N-acetyltransferase-dependent activation of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine: formation of 2-amino-1-methyl-6-(5-hydroxy)phenylimidazo [4,5-b]pyridine, a possible biomarker for the reactive dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mutagenic and c arcinogenic heterocyclic amine formed during ordinary cooking. PhIP is meta bolically activated to the ultimate mutagenic metabolite by CYP P450-mediat ed N-hydroxylation followed by phase II esterification, Incubation of N-hyd roxy-PhIP (N-OH-PhIP) with cytosol, acetyl coenzyme A (AcCoA) and 2'-deoxyg uanosine for 24 h resulted in the formation of three different adducts: N-2 -(deoxyguanosin-8-yl)-PhIP, N-2-(guanosin-8-yl) -PhIP and PhIP-xanthine. On e additional product, 5-hydroxy-PhIP (5-OH-PhIP), was also identified in th e incubation mixtures. 5-hydroxy-PhIP is formed as a degradation product of conjugates formed from N-acetoxy-PhIP and protein, glutathione or buffer c onstituents, A similar spectrum of products was obtained using 3'-phosphoad enosine-5'-phosphosulfate (PAPS) instead of acetyl CoA, Addition of glutath ione (3 mM) to the incubation mixture resulted in a 50% reduction in both a dducts and 5-hydroxy-PhIP formation in liver cytosol, The main product dete cted was PhIP, suggesting glutathione-dependent reduction of the N-acetoxy- PhIP, Addition of glutathione to incubation mixtures from the other cytosol ic preparations had less dramatic effects, In addition, increasing the amou nt of N-OH-PhIP in the incubation mixture resulted in proportional increase d amounts of total adducts and 5-OH-PhIP, Incubation of rat and human S9 wi th PhIP resulted in the formation of only traces of 5-OH-PhIP, Fortificatio n with AcCoA clearly increased the formation of 5-OH-PhIP, Addition of the CYP 450 1A2 inhibitor, furafylline, completely inhibited the formation of 5 -OH-PhIP in incubations with human S9, These results indicate that both PhI P adducts and 5-OH-PhIP are formed by similar routes of activation of N-OH- PhIP. 5-OH-PhIP may therefore serve as a biomarker for the formation of the ultimate mutagenic metabolite of PhIP, A rat dosed orally with PhIP excret ed 1% of the dose as 5-OH-PhIP in the urine at 24 h and 0.05 and 0.01% at 4 8 and 72 h, respectively. This shows that 5-OH-PhIP is also formed in vivo and indicates the possible use of 5-OH-PhIP as a urinary biomarker.