[Effect of monoclonal antibody against methylcholanthrene-induced cytochrome P-450 forms on benzo(a)pyrene metabolism in hepatic microsomes of C57BL/10 mice].
In this study, hepatic microsomes from 5,6-benzoflavone induced C57BL/10 mice were used. To inhibit monooxygenase activities, the monoclonal antibody MAb 1-7-1 recognizing two isoenzymes of methylcholanthrene-induced cytochrome P-450 was applied. Microsomes were incubated with tritium labeled benzo(a)pyrene [G-3H]BP for 10 min at 37 degrees C. The incubation mixture contained: 50 mM potassium phosphate buffer, pH 7.25; 30 mM KCl; 3 mM MgCl2; 2 mM NADPH; 80 microM [G-3H]BP (specific activity 50 mCi/mmol); and monoclonal antibody MAb 1-7-1 or ascites fluid (NBS) containing nonspecific IgG as a control. The ratio of antibody protein/microsomal protein was 2:5. BP metabolites were extracted from incubation mixtures by ethyl acetate. The organic layer was dried over sodium sulfate, and evaporated under a stream of nitrogen. To separate BP metabolites HPLC technology was used. The column was eluted with methanol gradient (60-100%) for 45 minutes. The radio-activity of collected samples was determined using liquid scintillation counter. Differential inhibitory effects of MAb 1-7-1 on BP-metabolites formation were found, e.g. 7,8-diol was inhibited by 86.1% and quinones by 62.5%. The predominant metabolite, 3-OH-BP, was inhibited by 80.4%. Moreover, it was found that MAb 1-7-1 inhibition of benzo(a)pyrene hydroxylase activity (by 75.8%, as measured by fluorescent technique) was very similar to the inhibition of 3-OH-BP along with 9-OH-BP formation (as measured by HPLC).
Brauze
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Polskiej Akademii Nauk w Poznaniu.
Mikstacka
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001564:Benzo(a)pyrene / Q000378:metabolism*; D003470:Culture Media; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000276:immunology*; D004790:Enzyme Induction / Q000187:drug effects / Q000502:physiology; D066298:In Vitro Techniques; D008297:Male; D008748:Methylcholanthrene; D051379:Mice; D008810:Mice, Inbred C57BL; D008862:Microsomes, Liver / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D003470:Culture Media; D001564:Benzo(a)pyrene; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System
Wpływ przeciwciała monoklonalnego skierowanego przeciwko formom cytochromu P-450, indukowanym przez metylocholantren, na metabolizm benzo(a)pirenu w mikrosomach watroby myszy C57BL/10.