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Mass Spectrometric Characterization of an Acid-Labile Adduct Formed with 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and Albumin in Humans.

ABSTRACT: 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a carcinogenic heterocyclic aromatic amine formed during the high-temperature cooking of meats. The cytochrome P450-mediated N-hydroxylation of the exocyclic amine group of PhIP produces 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine, an electrophilic metabolite that forms adducts with DNA and proteins. Previous studies conducted by our laboratory showed that the reaction of N-oxidized PhIP metabolites with human albumin in vitro primarily occurs at the Cys34 residue, to produce an acid-labile linked sulfinamide adduct. On the basis of these findings, we developed a sensitive ultraperformance liquid chromatography-mass spectrometry method to measure acid-labile albumin-PhIP adducts in human volunteers administered a dietary-relevant dose of 14C-labeled PhIP [Dingley, K. H., et al. (1999) Cancer Epidemiol., Biomarkers Prev. 8, 507-512]. Mild acid treatment of albumin (0.1 N HCl, 37 °C for 1 h) or proteolytic digestion with Pronase [50 mM ammonium bicarbonate buffer (pH 8.5) at 37 °C for 18 h] released similar amounts of covalently bound PhIP, which was characterized by multistage scanning and quantified by Orbitrap mass spectrometry. The amount of [14C]PhIP recovered by acid treatment of albumin 24 h following dosing accounted for 7.2-21.3% of the [14C]PhIP bound to albumin based on accelerator mass spectrometry measurements. 2-Amino-1-methyl-6-(5-hydroxy)phenylimidazo[4,5-b]pyridine, a hydrolysis product of the Cys34 S-N linked sulfenamide adduct of PhIP, was not detected in either acid-treated or protease-treated samples. These findings suggest that a portion of the PhIP bound to albumin in vivo probably occurs as an acid-labile sulfinamide adduct formed at the Cys34 residue.

SUBMITTER: Wang Y

PROVIDER: S-EPMC5375106 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Mass Spectrometric Characterization of an Acid-Labile Adduct Formed with 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and Albumin in Humans.

Wang Yi Y Villalta Peter W PW Peng Lijuan L Dingley Karen K Malfatti Michael A MA Turteltaub K W KW Turesky Robert J RJ

Chemical research in toxicology 20170118 2

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a carcinogenic heterocyclic aromatic amine formed during the high-temperature cooking of meats. The cytochrome P450-mediated N-hydroxylation of the exocyclic amine group of PhIP produces 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine, an electrophilic metabolite that forms adducts with DNA and proteins. Previous studies conducted by our laboratory showed that the reaction of N-oxidized PhIP metabolites with human albumin in vitro ...[more]

PMID: 27984695

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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.

Project description:2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic aromatic amine that is formed during the cooking of meats. PhIP is a potential human carcinogen: it undergoes metabolic activation to form electrophilic metabolites that bind to DNA and proteins, including serum albumin (SA). The structures of PhIP-SA adducts formed in vivo are unknown and require elucidation before PhIP protein adducts can be implemented as biomarkers in human studies. We previously examined the reaction of genotoxic N-oxidized metabolites of PhIP with human SA in vitro and identified covalent adducts formed at cysteine³⁴ (Cys³⁴); however, other adduction products were thought to occur. We have now identified adducts of PhIP formed at multiple sites of SA reacted with isotopic mixtures of electrophilic metabolites of PhIP and 2-amino-1-methyl-6-[²H₅]-phenylimidazo[4,5-b]pyridine ([²H₅]-PhIP). The metabolites used for study were 2-nitro-1-methyl-6-phenylimidazo[4,5-b]pyridine (NO₂-PhIP), 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP), or N-acetyloxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-acetoxy-PhIP). Following proteolytic digestion, PhIP-adducted peptides were separated by ultra performance liquid chromatography and characterized by ion trap mass spectrometry, employing isotopic data-dependent scanning. Analysis of the tryptic or tryptic/chymotryptic digests of SA modified with NO₂-PhIP revealed that adduction occurred at Cys³⁴, Lys¹⁹⁵, Lys¹⁹⁹, Lys³⁵¹, Lys⁵⁴¹, Tyr¹³⁸, Tyr¹⁵⁰, Tyr⁴⁰¹, and Tyr⁴¹¹, whereas the only site of HONH-PhIP adduction was detected at Cys³⁴. N-Acetoxy-PhIP, a penultimate metabolite of PhIP that reacts with DNA to form covalent adducts, did not appear to form stable adducts with SA; instead, PhIP and 2-amino-1-methyl-6-(5-hydroxy)-phenylimidazo[4,5-b]pyridine, an aqueous reaction product of the proposed nitrenium ion of PhIP, were recovered during the proteolysis of N-acetoxy-PhIP-modified SA. Some of these SA adduction products of PhIP may be implemented in molecular epidemiology studies to assess the role of well-done cooked meat, PhIP, and the risk of cancer.

Dataset Information

Mass Spectrometric Characterization of an Acid-Labile Adduct Formed with 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and Albumin in Humans.

Publications

Mass Spectrometric Characterization of an Acid-Labile Adduct Formed with 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and Albumin in Humans.

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