humans ; Liver Cirrhosis ; Methemoglobin ; Methemoglobinemia* ; Methylene Blue* ; NADP ; Oxidative Stress ; Oxidoreductases ; Oxygen ; Pneumonia ; Pulmonary Disease, Chronic Obstructive

OBJECTIVETo study the effect of the volatile oil from nutmeg on liver microsomal cytochrome P450 in mice.

METHODMice were administered the volatile oil from nutmeg at 0.4, 0.8 and 1.2 mg x g(-1), respectively, twice a day for 10 days. And then, the contents of liver microsomal cytochrome P450 (CYP), cytochrome b5 (Cytb5), MDA and GST in serum were examined by UV chromatography method.

RESULTThe contents of liver CYP, Cytb5 and GST in serum were increased significantly (P < 0.01) and the contents of MDA was reduced significantly (P < 0.01).

CONCLUSIONThe volatile oil from nutmeg showed induction effect on the hepatic microsomal CYP in mice.

Animals ; Cytochrome P-450 Enzyme System ; metabolism ; Cytochromes b5 ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Female ; Gene Expression Regulation, Enzymologic ; Male ; Mice ; Microsomes, Liver ; drug effects ; enzymology ; Myristica fragrans ; chemistry ; Oils, Volatile ; pharmacology ; Plant Oils ; pharmacology

OBJECTIVETo study the effects of salvianolic acid A on content of cytochrome P450,cytochrome b5 and CYP1A2, CYP2E1 activities of rats.

METHODThe rats were randomly divided into two groups and each group contained 5 male rats and 5 female rats. One is control group, another is dosage group. The dosage group was injected salvianolic acid A into a rat tail vein at doses of 20 mg x kg(-1) x d(-1) for 5 days. The control group was injected placebo into a rat tail vein at the same doses as the dosage group. The content of cytochrome P450 and cytochrome b5 of rats were assayed using UV and CYP1A2, CYP2E1 activities were evaluated using probe substrate.

RESULTAfter salvianolic acid A was injected into rats tail vein for 5 days, the total content of cytochrome P450 and cytochrome b5 and CYP1A2 and CYP2E1 activities have no statistical significance of differences than the control group.

CONCLUSIONSalvianolic acid A has no effects on CYP1A2 and CYP2E1 activities, indicating that there is no internation between salvianolic acid A and the drugs metabolized by CYP1A2 or CYP2E1.

Animals ; Caffeic Acids ; pharmacology ; Chromatography, High Pressure Liquid ; Cytochrome P-450 CYP1A2 ; metabolism ; Cytochrome P-450 CYP2E1 ; metabolism ; Cytochrome P-450 Enzyme System ; metabolism ; Cytochromes b5 ; metabolism ; Female ; Lactates ; pharmacology ; Male ; Microsomes, Liver ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study the modulatory effect of Panax gingseng and coadministration with Veratrum nigrum on the activity and mRNA expression of cytochrome P450 isoenzymes in rat liver.

METHODRat liver microsomal cytochrome P450, b5, aminopyrine N-demethylase(APND), p-nitrophenol-hydroxylase(pNPH)activities were quantitated by UV chromatography. The mRNA expression level of five CYP isoenzymes CYP1A1, CYP2B1/2, CYP2C11, CYP2E1 and CYP3A1 were detected by semi-quantitative reverse transcriptase-polymerase chain reaction(RT-PCR).

RESULTP. gingseng coadministrated with V. nigrum obviously decreased the P450 contents of liver microsomes, and the b5 contents. Both single and combined used inhibited the activities of aminopyrine N-demethylase. At the mRNA level, the expression of CYP2C11 markedly induced exposure to V. nigrum, but combinative groups decreased the expression of CYP2C11. The combination of P. gingseng and V. nigrum induced the expression of CYP1A1. P. gingseng has inhibitory effect on CYP2B1/2 and inductive effect used with V. nigrum. The combination of P. gingseng with V. nigrum also induced the expression of CYP3A1.

CONCLUSIONP. gingseng used singly has some different modulation effects compared with combinative used, which may occur because of drug-drug interaction based on cytochrome P450. To elucidate the drug-drug interaction, it needs further analysis and metabolism research.

Aminopyrine N-Demethylase ; metabolism ; Animals ; Cytochrome P-450 Enzyme System ; biosynthesis ; genetics ; Cytochromes b5 ; metabolism ; Drug Incompatibility ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Female ; In Vitro Techniques ; Isoenzymes ; biosynthesis ; genetics ; Male ; Microsomes, Liver ; metabolism ; Panax ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Veratrum ; chemistry