| Ochratoxin A (OTA), a mycotoxin mainly produced by ubiquitous Aspergillus and Penicillium, is one of the most abundant food-contaminating mycotoxins, particularly in cereals, grains and beans, as well as coffee, grape juice, beer and bread. OTA exhibits a wide range of toxic activities including nephrotoxicity, hepatotoxicity, neurotoxicity, immunotoxicity, teratogenecity, mutagenicity and carcinogenicity. Accordingly, OTA had been classified as"possibly carcinogenic to humans"by the International Agency for Research on Cancer since 1993.Our previous epidemiological study have showed that Zanhuang County of Hebei Province is a high incidence area of gastric carcinoma in China, where the average annual mortality rate of gastric cancer has been over 59 per 100,000 population. Our on-site investigations further confirmed that the OTA contamination in wheat was quite common in this area, and the highest content reached to 2.41μg/kg which was significantly higher than that of provisional tolerable weekly intake (PTWI, 100 ng/kg) defined by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). The high level of OTA contamination in wheat might be related to the high incidence of gastric carcinoma in rural area of China. However, there were no available data regarding the putative gastric toxicity of OTA up to now.In this study, we explored the effects of OTA treatment on the proliferation, apoptosis, cell cycle distribution and chromosome in a human gastric epithelial cell line (GES-1). First, the effects of OTA treatment on the cell cycle distribution, proliferation and apoptosis in GES-1 cells were studied. Furthermore, the effects of OTA treatment on chromosome damage in GES-1 cells were investigated. Finally, based on the results above, the changes of ATM-Chk2 and ATM-p53 signaling pathway, which was closely related to chromosome damage and cell cycle arrest, were studied to explore the putative molecular mechanism of OTA-induced G2 arrest and cell damage in GES-1 cells. The key point of this study is to provide some clues on the putative gastric toxicity and carcinogenic effects of OTA, and to improve the food security of rural residents, especially the residents of high incidence area of gastric cancer in China.Partâ… Effects of ochratoxin A on cell cycle distribution, proliferation and apoptosis in GES-1 cells in vitroObjective: To explore effects of ochratoxin A on cell cycle distribution, proliferation and apoptosis in human gastric epithelium cells (GES-1) in vitro.Methods: 1 Cell survival rates was tested after different concentration of OTA(0.01100μmol/L)treatment for 24 h by MTT assay. 2 The distribution of cell cycle after OTA treatment was analyzed with FCM, Giemsa staining and immunofluoresence staining. 3 The key factors of cell cycle transmission, such as Cdc25C, Cdc2 and CyclinB1 were detected both at mRNA and protein level by Western Blot and RT-PCR respectively. 4 The effect of OTA on the Cdc2-CyclinB1 complex was detected by immunoprecipitation. 5 The apoptosis induced by OTA was measured by flow cytometry using annexin V-FITC and propodium iodide staining, and the activity of apoptosis related factor Caspase-3 was determined with Western Blot.Results:1.1 Effects of OTA on the survival rates in GES-1 cells in vitroMTT assay showed that OTA had a potent inhibitory effect on the growth of GES-1 cells in vitro (P<0.05). The cell survival rates in OTA treated groups displayed a significant decrease in a dose-dependent manner ranging from 1 to 100μmol/L (r= -0.916, P<0.05), and the IC50 value was 67.72μmol/L.1.2 Effects of OTA on cell cycle distribution of GES-1 cells in vitroFlow Cytometric analysis showed that the proportions of cells in G2/M phase in 10μmol/L and 20μmol/L OTA treatment groups were significantly increased as compared with that in solvent control group (P<0.05). Giemsa staining results showed that the mitotic index (MI) was decreased in 20 μmol/L OTA treatment group with MI being 2.42±0.87%, which were significant lower than that in solvent control group (4.96±0.70%, P<0.05). Immunofluorescence