The water-soluble and protein-bound metabolites of benzo(a)pyrene formed by rat liver

An optimal incubation system to study benzo(a)pyrene metabolism to its polar derivatives is described. The system using the 20,000g supernatant enzymes of rat liver converts up to 80 per cent of micromolar concentrations of [¹⁴C]benzo(a)pyrene to water-soluble products of which about 10 per cent become covalently bound to protein, are trichloroacetic acid-precipitable, not extractable into organic solvents and are stable to polyacrylamide gel electrophoretic and gel filtration separation techniques.Four (I–IV) discrete[¹⁴C]benzo(a)pyrene-protein complexes have been purified by ion exchange Sephadex gel filtration from post-incubation cytosols. Protein I has the highest binding specificity, a molecular weight estimated at 44,500, migrates as a dimer in sodium dodecyl sulphate polyacrylamide gel, and has glutathione S-transferase activity. This binding protein is believed to be at least similar to, if not identical with, ligandin. Proteins II–IV are also active as glutathione S-transferases. This enzymic function of the binding proteins is markedly reduced by the covalent attachment of the metabolised benzo(a)pyrene, so that the de-activating conjugation with GSH of reactive metabolites is inhibited. The carcinogen, under these conditions hampers its own detoxification reaction.Certain metabolic inhibitors reduce specific binding of the carcinogen to protein I and enhance the binding to the other proteins. The less specific binding to proteins II–IV may be important to the carcinogenic process.