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 abund ant 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-Daw ely 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 le vels [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 i solated rat mammary epithelial cells (from untreated rats) were incuba ted in vitro with N-hydroxy-PhIP (45 mu M, 1 h, 37 degrees C), PhIP-DN A adducts were detected in cell DNA (RAL = similar to 97 x 10(7)); how ever, no adducts were detected in cells incubated with PhIP (200 mu M, 15 h, 37 degrees C), Incubating cells with pentachlorophenol, an inhi bitor of acetyltransferase, or incubating cells at 0-4 degrees C, redu ced N-hydroxy-PhIP adduct levels by 45 and 75% respectively, indicatin g that formation of N-hydroxy-PhIP adducts was largely due to metaboli c activation, Further studies showed that rat mammary gland microsomes had little capacity to N-hydroxylate PhIP, as assayed by the mutageni c activation of PhIP in the Ames Salmonella assay, In contrast, N-hydr oxy-PhIP was metabolically activated by rat mammary gland O-acetyltran sferase, 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-acetyltransferas e activation of N-hydroxy-PhIP was similar to 16- to 17-fold higher th an that observed with hepatic cytosol and at least two-fold higher tha n the mammary O-acetyltransferase activation of N-hydroxy-IQ or N-hydr oxy-MeIQx, All three N-hydroxylamines were activated via cytosolic pro line aminoacyl-tRNA synthetase and phosphorylase, although the activit ies of these enzymes were similar to 100-fold lower than O-acetyltrans ferase. No mammary cytosolic sulfotransferase activation could be dete cted with any of the N-hydroxylamines, Our results are consistent with the notion that PhIP-DNA adduct formation and initiation of carcinoge nesis 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-catalyze d activation of N-hydroxy-PhIP.