results showed that the number of sensitive mitotic cell marker Ser-10 phosphorylated histone H3 positive cells in 20μmol/L OTA treatment cells was significantly decreased as compared with that in solvent control group. In conclusion, these results confirmed OTA could induce G2 phase arrest in GES-1 cells.1.3 Effects of OTA on G2 phase key regulatory factor in GES-1 cells in vitroWestern Blot analysis showed that the expression of the G2 phase key regulatory proteins-Cdc25C, Cdc2, and CyclinB1 was significantly decreased after 10μmol/L and 20μmol/L OTA treatment. Similarly, the phosphorylation level of Cdc25C and Cdc2 was also decreased at OTA treatment groups. Immunoprecipitation results suggested that OTA could induce the decrease of Cdc2-CyclinB1 complex. RT-PCR analysis showed that the expression of Cdc25C, Cdc2 and CyclinB1 mRNA in 10μmol/L and 20μmol/L OTA treatment groups were significantly decreased as compared with that in solvent control group (P<0.05).1.4 Effects of OTA on cell apoptosis in GES-1 cells in vitroThe PI staining result showed that the apoptosis rate in 10μmol/L and 20μmol/L OTA treatment groups (16.11±2.51% and 21.27±1.23%, respectively) were significantly increased as compared with that in solvent control group (6.15±1.93%, P<0.05). Annexin V-PI staining showed that the apoptosis percentage of cells, including early and late apoptotic cells, were increased significantly in different OTA treatment groups as compared with that in solvent control group. Western Blot analysis showed that OTA could activate Caspase-3. The cleaved (activated) form of Caspase-3 could only be seen in OTA treated groups.Partâ…¡Effects of ochratoxin A on chromosome damage in GES-1 cells in vitroObjective: To explore the effect of OTA on chromosome in human gastric epithelium cells (GES-1) in vitro.Methods: 1 The situation of chromosome damage was measured by micronucleus test after different concentration OTA treatment (5, 10, 20μmol/L) for 24 h. 2 The type of chromosome aberration was further observed by chromosome karyotype analysis after OTA treatment.Results:2.1 Effect of OTA on micronucleus rate in GES-1 cells The micronucleus test result showed that the micronucleus rate in 10μmol/L and 20μmol/L OTA treatment groups was 2.90±0.54% and 3.84±1.06% respectively, which were significantly higher than that in solvent control group (1.23±0.27%, P<0.05). The results suggested that OTA could induce chromosome damage in GES-1 cells.2.2 Effect of OTA on chromosome karyotype in GES-1 cells Chromosome karyotype analysis showed that the number of different chromosomal structural aberrations (dicentric, gap, break, ring and deletion) and chromosomal quantitative aberrations (polyploidy) in 20μmol/L and 50μmol/L OTA treated groups were increased as compared with that in solvent control group, with the highest number seen in 50μmol/L OTA treatment group. The total frequencies of aberrations was 17% and 26% in 20μmol/L and 50μmol/L OTA treatment groups respectively, which were all significantly higher than that in solvent control group (7%, P<0.05). The result suggested that the higher concentration of OTA could induce chromosome aberration in GES-1 cells.Partâ…¢Studies on the putative molecular mechanisms of G2 Arrest in GES-1 cells induced by ochratoxin A in vitroObjective: To explore the possible molecular mechanisms on OTA induced G2 phase arrest in human gastric epithelium cells (GES-1) in vitro.Methods: 1 The expression of related molecules involved in the ATM-Chk2 and ATM-p53 signaling pathway were analyzed in OTA treatment cells with or without the ATM specific inhibitor-caffeine pretreatment using Western Blot and Real-time RT-PCR. 2 The key factors of cell cycle transmission including Cdc25C, Cdc2 and CyclinB1 and the cell cycle distribution were studied in OTA treatment cells with and without ATM specific inhibitor pretreatment.Results:3.1 Effects of OTA on ATM-Chk2 signaling pathway in GES-1 cellsFirstly, we found that ATM mRNA expression was significantly increased by 10μmol/L and 20μmol/L