Metabolic formation, synthesis and genotoxicity of the N-hydroxy derivative of the food mutagen 2-amino-1-methyl–6-phenylimidazo (4, 5–b) pyridine (PhIP)

Abstract

Hepatic microsomes from rats pretreated with PCB were found to metabolize the food mutagen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) to two major metabolites, one of which was identified as the N-hydroxy derivative, 2-hydroxy-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (N-OH-PhIP). This identification was based on mass spectral (MS), UV and HPLC data by comparison with N-OH-PhIP prepared by chemical synthesis, as well as the specific activity of the compound in the Ames Salmonella test. Synthetic N-OH-PhIP was prepared by catalytic reduction of the nitro derivative of PhIP, which was synthesized from PhIP by diazotization and reaction with sodium nitrite. N-OH-PhIP was mutagenic to Salmonella typhimurium TA98 without metabolic activation and had a specific mutagenic activity of 2700 revertants/nmol. N-OH-PhIP thus seems to be a proximate mutagenic metabolite of PhIP. Other direct acting mutagens were not detected in the microsomal incubation mixture after HPLC separation. N-OH-PhIP also induced sister chromatid exchange (SCE) in Chinese hamster ovary cells (CHO cells) without metabolic activation. The specific activity of N-OH-PhIP in this assay was approximately 3 times higher than the activity of PhIP with microsomal activation.

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

“…2-hydroxyamino-1-methyl-6-phenylimidazo [4,5-b]pyridine (N-hydroxylamino-PhIP) was prepared as described previously, by diazotization of PhIP and reaction with sodium nitrite followed by catalytic reduction of the nitro derivative thus obtained with Pd/C to the Nhydroxylamine. [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.…”

“…32 P-Postlabelling has been the most widely used method for the detection of PhIP-DNA adducts in animal and human tissues offering the advantage that only small amounts of DNA are required for analysis. The development of electrospray ionization (ESI) has permitted the coupling of liquid chromatography (LC) to mass spectrometry (MS), which has been used for the detection of numerous DNA adducts [26].…”

Heterocyclic amines: Chemistry and health

“…Most HAs are not mutagenic/carcinogenic in their native form but acquire the capability of forming DNA adducts after metabolic activation [117][118][119]. The major activation pathway of HAs involves phase I N-hydroxylation followed by phase II esterification, both of which take place at the exocyclic amino group.…”

Determination of 2-Amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) and Its Metabolite 2-Hydroxyamino-PhIP by Liquid Chromatography/Electrospray Ionization–Ion Trap Mass Spectrometry

“…Of these AIAs, PhIP is the most abundant (0.56 -69.2 ng/g) in well-done meat (1) and is also present in cigarette smoke condensate (16.4 ng per cigarette), wine (14.1 Ϯ 6.18 ng/L), and beer (30.4 Ϯ 16.4 ng/L) (2,3). Although PhIP is the least active AIA in the Ames Salmonella mutagenesis assay, it is the most mutagenic AIA in mammalian cell mutagenesis assays (4,5). Studies in rats have shown that PhIP induces cancers in the colon, mammary gland, and prostate (6 -8).…”