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

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Abstract
2-Amino-1-methyl-6-phenylimidazo[4,5- b ]pyridine (PhIP) is a mutagenic and carcinogenic heterocyclic amine formed during ordinary cooking. PhIP is metabolically activated to the ultimate mutagenic metabolite by CYP P450-mediated N -hydroxylation followed by phase II esterification. Incubation of N -hydroxy-PhIP ( N -OH-PhIP) with cytosol, acetyl coenzyme A (AcCoA) and 2′-deoxyguanosine for 24 h resulted in the formation of three different adducts: N2 -(deoxyguanosin-8-yl)-PhIP, N2 -(guanosin-8-yl)-PhIP and PhIP-xanthine. One additional product, 5-hydroxy-PhIP (5-OH-PhIP), was also identified in the incubation mixtures. 5-hydroxy-PhIP is formed as a degradation product of conjugates formed from N -acetoxy-PhIP and protein, glutathione or buffer constituents. A similar spectrum of products was obtained using 3′-phosphoadenosine-5′-phosphosulfate (PAPS) instead of acetyl CoA. Addition of glutathione (3 mM) to the incubation mixture resulted in a 50% reduction in both adducts and 5-hydroxy-PhIP formation in liver cytosol. The main product detected was PhIP, suggesting glutathione-dependent reduction of the N -acetoxy-PhIP. Addition of glutathione to incubation mixtures from the other cytosolic preparations had less dramatic effects. In addition, increasing the amount of N -OH-PhIP in the incubation mixture resulted in proportional increased amounts of total adducts and 5-OH-PhIP. Incubation of rat and human S9 with PhIP resulted in the formation of only traces of 5-OH-PhIP. Fortification 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 PhIP 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 excreted 1% of the dose as 5-OH-PhIP in the urine at 24 h and 0.05 and 0.01% at 48 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.

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