| Heterocyclic amines (HCAs) are a kind of low molecular organic amine compounds, which are formed in the pyrolysis process under high temperature. HCAs from food source are widely in the cooking meats, they also exist in our environment. Presently more than20kinds of HCAs have been identified.2-amino-l-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) is accompanied with the typical features of larger toxicity, easy absorption, and more tissue distribution one of the main compositions of the HCAs chemicals. The research shows that HCAs has mutagenicity, and its intake has a relationship with many diseases (such as gastric cancer, colorectal cancer, breast cancer and so on). However, molecular mechanisms of PhlP-induced gastric injury and gastric cancer are not very clear, p16is an important tumor suppressor gene, and methylation of p16gene promoter may inhibit its protein expression. This process causes p16protein inactivation, leading to the excessive proliferation of tumor cells. This pathway may be an important mechanism of p16protein inactivation in the development of gastric cancer. p16gene aberrant methylation is related to gastric mucosa dysplasia carcinoma, it is regarded as the specific early warning biomarker. Therefore, p16is used as a biological marker of rat gastric injury in early stage induced by HCAs in the present study.In this study, the healthy adult and clean grade Wistar rats were divided randomly into four equal groups of five animals each:(1) the control group (55%ethanol-saline, PH4.5),(2)5mg/kg body weight Ph1P group,(3)10mg/kg body weight Ph1P group, and (4)15mg/kg body weight Ph1P group. Different dose Ph1P or55%ethanol-saline were administrated to rats with5.0ml/kg body weight dose once by gavage. After24h, the stomach tissues from rats were used as experimental materials to analyze histopathological change, lipid peroxidation, protein oxidation damage, p16mRNA and protein expression as well as p16promoter methylation induced by Ph1P using the microscope technique, real time fluorescent quantitative RT-PCR, western blot, methylation specific PCR (MSP) and biochemical analysis method. The stomach histopathologic examination through HE staining methods were performed; superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) activities and malondialdehyde (MDA) contents were measured in stomach tissues from rat; protein carbonyl (PCO) contents and DNA-protein crosslinks (DPC) coefficients were measured in stomach tissues from rats.The experimental results show:(1) The gastric mucosa in the control group rats were normal, glands arranged neatly, with no obvious abnormalities and no inflammatory cell infiltration. Different degree of pathological changes of stomach tissues from rats exposed to PhIP were observed, especially15mg/kg PhIP induced inflammatory cell infiltration, gland damage and hyperemia phenomenon in gastric mucosa of rats.(2) PhIP induced the changes of SOD, CAT, GSH-Px enzyme activities of stomach tissues in different group rats. High dose of PhIP (15mg/kg) significantly inhibited the enzyme activities of SOD and GSH-Px, while PhIP at the doses of10mg/kg and15mg/kg markedly induced the CAT activity compared with the control group (P<0.05). With the elevated doses of PhIP, the MDA contents were significantly increased in a dose-dependent manner. PhIP at the doses of10mg/kg and15mg/kg induced the MDA contents compared with the control group (P<0.05)(3) PhIP with different doses significantly increased PCO contents and DPC coefficients compared with the control groups (P<0.05, P<0.01) Moreover, the PCO contents and DPC coefficients in the tissues of rats showed a dose-dependent manner with the elevated concentrations.(4) PhIP with different doses significantly decreased p16mRNA expressions in the stomachs of rats, and PhIP at the doses of10mg/kg and15mg/kg markedly inhibited p16protein levels compared with the control group (P<0.05, P<0.01). With the elevated doses of PhIP, the p16gene expressions were down-regulated in a dose-dependent manner. In addition, high doses of PhIP (15mg/kg) induced p16gene DNA promoter methylation in the stomachs of rats. The results show that PhIP poisonous induced histopathological changes in rat stomach tissues, and caused stomach tissue lipid peroxidation and protein oxidative damage. The pathology changes may be related to the oxidative damage in the stomach tissues of rats induced by PhIP. At the same time, PhIP significantly inhibited p16gene expressions and caused p16gene DNA promoter methylation in the stomachs of rats exposed to high doses of PhIP (15mg/kg). p16genes promoter methylation may be an important cause in p16protein inactivation, which may increase the potential risk of initiation of stomach cancer. These results indicate that PhIP can induce the toxic damage effects in the stomach tissue of rats. In the present paper, the molecular mechanisms of PhIP on histopathological changes, lipid peroxidation and protein oxidation injury, downregulation of tumor-suppressor gene p16expression in rat stomach tissues were investigated in order to indicate early toxic injury mechanism of PhIP. These results not only can provide the experimental evidences for understanding the early toxic injury mechanism of PhIP on stomach tissues, but also have positive significance to advocate people to reasonable diet and protect people health.It has been shown that when PhIP enter the body, it can produce oxygen radicals, which may be an important mechanism of Ph1P-induced gastric tissue damage. The work about the mechanism of PhIP on gastric damage and stomach cancer needs to be further studied. |
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