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A comprehensive investigation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) metabolism in the mouse using a multivariate data analysis approach.

ABSTRACT: 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a potent rodent carcinogen and a potential human carcinogen because of its existence in the normal human diet. N2-OH-PhIP, a major PhIP metabolite, has been identified as a precursor of genotoxic species. In vitro data supported the view that CYP1A2 is the major enzyme responsible for the formation of N2-OH-PhIP. However, disruption of the CYP1A2 gene in mouse failed to inhibit PhIP-induced carcinogenesis. To investigate the mechanism underlying this observation, the metabolism of PhIP in wild-type, Cyp1a2-null, and CYP1A2-humanized mice was examined in detail using a metabolomic approach. Following data acquisition in a high-resolution LC-MS system, urinary metabolomes of the control and PhIP-treated mice were characterized in a principal component analysis (PCA) model. Comprehensive metabolite profiles of PhIP in high dose (10 mg/kg) and low dose (100 microg/kg) were established through analyzing urinary ions contributing to the separation of three mouse lines in the multivariate model and by measuring radiolabled PhIP metabolite in a radio-HPLC assay, respectively. The genotoxicity of PhIP to three mouse lines was evaluated by measuring DNA adduction levels in liver, lung, colon, and mammary gland. On the basis of the chemical identities of 17 urinary PhIP metabolites, including eight novel metabolites, multivariate data analysis revealed the role of CYP1A2 in PhIP metabolism and a human-mouse interspecies difference in the catalytic activity of CYP1A2. In addition, the results also showed that Cyp1a2-null mice still possess significant N2-hydroxylation and DNA adduction activities, which may be partially attributed to mouse CYP2C enzymes according to the results from in vitro microsome and Supersome incubations and antibody inhibition experiments.

SUBMITTER: Chen C

PROVIDER: S-EPMC1850849 | biostudies-literature | 2007 Mar

REPOSITORIES: biostudies-literature

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A comprehensive investigation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) metabolism in the mouse using a multivariate data analysis approach.

Chen Chi C Ma Xiaochao X Malfatti Michael A MA Krausz Kristopher W KW Kimura Shioko S Felton James S JS Idle Jeffrey R JR Gonzalez Frank J FJ

Chemical research in toxicology 20070206 3

2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a potent rodent carcinogen and a potential human carcinogen because of its existence in the normal human diet. N2-OH-PhIP, a major PhIP metabolite, has been identified as a precursor of genotoxic species. In vitro data supported the view that CYP1A2 is the major enzyme responsible for the formation of N2-OH-PhIP. However, disruption of the CYP1A2 gene in mouse failed to inhibit PhIP-induced carcinogenesis. To investigate the mechanism und ...[more]

PMID: 17279779

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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.

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

A comprehensive investigation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) metabolism in the mouse using a multivariate data analysis approach.

Publications

A comprehensive investigation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) metabolism in the mouse using a multivariate data analysis approach.

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