| Objective: Ochratoxin A (OTA) is a widespread mycotoxin mainly produced by the Penicillium verrucosum, Aspergillus ochraceus and A.carbonarius. Previous studies have found that OTA is a frequently existed contaminant in a wide range of food and its products (cereal, corn and wheat, etc). Because of its long half-time, the residue of OTA can be detected in the blood serum, bile and urine of either humans or animals consuming contaminated food. According to numerous researches, OTA exhibits a wide range of toxic activities including nephrotoxicity, hepatotoxicity, neurotoxicity, immunotoxicity, teratogenecity, mutagenicity and carcinogenicity. OTA had been classified as"possibly carcinogenic to humans"by the International Agency for Research on Cancer since 1993.We demonstrated that in the mid south part of Hebei Province such as Ci and Zanhuang County, the incidence of gastric and esophageal carcinoma is very high. In 2006, we conducted an on-site investigation on the OTA contamination in the two areas, the detectable rate of OTA in wheat was 37.70%, with the lowest content of 0.23μg/kg and the highest content of 14.25μg/kg, both were markedly higher than the provisional tolerable weekly intake (PTWI, 100 ng/kg), which was regulated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA).These detections further verified that the exposure of OTA was very common in these areas. Brought together the possible carcinogenicity as well as the high contamination in areas with high incidence of gastric and esophageal carcinoma, we hypothesize OTA contamination might be involved in the prevalence of the two carcinomas.DNA is the most important genetic material in the life activities of the body. Chromosome is the carrying agent of the genetic material, being made up of a double helix structure DNA chain and the histone. As the chief target of the carcinogen, DNA can be injured directly or indirectly. Consequently, DNA appears a permanent change in nucleotide sequence during replication, and lead to chromosomal aberration with the consequence of the formation of tumor. Up to now, there was no available document accounting for the influence of OTA on DNA and chromosome of gastric epithelial cells and esophageal epithelial cells.In the current study, we explored the cytotoxic mechanism of OTA in human gastric epithelial line (GES-1) and esophageal mucosal epithelial cells (Het-1A). First, the effects of OTA treatment on the DNA damage in GES-1 cells and Het-1A cells were examined via single-cell gel electrophoresis (comet assay). Moreover, the effects of OTA treatment on chromosome damage in GES-1 cells and Het-1A cells were investigated through chromosome karyotype analysis. The key point of this study is to provide a scientific basis on the putative toxicity even carcinogenic effects of OTA on stomach and esophagus, and it plays a vital role in elevating the food security of rural residents, especially the residents of high prevalence area of gastric and esophageal carcinoma in China.Methods:1 Cell culture and treatment1.1 GES-1 Cell culture and treatmentGES-1 cells were grown in DMEM medium supplemented with 10% new-born calf serum (NBCS), streptomycin (100μg/ml), penicillin (100 U/ml) in an atmosphere of 5% CO2, 95% air at 37℃.GES-1 cells in logarithmic growth phase were randomly divided into five groups: solvent control group, 2.5μmol/L, 5μmol/L, 10μmol/L and 20μmol/L. The cells in solvent control group were treated with methanol (all in same volume). In addition, the 10μmol/L OTA group was treated at different time points, which were 6 h, 12 h, 24 h and 36 h, after the corresponding time in the experiment, collected cells and detected related indicators. 1.2 Het-1A Cell culture and treatmentHet-1A cells were grown in 1640 medium supplemented with 10% new-born calf serum (NBCS), streptomycin (100μg/ml), penicillin (100 U/ml) in an atmosphere of 5% CO2, 95% air at 37℃.Het-1A cells in logarithmic growth phase were randomly divided into five groups: solvent control group, 2.5μmol/L, 5μmol/L, 10μmol/L and 20μmol/L. The cells in solvent control group were treated with methanol (all in same volume). In addition, the 10μmol/LOTA group was treated at different time points, that were 6 h, 12 h, 24 h and 36 h, after the corresponding time in the experiment, collected cells and detected related indicators.2 comet assay2.1 Comet assay was used to observe the change of Tail DNA%, Tail Length, Olive Tail Moment of GES-1 cells and Het-1A cells after treated with OTA for different concentrations (2.5μmol/L, 5μmol/L, 10μmol/L, 20μmol/L).2.2 Comet assay was used to observe the change of Tail DNA%, Tail Length, Olive Tail Moment of GES-1 cells and Het-1A cells after treated with OTA for different time (6 h, 12 h, 24 h, 36 h).3 Chromosome karyotype analysis3.1 Chromosome karyotype analysis was used to observe the chromosome aberration in GES-1 cells and Het-1A cells treated with different concentration of OTA for 24 h.3.2 Chromosome karyotype analysis was used to further observe chromosome aberration in GES-1 cells and Het-1A cells after treated with 10μmol/L OTA for different time (6 h, 12 h, 24 h, 36 h).Result:1 Effect of OTA on DNA damage in GES-1 cells1.1 The concentration-effect of OTA on DNA damage in GES-1 cellsThe results of Comet assay showed that the comet rates were increased significantly in all the OTA treatment