RESUMO
Cytochrome P450 (P450) peroxygenase reactions are catalyzed by the ferric form of the enzyme and utilize hydroperoxides as oxidant donors. These reactions involve heterolytic cleavage of the hydroperoxide O-O bond and oxidation of the heme iron of P450 to a ferryl-oxyl complex, with subsequent transfer of the activated oxygen to a substrate. In the present study, we have demonstrated that arachidonic acid (AA) can serve as a substrate for P450 peroxygenase activity in the presence of cumene hydroperoxide (CuOOH) as a cosubstrate. AA is transformed into the same primary metabolite classes in both NADPH- and CuOOH-dependent oxidations. However, differences in the efficiency of the formation of the primary metabolite classes by CuOOH suggest that the disposition of AA in the presence of hydroperoxides is highly P450 isozyme dependent. Of particular note was the remarkable efficiency of AA epoxidation observed in the CuOOH-dependent reaction in guinea pig liver, whereas the formation of hydroxylated derivatives of AA at the thermodynamically less reactive positions C16 through C20 was more efficiently catalyzed via NADPH-dependent oxidation. Peroxygenase metabolism of AA and/or lipid hydroperoxide transformation reactions may be important functions for endothelial cells that contain P450 but are deficient in NADPH-P450 reductase, and they may also be involved in the cellular response to oxidative stress during NADPH depletion.
Assuntos
Ácido Araquidônico/metabolismo , Animais , Derivados de Benzeno/metabolismo , Cromatografia Líquida de Alta Pressão , Indução Enzimática/efeitos dos fármacos , Compostos Férricos , Cobaias , Ácidos Hidroxieicosatetraenoicos/metabolismo , Isoenzimas/metabolismo , Masculino , Microssomos Hepáticos/metabolismo , Oxigenases de Função Mista/metabolismo , NADP/metabolismo , Fenobarbital/farmacologia , beta-Naftoflavona/farmacologiaRESUMO
Arsenite is a potent toxin, a carcinogen, and an inducer of heat shock proteins. In this study we found that arsenite is also a novel inducer of NAD(P)H:quinone acceptor oxidoreductase (QOR) [EC 1.6.99.2] in both liver and kidney. The increases in activity were unlinked to those caused by prior treatment with the polyaromatic hydrocarbon inducer, beta-naphthoflavone suggesting different mechanisms of induction. A single dose of sodium arsenite (75 mumol/kg sc) caused a 4-fold and 2-fold increase in activity in kidney and liver, respectively, whereas beta-naphthoflavone (60 mg/kg ip once daily for 4 days) caused a 10-fold and 4.7-fold increase in kidney and liver, respectively. This is the first study of a metalloid inducing QOR activity. Arsenite is chemically unlike any other inducer described for QOR, which include phenolic antioxidants and Michael acceptors, polyaromatic hydrocarbons, and hydrogen peroxide. Arsenite also increased glutathione S-transferase [EC 2.5.1.18] activity in rat kidney. Arsenite could be inducing QOR in liver and kidney and the glutathione S-transferase activity in kidney by an oxidant stress mechanism.
