[Activity of enzymes of xenobiotics metabolism in peripheral blood lymphocytes in man]. / Aktivnost fermentov metabolizma ksenobiotikov v limphotsitakh perifericheskoi krovi cheloveka.

The activity of NADPH-cytochrome c-reductase, benzpyrene hydroxylase, epoxy-hydratase and glutathione-S-transferase in human peripheral blood lymphocytes was studied. In the presence of NADPH, native lymphocytes were unable to reduce cytochrome c. In order to improve the availability of substrates for enzymes, lymphocytes were degraded by single-stage freezing-melting. At the same time, the activities of NADPH-cytochrome c-reductase, epoxide hydratase, and glutathione-S-transferase were 1.7 +/- 0.6, 49.0 +/- 18.0, and 30.0 +/- 6.0 nmol/min per mg protein, respectively. The lymphocytic levels of cytochrome P-450 were approximately 0.1-0.2 nmol per mg microsomal protein, while those of cytochrome b5 were nearly 0.5 nmol/mg microsomal protein in the lymphocytes.

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.

[The role of vitamin K3 in the hydroxylation of benz(a)pyrene in rat liver mitochondria after induction with 3-methylcholanthrene and phenobarbital]. / Uchastie vitamina K3 v gidroksilirovanii benz(a)pirena v mikrosomakh pecheni krys pri induktsii 3-metilkholantrenom i fenobarbitalom.

The "fast" phase reduction of microsomal cytochromes P-450 and P-448 and their benz(a)pyrene (BP) hydroxylase activity was investigated as a function of menadione concentrations. Within a narrow concentration range (1.5-3 microM) menadione activates cytochrome P-448 reduction and the BP hydroxylase activity. At higher concentrations menadione inhibits cytochromes P-450 and P-448 reduction and BP hydroxylation with participation of the both cytochromes. These data suggest that menadione molecules present in membrane lipids serve as an additional electron carrier to cytochrome P-448, the active site of which is embedded into lipids. The activating effect is unobserved is case of cytochrome P-450 with an active site localized in the aqueous phase. The number of different BP metabolites formed at low (3 microM) menadione concentrations in the microsomes of rats induced with 3-methylcholanthrene (MC) and phenobarbital (PB) was compared. In PB-induced microsomes the amount of 7,8-dihydrodiol rises whereas the total content of BP metabolites decreases. Contrariwise, in MC-induced microsomes the synthesis of all BP metabolites is augmented. Menadione has a very weak effect on the ratio of different BP metabolites in PB- and MC-microsomes, but strongly inhibits the formation of more polar metabolites. This results in a marked reduction of the number of "dangerous" BP diolepoxides.

[Mechanism of the inhibiting effect of the Cu-tyrosine complex on the hydroxylation reactions]. / Issledovanie mekhanizma ingibiruiushchego deistviia med'-tirozinovogo kompleksa na gidroksilaznye reaktsii.

The Cu-tyrosine complex, a low molecular weight analog of superoxide dismutase, exerts an inhibiting effect on cytochrome P-450. The inactivation of cytochrome Y-450 with its transition to cytochrome Y-420 can cause the inhibition by the Cu2+ - Tyr2 complex of dimethylaniline N-demethylation and p-nitroanisol O-demethylation. In case of p-hydroxylation of aniline the inhibiting effect of the Cu-tyrosine complex is much more pronounced than its inactivating effect on cytochrome P-450. In the presence of albumin the complex produced no inactivating effect on cytochrome P-450; under these conditions the inhibiting effect of Cu2+ - Tyr2 on N- and O-demethylation is removed. In case of aniline p-hydroxylation albumin partly decreases the inhibiting effect of the complex on this reaction. In a soluble system containing isolated cytochrome P-450 and cumole hydroperoxide only the aniline p-hydroxylation reaction was found sensitive to the effect of superoxide dismutase. The data obtained suggest participation of the superoxide radical only in aniline p-hydroxylation but not in the reactions of N-demethylation of dimethylaniline and O-dealkylation of p-nitroanisol.

[Cytochrome P450 inactivation on a cathode]. / Inaktivatsiia tsitokhroma P450 na katode.

It was concluded on the grounds of spectrophotometric analysis of cytochrome P450 electrolysis products that cytochrome P450 unlike other hem-containing proteins is not reduced on the cathode. At the potential -1.5V it is reduced simultaneously with the transformation into cytochrome P420. Inactivation in the electric field on the electrode surface is irreversible. When the potential is above -1.4V no inactivation of cytochrome P450 takes place.

[Microsomal hemoprotein reduction by superoxide radical formed on NADPH-specific flavoprotein]. / Vosstanovlenie mikrosomal'nykh gemoproteidov superokisnym radikalom, obrazuiushchimsia na NADPH-spetsifichnom flavoproteide.

The superoxide radicals formed on NADPH-specific flavoprotein of liver microsomes can reduce cytochromes c, b5, and P-450. This reaction is inhibited under aerobic conditions by a low molecular weight analog of superoxide dismutase, e.g. the copper-tyrosine complex. The inhibitory effect of the complex is not observed under anaerobic conditions. Based on the results obtained a scheme of the electron transfer between the flavoprotein and haemoproteins involving superoxide radicals is proposed.

[Comparative study of effects of the tyrosine-copper(II) complex on xenobiotic hydroxylation and lipid peroxidation]. / Sravnitel'noe issledovanie deitviia med'-tirozinobogo kompleksa na reaktsii gidroksilirovaniia ksenobiotikov i perekisnogo okisleniia lipidov.

It has been found that NADPH-dependent hydroxylation of dimethylaniline, aniline, p- and o-nitroanisol and lipid peroxidation is inhibited by the tyrosine-copper (II) complex (low molecular weight analog of superoxide dismutase), which is indicative of a possibility of superoxide radicals formation in these reactions. The inhibition of the above-mentioned reactions with Tyr2-Cu2+ is less pronounced or absent, if cumole hydroperoxide is used as cosubstrate instead of NADPH. Differences in the Tyr2-Cu2+ complex effects on the cumule hydroperoxide-dependent xenobiotics hydroxylation and lipid peroxidation catalyzed by various forms of cytochrome P-450, e. g. microsomal, soluble and incorporated into liposomes, have been found. The data obtained suggest that the efficiency of the inhibitory effect of the Tyr2-Cu2+ complex depends on the type of cosubstrates (NADPH, cumole hydroperoxide) and substrates used as well as on the form of cytochrome P-450.

[Electrochemical analysis of microsomal cytochromes]. / Elektrokhimicheskoe issledovanie mikrosomal'nykh tsitokhromov.

The electrochemical behaviour of the electron transfer proteins -- cytochromes b5, c and P-450 was studied by classical polarography and electrolysis with spectrophotometric monitoring. It was shown electrons are directly transferred from the electrode to oxidized cytochromes c and b5. Cytochrome P-450 is not reduced on the electrodes. However, the reduced inactivated from of cytochrome P-420 was detected at high potential values (-1,5 v).

[Modeling the oxidative detoxication function of the liver using electrochemical oxidation reactions]. / Modelirovanie okislitel'noi detoksitsiruiushchei funktsii pecheni s pomoshch'iu reaktsii élektrokhimicheskogo okisleniia.

An electrochemical system is developed, which permits to simulate oxidative reactions, processing in presence of cytochrome P-450. The system, combined with a dialyzing apparatus, may be used for oxidation and elimination of various hydrophobic substances of exoand endogenous origin from animal and human organisms.