groups at 24hs when compared to that in the solvent control group, respectively(P<0.05). Further analysis revealed that the comet rate was getting increased as the concentration of OTA ranging from 2.5μmol/L to 20μmol/L, indicating a statistical concentration dependent manner(r=0.954, P<0.05).Then, the difference in Tail DNA%, Tail Length and Olive Tail Moment of the comets was analysed further between the OTA treatment groups and the control group. The value of comet Tail DNA% was significantly higher in each OTA treated groups than that in the solvent control group (P<0.05). Moreover, the value of comet Tail DNA% was increased within the concentration of OTA increasing from 2.5 to 20μmol/L (Tail DNA%: r=0.887, P<0.05). Consistant with the change of comet Tail DNA, Tail Length and Olive Tail Moment presented the same effect after OTA treatment in a concentration dependent manner (Tail Length: r=0.912, P<0.05; Olive Tail Moment: r=0.942, P<0.05).1.2 The time-effect of 10μmol/L OTA on DNA damage in GES-1 cellsThe results of Comet assay showed that the comet rates were increased significantly in a time-dependent manner from 6 h to 36 h (r=0.977, P<0.05).Then, the value of comet Tail DNA%, Tail Length and Olive Tail Moment was getting increased as the time of OTA treatment ranging from 6 h to 36 h. And there was a significant time-dependent manner (Tail DNA%: r=0.936, P<0.05; Tail Length: r=0.955, P<0.05; Olive Tail Moment: r=0.976, P<0.05).2 Effect of OTA on DNA damage in Het-1A cells2.1 The concentration-effect of OTA on DNA damage in Het-1A cellsIn comparison with the solvent control, the comet rates were increased significantly in the OTA treatment at different concentrations for 24 h. Results from concentration-dependent studies revealed that OTA could increase the comet rate in Het-1A cells in a concentration-dependent manner (r=0.968, P<0.05).The value of comet Tail DNA%,Tail Length and Olive Tail Moment was significantly higher in each OTA treated group than that in the solvent control group in a concentration-dependent manner (Tail DNA%: r=0.909, P<0.05; Tail Length: r=0.887, P<0.05; Olive Tail Moment: r=0.995, P<0.05). 2.2 The time-effect of 10μmol/L OTA on DNA damage in Het-1A cellsThe results of Comet assay showed that the comet rates were increased significantly in a time-dependent manner from 6 h to 36 h, when compared to that in the solvent control group (r=0.977, P<0.05).As the time of OTA treatment ranging from 6 h to 36 h, the value of comet Tail DNA%, Tail Length and Olive Tail Moment was significantly higher in each OTA treated group than that in the solvent control group (P<0.05). And there was a significant time-dependent manner (Tail DNA%: r=0.959, P<0.05; Tail Length: r=0.977, P<0.05; Olive Tail Moment: r=0.968, P<0.05)3 Effect of OTA on chromosome karyotype in GES-1 cells3.1 The concentration-effect of OTA on chromosome karyotype in GES-1 cellsChromosome karyotype analysis showed that the number of different chromosomal structural aberrations (break, gap, dicentric, and ring) and chromosomal quantitative aberrations (polyploidy) were induced in 2.5μmol/L, 5μmol/L, 10μmol/L OTA treated groups, respectively, with the highest number seen in 10μmol/L OTA treatment group (P<0.05).3.2 The time-effect of 10μmol/L OTA on chromosome karyotype in GES-1 cellsThe total frequency of the chromosomal structural aberrations was induced after 10μmol/L OTA treatment for 6 h, 12h, 24 h and 36 h, respectively. Further analysis confirmed that the number of chromosomal structural aberrations and chromosomal quantitative aberrations were all increased compared with that in solvent control group (P<0.05).4 Effect of OTA on chromosome karyotype in Het-1A cells4.1 The concentration-effect of OTA on chromosome karyotype in Het-1A cellsThe total number of chromosomal structural aberrations were 9.00%, 9.00%, 10.00% and 15.00% in 2.5μmol/L, 5μmol/L, 10μmol/L and 20μmol/L OTA treated groups respectively, which were all significantly higher than that in solvent control group ( 2.00%, P<0.05). 4.2 The time-effect of 10μmol/L OTA on chromosome karyotype in Het-1A cellsThe number of different chromosomal structural aberrations (break, gap, dicentric, and ring) and chromosomal quantitative aberrations (polyploidy) were increased compared with that in solvent control group from 6 h to 36 h (P<0.05).5 Comparision of DNA and chromosome damage induced by OTA in GES-1 cells and Het-1A cellsIn GES-1 cells and Het-1A cells, the comet rate, comet Tail DNA, Tail Length and Olive Tail Moment were significantly higher in OTA treated cells than the control cells, which may suggest that there was no significant difference between GES-1 cells and Het-1A cells in the effect of OTA on DNA damage. But low doses of OTA (2.5μmol/L, 5μmol/L) could only induce chromosome aberration in Het-1A cells, it showed that Het-1A cells were more sensitive than GES-1 cells.Conclusion:1 OTA could induce DNA injury in GES-1 cells and Het-1A cells in vitro.2 OTA could induce chromosome damage in GES-1 cells and Het-1A cells in vitro.3 There was no significant difference between GES-1 cells and Het-1A cells in the effect of OTA on DNA damage. But in Het-1A cells, chromosome aberration could be induced at lower doses of OTA. It showed that Het-1A cells were more sensitive to OTA than GES-1 cells. |
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