The Adaptive Response Of The DNA Damage In The Body Induced By Pretreatment With Low Dose Of MTBE And The Role Of PARP-1 Protein In The Adaptive Response

Research Aim:To study the problem if low dose of methyl tertiary butyl ether(MTBE) can induce the adaptive response of DNA damage in the body caused by higher dose of MTBE,and how the dose-effect relationship is.And try to demonstrate the role of poly(ADP-ribose) polymerase-1(PARP-1) in the adaptive response.Methods:1.The MTT-test was used to detect the cytotoxic effect of MTBE in Chinese hamster lung cells(CHL cells),and the median inhibit concentration(IC₅₀) and no observed acute effect level(NOAEL) would confirmed.At the same time,to observe if the hormesis could be induced by MTBE in CHL cells and the pretreatment dose would determined by the hormetic zone.2.The adaptive response of the DNA damage in CHL cells induced by MTBE and its dose-response relationship were observed by the method of single cell gel electrophoresis(SCGE) technique.And 3-aminobenzamide(3-AB),which is a specific inhibitor of PARP,would added to the cultured CHL cells to observed whether the adaptive response of DNA damage induced by low dose of MTBE could be blocked.3.The adaptive response of the apoptosis in CHL cells induced by MTBE and its dose-response relationship were observed by the method of flow cytometry of apoptosis and cell cycle and whether it could be blocked by 3-AB.4.The expression of PARP-1 protein was tested with Western blotting test when CHL cells exposing to different dose of MTBE for 6 hours.5.The Kunming mice would expose to different dose of gaseous MTBE for 2 hours a day, 30 days,after that,high does of MTBE exposing for 2 hours,then the adaptive response of DNA damage in their leucocytes of blood and liver cells would be tested by SCGE.Result1.MTT test reveals that the median inhibit concentration(IC₅₀) of MTBE to CHL cell is 32.1g/L(95%CI is 27.6～38.7g/L) and the no observed active effect level(NOAEL) is 20.0g/L.The hormesis doesn't be observed in the test.2.MTBE cannot cause DNA damage in CHL cells when the dose is under 20.0g/L and the treatment time is 18 hours.And when the dose is higher than 30.0g/L,they can cause DNA damage.However,after CHL cells are treated with low dose(0.5g/L～2.5g/L) of MTBE for 6 hours,it can induce the adaptive response of DNA damage caused by 50.0g/L MTBE.3-AB can't block the adaptive response induced by low dose of MTBE when pretreatment is taking for 6 hours.While adaptive response can be blocked by 3-AB before the pretreatment has taken.3.The apoptosis and cell cycle of CHL cells will not be changed by MTBE when the dose is 1.0g/L.But the apoptosis is enhanced by hige dose of MTBE(over 35g/L,18 hours) and it can cause the cells' blockage in G₂ stage.The adaptive response of apoptosis can be induced by pretreating cells for 6 hours with low dose MTBE(0.5g/L～10.0g/L),and the cells' blockage in G₂ stage will be lightened.When the activity of PARP-1 is inhibited by 3-AB,the adaptive response of apoptosis is disappeared,but the cells are still blocked in G₂ stage.It indicate that 3-AB can affect the CHL cells' apoptosis,but little in cell cycle.4.Western blotting test reveals that the strap of PARP-1 protein 116KD molecular weight hasn't distinct when the CHL cells exposing to deffirent dose of MTBE for 6 hours.But the strap of degment of PARP-1 protein which molecular weight is 89KD is denser in the doses of 40.0g/L and 10.0g/L than other doses.It indicate that the degradation of PARP-1 protein in exposing to 40.0g/L or 10.0g/L MTBE are more than other doses.5.Afer exposing in 10 000mg/m³ MTBE for 2 hours one day,the DNA damage of Kunming mice' leucocytes of blood and liver cells(test by SCGE ) have taken place. While the mice pretreat with 126～1260 mg/m³ MTBE for 30 days,it can induce the deffirent level of adaptive response of the DNA damage in the mice' leucocytes of blood and liver cells.And the adaptive response in dose of 1260mg/m³ MTBE is obvious.Conclusion:1.The low dose pretreatment of MTBE which is between 0.5g/L and 2.5g/L can induce the adaptive response of cultured CHL cell to DNA damage caused by MTBE of 50.0g/L, and the pretreatment of 126～1260 mg/m³ MTBE can induce the adaptive response of DNA damage in Kunming mice' leucocytes of blood and liver cells.Accordingly,we conclude that low dose of MTBE hasn't genotoxicity to the body although it has genotoxicity in high dose.The genotoxicity of MTBE may be not the main risk for human.2.PARP-1 is a critical factor in the formation of the adaptive response induced by low dose of MTBE.And the dose that can induce obvious adaptive response will not damage cells' DNA.PARP-1 may be activated through the phosphorylated ERK₂ route when CHL cells are treated with low dose MTBE.Then the function of immunity and DNA repair in the cells are enhanced.3.In these research,the receptive biosystem is cultured CHL cells and mice.The above conclusion cannot be extrapolated to human conditions because of the deffirences between the specices.Such problem should be further studied in vitro of human somatic cell.