OTA treatment groups as compared with that in control group by Real-time RT-PCR analysis (P<0.05). Simultaneously, the level of p-ATM was up-regulated dramatically after OTA treatment at different concentrations (P<0.05). In addition, the Chk2 showed the same change as ATM kinase by OTA treatment. The results showed that ATM-Chk2 signaling pathway could be activated by OTA treatment in GES-1cells.We further explored the effect of ATM-Chk2 signaling pathway after OTA treatment with or without the ATM specific inhibitor-caffeine pretreatment. The results showed that the level of p-ATM and p-Chk2 in caffeine pretreatment group was decreased significantly as compared with that in OTA treatment without caffeine pretreatment (P<0.05), while the expression of ATM and Chk2 at the protein level in caffeine pretreatment group was higher than that in OTA treatment groups (P<0.05). The results suggested that caffeine could inhibit OTA-induced activation of ATM-Chk2 signaling pathway.3.2 Effects of OTA on ATM-p53-p21WAF1/CIP1 signaling pathway in GES-1 cellsWestern Blot results showed that both the level of p-p53 and the expression of p21WAF1/CIP1 were increased in OTA treatment at different concentrations for 24 h as compared with that in solvent control group (P<0.05). It suggested that OTA could active the p53-p21WAF1/CIP1 signaling pathway.To clarify that ATM was the upstream regulatory for the p53-p21WAF1/CIP1 signaling pathway, we observed the level of p-p53 and the expression of p21WAF1/CIP1 after the ATM specific inhibitor-caffeine pretreatment. The results showed that the level of p-p53 in caffeine pretreatment group was significantly decreased as compared with that in OTA treatment alone (P<0.05). The expression of p21WAF1/CIP1 also showed the same change as p-p53. Thus, it suggested that caffeine could inhibit OTA-induced the activation of p53-p21WAF1/CIP1 signaling pathway, and ATM was involved in the activation of p53-p21WAF1/CIP1 signaling pathway by OTA treatment.3.3 Effects of OTA on the key regulatory factors of G2 phase in GES-1 cells, with caffeine pretreatmentThe results of Western Blot showed that the expression of Cdc25C, Cdc2 and CyclinB1 in caffeine pretreatment group was significantly increased as compared with that in OTA treatment alone (P<0.05). Meanwhile, the level of p-Cdc25C and p-Cdc2 in caffeine pretreatment group were also significantly up-regulated as compared with that in cells with OTA treatment alone (P<0.05). The above data indicated that ATM specific inhibitor-caffeine pretreatment could reverse the inhibition of Cdc25C, Cdc2 and CyclinB1 expression by OTA.3.4 Effects of OTA on cell cycle distribution in GES-1 cells, with caffeine pretreatmentFlow Cytometric analysis showed that the proportion of cells in G2/M phase in caffeine pretreatment group was reduced as compared with that in 10μmol/L OTA treatment group (P<0.05). The results showed that caffeine pretreatment could inhibit OTA-induced G2 phase arrest in GES-1 cells.All the above data indicated that OTA could induce the activation of ATM-Chk2 and ATM-p53-p21WAF1/CIP1 signaling pathway, and these related proteins were involved in the process of G2 phase arrest by OTA treatment in GES-1 cells.Conclusions:1 OTA could induce GES-1 cell cycle to be arrested at G2 phase, and the reduced expressions of Cdc25C, Cdc2, CyclinB1 and Cdc2-CyclinB1 complex by OTA may contribute to the G2 phase arrest.2 The induction of apoptosis through the activation of Caspase-3, and the inhibition of proliferation of GES-1 cells in vitro might be the out comings of G2 phase arrest induced by OTA.3 OTA could induce chromosome damage in GES-1 cells in vitro.4 OTA could activate ATM-Chk2 and ATM-p53- p21WAF1/CIP1 signal pathway in GES-1 cells in vitro.5 The activation of ATM-Chk2 and ATM-p53-p21WAF1/CIP1 signaling pathways were all involved in the G2 arrest by OTA in GES-1 cells in vitro. This may be one of the putative mechanisms for OTA-induced chromosome damage and the cell cycle G2 phase arrest. |
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