title: Mapping serum albumin adducts of the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by data-dependent tandem mass spectrometry.
abstract: 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.
authors: Division of Environmental Health Sciences, Wadsworth Center, New York State Department of Health , Albany, New York 12201, USA.
authors: Lijuan
authors: L
authors: Peng
authors: Surendra
authors: S
authors: Dasari
authors: David L
authors: DL
authors: Tabb
authors: Robert J
authors: RJ
authors: Turesky
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002273:Carcinogens / Q000737:chemistry / Q000378:metabolism*; D003296:Cooking; D004150:Dipeptidases / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000737:chemistry / Q000378:metabolism*; D007931:Leucyl Aminopeptidase / Q000378:metabolism; D008460:Meat / Q000032:analysis; D008969:Molecular Sequence Data; D009593:Nitroimidazoles / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D011402:Pronase / Q000378:metabolism; D011725:Pyridines / Q000737:chemistry / Q000378:metabolism; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism
chemical_list: C096092:2-(acetoxyamino)-1-methyl-6-phenylimidazo(4,5-b)pyridine; D002273:Carcinogens; D007093:Imidazoles; D009593:Nitroimidazoles; D011725:Pyridines; D012709:Serum Albumin; C060533:2-hydroxyamino-1-methyl-6-phenylimidazo(4,5-b)pyridine; C069031:2-nitro-1-methyl-6-phenylimidazo(4,5-b)pyridine; C049584:2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine; D007931:Leucyl Aminopeptidase; D004150:Dipeptidases; C022449:proline dipeptidase; D014357:Trypsin; D011402:Pronase