Improvement of the correlation of bacterial mutagenicity with carcinogenicity of benzo(a)pyrene and four of its major metabolites by activation with intact liver cells instead of cell homogenate.

Abstract

Benzo( a )pyrene (BP) and four main metabolites were investigated for mutagenicity with Salmonella typhimurium TA 100 using either intact or homogenized hepatocytes from Aroclor 1254-treated rats for metabolism of the test compounds. Homogenates were supplemented with various cofactor systems. BP was strongly mutagenic with both intact and broken cells. In the latter case, a reduced nicotinamide adenine dinucleotide phosphate-generating system was necessary for activation, and much lower doses of compound were required than with intact cells. 3-Hydroxybenzo( a )pyrene (3-OH-BP), 9-hydroxybenzo( a )pyrene (9-OH-BP), and (±)- trans -9,10-hydroxy-9,10-dihydrobenzo( a )pyrene (BP-9,10-dihydrodiol) showed marked mutagenic effects in the presence of cell homogenate when a reduced nicotinamide adenine dinucleotide phosphate-generating system was added. However, these BP metabolites showed only very weak mutagenic effects in the presence of intact hepatocytes. (±)- trans -7,8-Dihydroxy-7,8-dihydrobenzo( a )pyrene (BP-7,8-dihydrodiol) was the most powerful mutagen of the compounds tested with both intact and broken cells. Thus, the correlation of mutagenicity with whole-animal carcinogenicity was substantially better with intact hepatocytes than with cell homogenate as metabolizing system. The weak carcinogens or tumor initiators, 3-OH-BP, 9-OH-BP, and BP-9,10-dihydrodiol, were weak mutagens; whereas the strong carcinogens and initiators, BP and BP-7,8-dihydrodiol, showed strong mutagenic effects with intact hepatocytes. Experiments conducted with the commonly used postmitochondrial fraction as a metabolizing system showed results that were in essence very similar to those with hepatocyte homogenate. When cofactors for conjugation reactions were added to the homogenate along with a reduced nicotinamide adenine dinucleotide phosphate-generating system, the mutagenicity of all five compounds was decreased. The mutagenicity of BP and 3-OH-BP was reduced 45 to 69% upon addition of either glutathione, a 3′-phosphoadenosine 5′-phosphosulfate-generating system, or UDP (uridine diphosphate)-glucuronic acid in the presence of the UDP-glucuronosyltransferase activator UDP- N -acetylglucosamine. Addition of pure epoxide hydrolase showed no significant effect. With BP-9,10-dihydrodiol, two cofactor systems, glutathione and UDP-glucuronic acid, decreased its mutagenicity as did epoxide hydrolase. The mutagenicity of 9-OH-BP was reduced to the greatest extent by UDP-glucuronic acid. It was also reduced slightly in the presence of either the 3′-phosphoadenosine 5′-phosphosulfate-generating system or glutathione, but not by addition of epoxide hydrolase. The mutagenic activity of BP-7,8-dihydrodiol was reduced by glutathione, whereas the other metabolic alterations had little or no inactivating effect. The 3′-phosphoadenosine 5′-phosphosulfate-generating system even somewhat increased its mutagenicity. Addition of all the conjugation cofactors together resulted in the greatest inactivation with 3-OH-BP and BP-9,10-dihydrodiol and the least inactivation with BP-7,8-dihydrodiol. The inverse approach, inhibition of inactivation mechanisms in intact cells by depletion of glutathione and the use of competitive substrates for conjugation, was less successful. Increases in the mutagenicity of BP and BP-7,8-dihydrodiol were observed in the presence of diethylmaleate or salicylamide, but the mutagenicity of the three compounds with low carcinogenicity remained extremely low. These results show that the correlation of bacterial mutagenicity with carcinogenicity of BP and four major metabolites is substantially better when these compounds are activated by intact hepatocytes as compared to commonly used broken cell preparations; the results suggest that the relatively poor correlation between whole-animal carcinogenicity and mutagenicity in the standard Ames assay of BP metabolites is due to differences in metabolism of the compounds in the two systems rather than to differences in the biological end points, i.e. , bacterial mutagenicity versus mammalian carcinogenicity.