Journal Article

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

Henrik Frandsen and Jan Alexander

in Carcinogenesis

Volume 21, issue 6, pages 1197-1203
Published in print June 2000 | ISSN: 0143-3334
Published online June 2000 | e-ISSN: 1460-2180 | DOI:
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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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.

Keywords: AcCoA, acetyl coenzyme A; dG, 2′-deoxyguanosine; dG-C8-PhIP, N2-(deoxyguanosin-8-yl)-PhIP; DMF, dimethylformamide; GSH, glutathione (reduced); G-C8-PhIP, N2-(guanosin-8-yl)-PhIP; N-OH-PhIP, N-hydroxy-PhIP, hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine; 4'-OH-PhIP, 2-amino-1-methyl-b-(4-hydroxyphenyl)imidazo[4,5-b]pyridine; 5-OH-PhIP, 5-hydroxy-PhIP, 2-amino-1-methyl-6-(5-hydroxy)phenylimidazo [4,5-b]pyridine; PAPS, 3′-phosphoadenosine-5′-phosphosulphate; PCB, Aroclor 1254; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

Journal Article.  4636 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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