Dataset Information

A comprehensive approach to the profiling of the cooked meat carcinogens 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, and their metabolites in human urine.

ABSTRACT: A targeted liquid chromatography/tandem mass spectrometry-based metabolomics type approach, employing a triple stage quadrupole mass spectrometer in the product ion scan and selected reaction monitoring modes, was established to profile 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and their principal metabolites in the urine of omnivores. A mixed-mode reverse phase cation exchange resin enrichment procedure was employed to isolate MeIQx and its oxidized metabolites, 2-amino-8-(hydroxymethyl)-3-methylimidazo[4,5-f]quinoxaline (8-CH(2)OH-IQx) and 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid (IQx-8-COOH), which are produced by cytochrome P450 1A2 (P450 1A2). The phase II conjugates N(2)-(beta-1-glucosiduronyl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and N(2)-(3,8-dimethylimidazo[4,5-f]quinoxalin-2-yl)-sulfamic acid were measured indirectly, following acid hydrolysis to form MeIQx. The enrichment procedure permitted the simultaneous analysis of PhIP, N(2)-(beta-1-glucosidurony1)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, N3-(beta-1-glucosidurony1)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 2-amino-1-methyl-6-(4'-hydroxy)-phenylimidazo[4,5-b]pyridine (4'-HO-PhIP), and the isomeric N(2)- and N3-glucuronide conjugates of the carcinogenic metabolite, 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP), which is formed by P450 1A2. The limit of quantification (LOQ) for MeIQx, PhIP, and 4'-HO-PhIP was approximately 5 pg/mL; the LOQ values for 8-CH(2)OH-IQx and IQx-8-COOH were, respectively, <15 and <25 pg/mL, and the LOQ values for the glucuronide conjugates of PhIP and HONH-PhIP were 50 pg/mL. The metabolism was extensive; less than 9% of the dose was eliminated in urine as unaltered MeIQx, and <1% was eliminated as unaltered PhIP. Phase II conjugates of the parent amines accounted for up to 12% of the dose of MeIQx and up to 2% of the dose of PhIP. 8-CH(2)OH-IQx and IQx-8-COOH accounted for up to 76% of the dose of MeIQx, and the isomeric glucuronide conjugates of HONH-PhIP accounted for up to 33% of the dose of PhIP that were eliminated in urine within 10 h of meat consumption. P450 1A2 significantly contributes to the metabolism of both HAAs but with marked differences in substrate specificity. P450 1A2 primarily catalyzes the detoxification of MeIQx by oxidation of the 8-methyl group, whereas it catalyzes the bioactivation of PhIP by oxidation of the exocyclic amine group.

SUBMITTER: Gu D

PROVIDER: S-EPMC3196554 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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A comprehensive approach to the profiling of the cooked meat carcinogens 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, and their metabolites in human urine.

Gu Dan D McNaughton Lynn L Lemaster David D Lake Brian G BG Gooderham Nigel J NJ Kadlubar Fred F FF Turesky Robert J RJ

Chemical research in toxicology 20100401 4

A targeted liquid chromatography/tandem mass spectrometry-based metabolomics type approach, employing a triple stage quadrupole mass spectrometer in the product ion scan and selected reaction monitoring modes, was established to profile 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and their principal metabolites in the urine of omnivores. A mixed-mode reverse phase cation exchange resin enrichment procedure was employed to isolate ...[more]

