Dataset Information

Mass spectrometric characterization of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine N-oxidized metabolites bound at Cys34 of human serum albumin.

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 and poultry. PhIP is a carcinogen in rodents and a potential human carcinogen. Several short-term biomarkers of PhIP have been established for human biomonitoring, but validated long-term biomarkers of the biologically effective dose of PhIP remain to be developed. Metabolites of PhIP have been reported to covalently bind to human serum albumin (SA), which is the most abundant protein in plasma; however, the chemical structures of PhIP-SA adducts are unknown. Cysteine(34) is one of 35 conserved Cys residues in SA across species. Thirty-four of these Cys are involved in 17 disulfide bonds. The single unpaired Cys(34) residue in SA is well-known to react with carcinogenic metabolites and toxic electrophiles. 2-Nitro-1-methyl-6-phenylimidazo[4,5-b]pyridine (NO(2)-PhIP), 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP), and 2-nitroso-1-methyl-6-phenylimidazo[4,5-b]pyridine (NO-PhIP), three genotoxic metabolites of PhIP, were reacted with purified human SA or human plasma, and the SA adduction products, following enzymatic digestion, were separated by ultra performance liquid chromatography and characterized with a linear quadrupole ion trap mass spectrometer. The major adduct of NO(2)-PhIP was formed at the Cys(34) of SA with bond formation occurring between the sulfhydryl group of Cys and the C-2 imidazole atom of PhIP. The major adducts formed between SA and HNOH-PhIP or NO-PhIP were identified as acid-labile sulfinamide linkages at Cys(34). These PhIP-SA adducts represent a measure of bioactivation of PhIP and may serve as long-term biomarkers of the biologically effective dose of PhIP.

SUBMITTER: Peng L

PROVIDER: S-EPMC3221794 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Mass spectrometric characterization of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine N-oxidized metabolites bound at Cys34 of human serum albumin.

Peng Lijuan L Turesky Robert J RJ

Chemical research in toxicology 20111004 11

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic aromatic amine that is formed during the cooking of meats and poultry. PhIP is a carcinogen in rodents and a potential human carcinogen. Several short-term biomarkers of PhIP have been established for human biomonitoring, but validated long-term biomarkers of the biologically effective dose of PhIP remain to be developed. Metabolites of PhIP have been reported to covalently bind to human serum albumin (SA), which is the mos ...[more]

PMID: 21916490

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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 cysteine³? (Cys³?); 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-[²H?]-phenylimidazo[4,5-b]pyridine ([²H?]-PhIP). The metabolites used for study were 2-nitro-1-methyl-6-phenylimidazo[4,5-b]pyridine (NO?-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 NO?-PhIP revealed that adduction occurred at Cys³?, Lys¹??, Lys¹??, Lys³?¹, Lys??¹, Tyr¹³?, Tyr¹??, Tyr??¹, and Tyr?¹¹, whereas the only site of HONH-PhIP adduction was detected at Cys³?. 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.

Project description:More than 85% of breast cancers are sporadic and attributable to long-term exposure to environmental carcinogens, such as those in the diet, through a multistep disease process progressing from non-cancerous to premalignant and malignant stages. The chemical carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is one of the most abundant heterocyclic amines found in high-temperature cooked meats and is recognized as a mammary carcinogen. However, the PhIP's mechanism of action in breast cell carcinogenesis is not clear. Here, we demonstrated, for the first time, that cumulative exposures to PhIP at physiologically achievable, pico to nanomolar concentrations effectively induced progressive carcinogenesis of human breast epithelial MCF10A cells from a non-cancerous stage to premalignant and malignant stages in a dose- and exposure-dependent manner. Progressive carcinogenesis was measured by increasingly- acquired cancer-associated properties of reduced dependence on growth factors, anchorage-independent growth, acinar-conformational disruption, proliferation, migration, invasion, tumorigenicity with metastasis and increased stem-like cell populations. These biological changes were accompanied by biochemical and molecular changes, including upregulated H-Ras gene expression, extracellular signal-regulated kinase (ERK) pathway activation, Nox-1 expression, reactive oxygen species (ROS) elevation, increased HIF-1?, Sp1, tumor necrosis factor-?, matrix metalloproteinase (MMP)-2, MMP-9, aldehyde dehydrogenase activity and reduced E-cadherin. The Ras-ERK-Nox-ROS pathway played an important role in not only initiation but also maintenance of cellular carcinogenesis induced by PhIP. Using biological, biochemical and molecular changes as targeted endpoints, we identified that the green tea catechin components epicatechin-3-gallate and epigallocatechin-3-gallate, at non-cytotoxic doses, were capable of suppressing PhIP-induced cellular carcinogenesis and tumorigenicity.

Dataset Information

Mass spectrometric characterization of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine N-oxidized metabolites bound at Cys34 of human serum albumin.

Publications

Mass spectrometric characterization of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine N-oxidized metabolites bound at Cys34 of human serum albumin.

Similar Datasets

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