Abstract
A simple method is described for the simultaneous determinations of total water-soluble metabolites of [14C] benzo(a)pyrene and their irreversible binding to DNA, RNA and proteins. [14C]Benzo(a)pyrene, DNA, microsomes and NADPH were incubated together in a suitable buffer. Reactions were stopped by adding acetone and hexane, and unmetabolized benzo(a)pyrene and organic solvent-soluble metabolites were extracted into the hexane layer. An aliquot of the mixture was transferred to a small centrifuge tube and placed at -70℃ after centrifugation. Only the aqueous phase was frozen and the organic solvent was discarded by decantation. This procedure was repeated three times. An aliquot of the remaining aqueous phase was employed directly to determine the amounts of water-soluble metabolites of benzo(a)pyrene. Another aliquot was spread on filter paper and the filter was soaked into cold 10% trichloroacetic acid to remove acid-soluble materials. The filter papers were washed sequentially in batchweise with 10% trichloroacetic acid and acetone. The radioactivities remaining on the filter papers were counted as the binding-products. Total water-soluble metabolites of benzo(a)pyrene with liver microsomes from polychloro biphenyl-treated mice were much greater than those with microsomes from normal liver. It was found, however, that approximately 35 to 45% of water-soluble metabolites were existing as binding-adducts to macromolecules in both induced and uninduced cases. The present method is convinient for quantitative estimation of the activating system of carcinogens in some biological sources.
