Reaction of the N²-acetoxy derivative of 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP) with 2'-deoxyguanosine and DNA. Synthesis and identification of N²-(2'-deoxyguanosin-8-yl) - PhIP

Abstract

The direct acting mutagenic N2-hydroxylated metabolite of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) does not react with DNA. Upon acetylation of the N2-hydroxy-PhIP with acetic anhydride two products could be detected. Mass spectrometric analysis showed that both products were monoacetyl derivatives of N2-hydroxy-PhIP. One of the products did not show any reactivity towards DNA and is probably the N-acetyl derivative of N2-hydroxy-PhIP. The other product which is most likely to be N2-acetoxy-PhIP reacted with DNA and 2'-deoxyguanosine but not with 2'-deoxycytidine, 2'-deoxyadenosine or 2'-deoxythymidine. The PhIP-2'-deoxyguanosine adduct was purified and characterized by mass spectral, 1H and [13C]NMR analysis, showing that PhIP like the other cooked food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline, had reacted with C-8 of guanine forming N2-(2'-deoxyguanosin-8-yl)-PhIP. HPLC analysis of enzymatically hydrolyzed calf thymus DNA which had been reacted with N2-acetoxy-PhIP showed one adduct which was chromatographically and spectroscopically identical to N2-(2'-deoxyguanosin-8-yl)-PhIP. HPLC separation followed by liquid scintillation counting of hydrolyzed liver DNA from a rat dosed with [3H]PhIP showed that radioactivity coeluted with the hydrolysis product of the synthetic PhIP-2-deoxyguanosine adduct, indicating that PhIP in vivo also forms an N2-(2'-deoxyguanosin-8-yl)-PhIP adduct.

Detection and quantitation of N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine adducts in DNA using online column-switching liquid chromatography tandem mass spectrometry

“…[32] The hydroxylamine was acetylated as described previously to afford Nacetoxy-PhIP. [14] Given the expected instability of N-acetoxy-PhIP, it was used without any further purification. The PhIP-C8-dG adduct standards were prepared as described previously [14], by reacting the crude acetylation mixtures containing N-acetoxy-PhIP with solutions of 2′-deoxyguanosine or [ 13 C 10 ]-2′-deoxyguanosine.…”

“…[14] Given the expected instability of N-acetoxy-PhIP, it was used without any further purification. The PhIP-C8-dG adduct standards were prepared as described previously [14], by reacting the crude acetylation mixtures containing N-acetoxy-PhIP with solutions of 2′-deoxyguanosine or [ 13 C 10 ]-2′-deoxyguanosine. Unlabelled and [ 13 C 10 ]-labelled PhIP-C8-dG thus obtained were purified by reverse-phase HPLC [14].…”

“…The PhIP-C8-dG adduct standards were prepared as described previously [14], by reacting the crude acetylation mixtures containing N-acetoxy-PhIP with solutions of 2′-deoxyguanosine or [ 13 C 10 ]-2′-deoxyguanosine. Unlabelled and [ 13 C 10 ]-labelled PhIP-C8-dG thus obtained were purified by reverse-phase HPLC [14]. The identity and purity of the unlabelled PhIP-C8-dG was confirmed by 1 H NMR and ESI-MS/MS (data not shown).…”

“…Subsequent metabolism by N-acetyltransferases (NAT) or sulfotransferases (SULT) converts the N-hydroxylaminoPhIP into an ester capable of undergoing heterolytic cleavage to produce a PhIP-nitrenium ion, which is the ultimate reactive species that reacts with DNA (Scheme 1) [5,13,14]. The major covalent DNA adduct detected in vivo resulting from the exposure of experimental animals and humans to PhIP is N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP-C8-dG) [14,15].…”

Human Exposure to Carcinogenic Heterocyclic Amines and Their Mutational Fingerprints in Experimental Animals

“…In the rat Apc gene, this 5'-GTGGGAT-3' sequence is present at only two sites where codons 635 and 1413 are included, and two mutations were detected at each of these two sites (24). Because PhIP forms DNA adducts by covalent binding mainly at guanine-C8 (25,26), these mutations might be directdy induced by the PhIP-DNA adduct.…”