Assuntos
Arsênio/farmacologia , Arsenitos , Benzoflavonas/farmacologia , Compostos Policíclicos/farmacologia , Quinona Redutases/biossíntese , Animais , Citosol/metabolismo , Indução Enzimática/efeitos dos fármacos , Glutationa Transferase/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , beta-NaftoflavonaRESUMO
Characterization of cytochrome P450 1A1 dependent monooxygenases in guinea pig heart revealed low rates of 7-ethoxyresorufin O-deethylation, which are markedly increased (20-fold) by treatment with beta-naphthoflavone, a polycyclic aromatic hydrocarbon. Both 7-ethoxyresorufin O-deethylation and 7-methoxyresorufin O-demethylation were found to be approximately 4-fold higher in microsomes prepared from the ventricle than the atrium of beta-naphthoflavone-induced guinea pigs. The low rates of 7-ethoxyresorufin O-deethylation in cardiac microsomes were due, at least in part, to a deficiency of the flavoprotein NADPH--cytochrome P450 reductase; addition of exogenous NADPH--cytochrome P450 reductase; addition of exogenous NADPH--cytochrome P450 reductase dramatically increased 7-ethoxyresorufin O-deethylation in cardiac microsomes of guinea pigs, before and after treatment with beta-naphthoflavone. N-Benzyl-1-aminobenzotriazole, a suicide substrate of cytochrome P450 1A1 in guinea pig, was able to inhibit almost all of the 7-ethoxyresorufin O-deethylase and 7-methoxyresorufin O-demethylase activities in polycyclic aromatic hydrocarbon induced guinea pig heart (88 and 71%, respectively), suggesting that cytochrome P450 1A1 coupled to NADPH--cytochrome P450 reductase in these microsomes inactivates itself by a suicidal mechanism. Addition of alpha-naphthoflavone, an inhibitor of cytochrome P450 1A isozymes, to cardiac microsomes from beta-naphthoflavone-induced guinea pigs resulted in greater than 95% inhibition of 7-ethoxyresorufin O-deethylase activity. The biological significance of these low levels of cytochrome P450 1A1 monooxygenase activity in guinea pig heart and their induction by polycyclic aromatic hydrocarbons are not currently understood.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Miocárdio/enzimologia , NADPH-Ferri-Hemoproteína Redutase/farmacologia , Oxigenases/metabolismo , Animais , Benzoflavonas/farmacologia , Citocromo P-450 CYP1A1 , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/biossíntese , Ativação Enzimática , Indução Enzimática , Cobaias , Coração/efeitos dos fármacos , Átrios do Coração/enzimologia , Ventrículos do Coração/enzimologia , Masculino , Microssomos/enzimologia , NADPH-Ferri-Hemoproteína Redutase/deficiência , Oxirredutases/antagonistas & inibidores , Oxirredutases/metabolismo , Oxigenases/antagonistas & inibidores , Oxigenases/biossíntese , Triazóis/farmacologia , beta-NaftoflavonaRESUMO
Tissue specific changes in the cytochrome P-450 (P-450) monooxygenase system were observed following a single subcutaneous dose of sodium arsenite (75 mumol/kg), a known inducer of stress proteins. P-450 monooxygenase activities were assayed with several isozyme selective substrates; 7-ethoxyresorufin, 7-pentoxyresorufin, 4-aminobiphenyl and erythromycin. Both tissue selective and isozyme selective changes in monooxygenase activity were noted. For example, the rate of 4-aminobiphenyl N-hydroxylation (ABH) was increased by arsenite administration in lung but not in liver. Arsenite inhibited 7-ethoxyresorufin O-deethylation (ERF) in all tissues of control animals, but to a lesser extent in lung. However, increases of ERF activity occurred after arsenite treatment in lung of beta-naphthoflavone (beta NF)-treated guinea pigs whereas arsenite decreased ERF activities in the kidney and liver of these animals. These complex effects on ERF activity may in part be modulated by induction of heme oxygenase, whose activity was increased 2.5-3.5-fold in these organs by arsenite. The highest heme oxygenase activity was found in kidney with lower activities being present in liver and lung, respectively. These data are consistent with the decreased P-450 content observed in kidney and liver microsomes of arsenite treated guinea pigs. On the other hand there was either no change or a slight increase (about 2-fold) in the pulmonary microsomal P-450 content of these animals. A complex pattern of induction for the non-heme, Ah locus associated enzyme, NAD(P)H:quinone acceptor oxidoreductase (QOR) was also observed. With menadione as substrate arsenite treatment increased QOR activity in all tissues studied. However, with dichlorophenolindophenol (DCPIP) as substrate a significant arsenite effect was observed only in the kidney. Significant differences between the QOR substrates were also observed in beta NF-treated guinea pigs and control animals. Our results are consistent with the presence of more than one form of QOR in the guinea pig. Arsenite treatment also caused an increase in glutathione S-transferase activity, with 2,4-dinitro-1-chlorobenzene (DNCB) as substrate, of guinea pig kidney but not liver or lung.