POSSIBLE MECHANISMS FOR PHIP-DNA ADDUCT FORMATION IN THE MAMMARY-GLAND OF FEMALE SPRAGUE-DAWLEY RATS

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant heterocyclic amine in fried beef, is a mammary gland carcinogen in rats. Using the P-32-postlabeling method, PhIP-DNA adduct levels were measured in mammary epithelial cells isolated from female Sprague-Dawely rats given 10 daily doses of PhIP (75 mg/ kg, p.o.) according to a protocol previously shown to induce mammary gland cancer. At 24 h, 48 h, 1 week and 5 weeks after the last dose of PhIP, PhIP-DNA adduct levels [relative adduct labeling (RAL) x 10(7), mean +/- SD] were 10.2 +/- 0.7, 7.9 +/- 2.7, 2.2 +/- 0.6 and 0.9 +/- 0.03 respectively, When isolated rat mammary epithelial cells (from untreated rats) were incubated in vitro with N-hydroxy-PhIP (45 mu M, 1 h, 37 degrees C), PhIP-DNA adducts were detected in cell DNA (RAL = similar to 97 x 10(7)); however, no adducts were detected in cells incubated with PhIP (200 mu M, 15 h, 37 degrees C), Incubating cells with pentachlorophenol, an inhibitor of acetyltransferase, or incubating cells at 0-4 degrees C, reduced N-hydroxy-PhIP adduct levels by 45 and 75% respectively, indicating that formation of N-hydroxy-PhIP adducts was largely due to metabolic activation, Further studies showed that rat mammary gland microsomes had little capacity to N-hydroxylate PhIP, as assayed by the mutagenic activation of PhIP in the Ames Salmonella assay, In contrast, N-hydroxy-PhIP was metabolically activated by rat mammary gland O-acetyltransferase, as assayed by cytosol-catalyzed PhIP-DNA adduct formation to calf thymus DNA incubated in vitro with N-hydroxy-PhIP (2 mu M) in the presence of acetyl CoA, Notably, mammary cytosolic O-acetyltransferase activation of N-hydroxy-PhIP was similar to 16- to 17-fold higher than that observed with hepatic cytosol and at least two-fold higher than the mammary O-acetyltransferase activation of N-hydroxy-IQ or N-hydroxy-MeIQx, All three N-hydroxylamines were activated via cytosolic proline aminoacyl-tRNA synthetase and phosphorylase, although the activities of these enzymes were similar to 100-fold lower than O-acetyltransferase. No mammary cytosolic sulfotransferase activation could be detected with any of the N-hydroxylamines, Our results are consistent with the notion that PhIP-DNA adduct formation and initiation of carcinogenesis in the rat mammary gland may be associated with N-hydroxylation of PhIP outside the mammary gland, transport of the N-hydroxylamine to the mammary gland and subsequent in situ O-acetyltransferase-catalyzed activation of N-hydroxy-PhIP.