Dataset Information

Biomonitoring of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and its carcinogenic metabolites in urine.

ABSTRACT: 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a carcinogenic heterocyclic aromatic amine that is produced in cooked meats. The simultaneous analysis of PhIP and its metabolites in human urine is a challenge, because these biomarkers only occur in urine at parts per billion or lower concentrations and must be selectively purifed from thousands of other urinary constituents. We have developed a facile solid-phase extraction method, employing a mixed-mode reverse-phase cation exchange resin, to simultaneously isolate PhIP, its glucuronide conjugates, and the glucuronide conjugates of the genotoxic metabolite 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine from the urine of meat eaters. PhIP and its metabolites were quantified by liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI/MS/MS), using a triple stage quadrupole mass spectrometer in the selected reaction monitoring scan mode. The lower limit of quantification (LOQ) of PhIP is 5 parts per trillion (ppt), and the LOQ values for the glucuronide conjugates are 50 ppt, when 25 microL of urine is employed for assay. The extraction scheme is versatile and has been employed to isolate other ring-hydroxylated and glucuronidated metabolites of PhIP, for characterization by LC-ESI/MS/MS.

SUBMITTER: Fede JM

PROVIDER: S-EPMC3215276 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Biomonitoring of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and its carcinogenic metabolites in urine.

Fede Jean-Marie JM Thakur Anup P AP Gooderham Nigel J NJ Turesky Robert J RJ

Chemical research in toxicology 20090601 6

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a carcinogenic heterocyclic aromatic amine that is produced in cooked meats. The simultaneous analysis of PhIP and its metabolites in human urine is a challenge, because these biomarkers only occur in urine at parts per billion or lower concentrations and must be selectively purifed from thousands of other urinary constituents. We have developed a facile solid-phase extraction method, employing a mixed-mode reverse-phase cation exchange r ...[more]

PMID: 19441775

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Project description: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.

Project description:Background2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), a heterocyclic aromatic amine (HCA) formed in meat that is cooked at high temperatures and then ingested, can potentially be retained in human adipose tissues.MethodsTo determine if PhIP is bioactive in the adipocyte, we exposed a human adipocyte cell line,HepG2 and Caco-2 cells to low dose PhIP. Uptake and retention of PhIP was determined and cytotoxicity was assessed by the TUNEL assay. Relative expression of PhIP-activating genes (CYP1A1, CYP1A2, SULT1A1 and UGT1A1) was determined by RT-PCR and global expression changes were also examined.ResultsThe percent retention of 0.1 μCi [(14)C]-PhIP over a 24 h period was significantly higher in the adipocyte than the HepG2 (p = 0.0001) and Caco-2 (p = 0.0007) cell lines. Cytotoxicity rates were 14.4 and 2.6 % higher compared to controls in Caco-2 and HepG2 cells (p < 0.001 and 0.054, respectively); no significant differences were detected in adipocyte cells (p = 0.18). Caco-2 and HepG2 cells, respectively, had significantly higher basal expression of CYP1A1 (p = 0.001, p = 0.003), SULT1A1 (p = 0.04, p < 0.001) and UGT1A1 (p < 0.001, p = 0.01) compared to the adipocyte. Exposure to 5nM PhIP did not significantly induce expression of these genes in any of the cell lines. Global gene expression analysis of mature adipocytes exposed to 5nM PhIP for 72 h resulted in statistically significant changes in 8 genes (ANGPTL2, CD14, CIDEA, EGR1, FOS, IGFBP5, PALM and PSAT1). Gene-gene interaction and pathway analysis indicates that PhIP modulates genes controlled by the STAT3 transcriptional factor and initiates leptin signaling via the JAK/STAT and MAPK pathway cascades. Early growth response 1 (EGR1) and prostaglandin synthase 2 (COX-2) were down-regulated via c-Fos, while insulin binding protein 5 (IBP5) was up regulated. Expression of transcription factors (ANGPTL2, HP, LEP, SAA1, SAA2), genes related to inflammation (SAA1, LEP), diabetes (IGFBP5) and cancer risk (SAA2) were also elevated upon exposure to 5 nM PhIP..ConclusionsPhIP mediates gene expression changes within the adipocyte, and the pathways most affected are related to cancer and other chronic diseases. Further studies are needed on the relationship between dietary carcinogens such as PhIP with cancer, obesity and diabetes.

Dataset Information

Biomonitoring of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and its carcinogenic metabolites in urine.

Publications

Biomonitoring of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and its carcinogenic metabolites in urine.

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