Application of electron-donor properties of glucose oxidase and xanthine oxidase for reduction of microsomal NAD(P)H-dependent electron-transport chains.

The reduction of cytochromes b5 and P-450 in mammalian hepatic microsomes by glucose oxidase and xanthine oxidase has been investigated. Under anaerobic conditions cytochrome b5 is reduced by glucose oxidase to the "dithionite" level, while cytochrome P-450 remains oxidized. Under the same conditions xanthine oxidase completely reduces both hemoproteins. Besides, neither glucose oxidase nor xanthine oxidase reduces isolated cytochromes. They can be reduced only after addition of microsomes to incubation media. Only in this case are the cytochromes, both isolated and included in microsomal membranes, reduced. The participation of microsomal flavoproteins in the reduction reaction is discussed. The method suggested makes it possible to substantially decrease the rates of reduction of microsomal hemoproteins, thus permitting the investigation of interactions between microsomal NADH- and NADPH-dependent electron-transport chains and electron carriers.

[The role of secondary metabolism in formation of benz(a)pyrene dihydrodiol profile in microsome membranes]. / Rol' vtorichnogo metabolizma v formirovanii profilia digidrodiolov benz(a)pirena v membranakh mikrosom.

4,5-, 7,8- and 9,10-dihydrodiols of benz(a)pyrene (BP) were separated by thin-layer chromatography and their influence on BP-hydroxylase activity was studied in liver microsomes isolated from rats treated with phenobarbital (PB-microsomes) and 3-methylcholanthrene (MC-microsomes). All diols studied inhibited hydroxylation of BP by the competitive type. Accumulation of BP-diols in the incubation media correlated with their affinity to cytochrome P-450 isoenzymes which catalyzed the secondary metabolism of these diols. This correspondence allowed to formulate the kinetic and temperature dependence of BP oxidation suggesting that two main groups of hemoprotein isoforms were contained which were dissimilar in the active site orientation. Treatment with 3-methylcholanthrene induced specifically those hemoproteins which had the active site directed inside the membrane lipids; treatment with phenobarbital involved induction of two groups of hemoproteins active site of which was directed both to lipid and to water. The primary metabolism of the hydrophobic BP involved cytochrome P-450 isoenzymes which had the active site directed inside the lipids; the secondary metabolism of more polar diols was realized using both groups of hemoprotein isoenzymes with active sites oriented into lipids and water.

The ratio of two isozyme groups in microsomal cytochrome P-450 under exogenous influence of carbon tetrachloride and cyclophosphamide.

A method for measuring the content of two groups of microsomal cytochrome P-450 isozymes--cytochromes P-450W and P-450L--with the active sites directed into the water phase and membrane lipids, respectively, has been developed. The method is based on the ability of the xanthine oxidase-menadione complex to reduce microsomal cytochromes b5 and P-450 under anaerobic conditions by transferring electrons to hemoproteins with the active sites directed into the water phase. Cytochrome b5 is completely reduced (to the dithionite level) and cytochrome P-450 is reduced partially (only a group of cytochromes P-450W). The amount of cytochromes P-450L is estimated using the difference between the total content of cytochrome P-450 reduced by sodium dithionite and the content of cytochromes P-450W. The possibility of controlling the ratio of these two isozyme groups in cytochrome P-450 in vivo in membranes of the endoplasmic reticulum by pretreatment of animals with a variety of chemicals has been demonstrated. The ratio of cytochromes P-450W and P-450L has been shown to decrease two-fold 18 days after three injections of phenobarbital into mice. Carbon tetrachloride and cyclophosphamide also decrease this ratio in vivo.

[Disulfiram and butylhydroxyanisole induction of glutathione-S- and UDP glucuronosyltransferase activities in the liver and its role in preventing the toxic action of diethylnitrosamine in rats]. / Induktsiia disul'firamom i butilgidroksianizolom aktivnosti glutation-S- i UDF-gliukuronoziltransferaz v pecheni i ee rol' v predotvrashchenii toksicheskogo deistviia diétilnitrozamina u krys.

Pretreatment of Wistar male rats with disulfiram (DSF, 500 mg/kg, three times, orally) or butylhydroxyanisole (BHA, 400 mg/kg, three times, orally) was shown to protect animals completely against intoxication with diethylnitrosamine (DENA) in the dose equal to LD50 (280 mg/kg, once, orally). DSF and BHA were also found to produce a significant modification of the 2nd stage of metabolism of xenobiotics: to induce the activity of cytosol and microsomal glutathione-S-transferases (GST) (by 1.7 and 2 times, 2.3 and 3.1 times, respectively), the content of glutathione (by 1.6 and 2.3 times), the activity of UDP glucuronosyltransferase (UDP-GT) (by 2 and 3.2 times) but to decrease the activity of sulfotransferase (by 22 and 35%). Administration of DENA decreased the activity of GST, UDP-GT and the content of glutathione, however they remain significantly higher than the corresponding parameters in intact animals. It is concluded that the protective action of DSF and BHA is related greatly to the induction of the systems of conjugation with glutathione and UDP-glucuronic acid.