Hormesis Of Cell Proliferation In L929 Cells Stimulated By Low Dose MTBE

Objective:To observe whether hormesis of the cells proliferation could be stimulated by MTBE (methyl tert-butyl ether) in L929. Determine the hormetic zone and judge the health significance.Methods:1 Used the colorimetric MTT method to observe whether hormesis and adaptive response of the cells proliferation could be stimulated by MTBE in L929, and determine the hormetic zone and adaptability reaction dose of the cell proliferation.2 Used the hematoxylin-eosin staining method to observe the change of cells form stimulated by MTBE in L929.3 Used xanthine oxidase method to detect the activity of superoxide dismutase (SOD) stimulated by MTBE in L929.4 Used Single-Cell Gel Electrophoresis (SCGE) method to detect the DNA damage stimulated by MTBE in L929.5 Used KCl-SDS deposition method to detect the DNA-protein crosslinks stimulated by MTBE in L929.Results:1 In the MTT assay,the cells proliferation of two treatment groups(1.25g/L and 5g/L MTBE) are higher than that of the control group after exposed to MTBE 24h, displaying hormesis, but it can't be seen in 48,72h.The range of IC50 were 20g/L to 26g/L after exposed to MTBE 24h to 72h, it calculated that LD50 were 15.52g/kg～17.40g/kg. We also found that when L929 of the treatment groups exposed to MTBE in advance 9h and 12h,the cells proliferation of two treatment groups(0.039g/L and 0.078g/L MTBE) are higher than that of the positive group after exposed to a large doses of MTBE(25g/L) 24h, displaying adaptation reaction.2 In the hematoxylin-eosin staining assay, the treatment group of flushing dose (20g/L MTBE) appeared the cells form of apoptosis after exposed to MTBE 12h, and appeared the cells form of necrocytosis after exposed to MTBE 72h. The cells form of two treatment groups (1.25g/L and 5g/L MTBE) had a little change after exposed to MTBE 72h.3 In the xanthine oxidase assay, the relative activity of superoxide dismutase in the 20g/L are higher than that in the control group after exposed to MTBE 6h, and the relative activity of SOD in the 0.312g/L are higher than that in the control group after exposed to MTBE 9h. When we maked the logarithm of dosage and the relative activity of SOD in quadratic function curve fitting, it appeared a inverted U-shaped curve after exposed to MTBE 6h and 9h.4 In the Single-Cell Gel Electrophoresis (SCGE) assay, each treatment group (0.078～20g/L) developed DNA damage after exposed to MTBE 1h, and we observed a significant increase of DNA damage in dose-dependent manner. After exposed to MTBE 3h, the DNA damage of each treatment group reduced comparing with exposure 1h; After exposed to MTBE 6h, DNA damage of each group had repaired, and the DNA Damage of 5g/L group was lower than that of the control group, displaying hormesis; After exposed to MTBE 9h, DNA damage of each treatment group appeared again.5 In the KC1-SDS deposition assay, the DPC coefficient of the 20g/L treatment group is lower than that of the control group after exposed to MTBE 1h,6h and 9h; The DPC coefficient of other treatment groups had no difference with that of the control group.Conclusion:1 It is feasible to use MTT to measure hormesis of the cells proliferation.2 Hormesis of the cells proliferation and hormesis of DNA damage repair could be stimulated by MTBE in L929, both hormesis had a time-dependent effect. The two hormetic zone had a overlap section (5g/L), it hinted that hormesis of cell multiplication and hormesis of DNA damage repair existed a closed contact.3 The hormetic zone of the cells proliferation was not a safe doses range. It appeared DNA damage after exposed to MTBE in the hormetic zone of the cells proliferation 1h, although the DNA damage was repaired after exposed to MTBE 6h, but it was not a complere repair that there are different degree of DNA damage after exposed to MTBE 9h. And the cells suffered oxidative damage after exposed to MTBE bolow the hormetic zone of the cells proliferation 9h. The cells form changed after exposed to MTBE in the hormetic zone of the cells proliferation 72h,4 Adaptation reaction of the cells proliferation could be stimulated by MTBE in L929, and it had a time-dependent effect. Althoug in the range of adaptation reaction it didn't appear oxidative damage and the change of cell form after exposed to MTBE 9h and 72h, but it need more experiments to prove the safety of MTBE in this range.5 IC50 detected by the MTT method could be used to predict LD50.