PMID: 20192249

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Project description:2-Amino-1,7-dimethylimidazo[4,5-g]quinoxaline (MeIgQx) is a recently discovered heterocyclic aromatic amine (HAA) that is formed during the cooking of meats. MeIgQx is an isomer of 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), a rodent carcinogen and possible human carcinogen that also occurs in cooked meats. MeIgQx is a bacterial mutagen, but knowledge about its metabolism and carcinogenic potential is lacking. Metabolism studies on MeIgQx and MeIQx were conducted with human and mouse liver microsomes, and recombinant human P450s. DNA binding studies were also investigated in mice to ascertain the genotoxic potential of MeIgQx in comparison to MeIQx. Both HAAs underwent comparable rates of N-oxidation to form genotoxic N-hydroxylated metabolites with mouse liver microsomes (0.2-0.3 nmol/min/mg protein). The rate of N-oxidation of MeIQx was 4-fold greater than the rate of N-oxidation of MeIgQx with human liver microsomes (1.7 vs 0.4 nmol/min/mg protein). The rate of N-oxidation, by recombinant human P450 1A2, was comparable for both substrates (6 pmol/min/pmol P450 1A2). MeIgQx also underwent N-oxidation by human P450s 1A1 and 1B1 at appreciable rates, whereas MeIQx was poorly metabolized by these P450s. The potential of MeIgQx and MeIQx to form DNA adducts was assessed in female C57BL/6 mice given [(14)C]-MeIgQx (10 μCi, 9.68 mg/kg body wt) or [(14)C]-MeIQx (10 μCi, 2.13 mg/kg body wt). DNA adduct formation in the liver, pancreas, and colorectum was measured by accelerator mass spectrometry at 4, 24, or 48 h post-treatment. Variable levels of adducts were detected in all organs. The adduct levels were similar for both HAAs, when adjusted for dose, and ranged from 1 to 600 adducts per 10(7) nucleotides per mg/kg dose. Thus, MeIgQx undergoes metabolic activation and binds to DNA at levels that are comparable to MeIQx. Given the high amounts of MeIgQx formed in cooked meats, further investigations are warranted to assess the carcinogenic potential of this HAA.

Project description:Heterocyclic aromatic amines (HAAs) are carcinogens formed during the cooking of meats or arise in tobacco smoke. The genotoxic N-oxidized metabolites of HAAs bind to Cys residues of proteins to form arylsulfinamide adducts. However, these adducts are unstable and undergo hydrolysis during enzymatic digestion, and thus have been precluded as biomarkers of exposure to HAAs. Arylsulfinamide adducts of HAAs can undergo oxidation to form stable arylsulfonamide linkages, which are chemically stable and amenable for analysis. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a carcinogen present in cooked meat. We established a quantitative MS-based method to measure the sulfinamide adduct of PhIP formed at the cysteine(34) (Cys(34)) residue of human serum albumin (SA), following chemical oxidation of PhIP-modified SA with m-chloroperoxybenzoic acid. Different enzyme systems (trypsin; chymotrypsin; trypsin/chymotrypsin; proteinase K; pronase E; and pronase E/leucine aminopeptidase/prolidase) were evaluated for their proficiency of digestion of SA modified with PhIP. The strongest signal was observed for the L(31)QQC*PFEDHVK(41) peptide, by ultraperformance liquid chromatography and ion trap MS. A limit of quantification value was 0.3fmol of LQQC*PFEDHVK per μg SA, or 2.5 adducts per 10(5) SA molecules, when assaying 0.75μg of SA.Biological significanceThis article describes a mass spectrometric based method to characterize and measure human serum albumin (SA) adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogenic heterocyclic aromatic amine formed in cooked meats and tobacco smoke. PhIP undergoes metabolic activation to form reactive N-oxidized intermediates that bind to DNA and proteins. N-oxidized PhIP metabolites bind to the Cys(34) residue of SA to form a sulfinamide linkage. However, the linkage undergoes hydrolysis during proteolysis, precluding the employment of this adduct as a biomarker in human studies. We have shown that the sulfinamide linkage undergoes oxidation to form the [cysteine-S-yl-PhIP]-S-dioxide, a sulfonamide linked adduct which is stable toward proteolysis. The specificity and efficiency of several different proteases toward the digestion of the SA-Cys(34)-PhIP adduct were examined. The combination of trypsin and chymotrypsin produced the single-missed cleaved peptide LQQC*PFEDHVK in high yield. Moreover, denaturation and chemical reduction of the internal Cys disulfide bonds of SA were not required for the recovery of LQQC*PFEDHVK. The novel chemistry and proteomic approaches developed in this study may be applied to monitor biologically reactive N-oxidized intermediates of arylamines through their adduction products formed at nucleophilic Cys residues of proteins.

Dataset Information

A comprehensive approach to the profiling of the cooked meat carcinogens 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, and their metabolites in human urine.

Publications

A comprehensive approach to the profiling of the cooked meat carcinogens 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, and their metabolites in human urine.

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