The Adaptive Response Induced By Pretreatment With Low Dose NaNO₂ To The Oxidative Damage Caused By Higher Dose NaNO₂ In Cultured CHL Cells And Its Mechanism

Research Aim:To study the problem if low dose NaNO₂ can induce the adaptive response of cultured Chinese hamster lung cells(CHL cells) to oxidative damage caused by higher dose NaNO₂ and how the dose-effect relationship is.Try to explain the function of polyADP-ribose polymerases(PARP-1) in the adaptive response.Methods:1.The MTT-test was used to detect the cytotoxic effect of NaNO₂ in CHL cells.And the highest dose in which NaNO₂ cannot harm the cells would be found.This dose is also called "no observed active effect lever,NOAEL".The adaptive response of CHL cells to cytotoxicity of NaNO₂ induced by NaNO₂ pretreatment with its dose below the NOAEL and its dose-response relationship were observed.2.The adaptive response of CHL cells to the genotoxicity of NaNO₂ and its dose-response relationship was observed by the methods of the chromosomal aberration test,single cell gel electrophoresis technique and flow cytometry of apoptosis and cell cycle.3.3-aminobenzamide(3AB) is the specific inhibitor of PARP-1.After the activity of PARP-1 was inhibited by 3AB,it was observed whether the adaptive response induced by low dose NaNO₂ pretreatment could be blocked.And the function of PARP-1 was revealed according to the research results.4.The expression of PARP-1 protein was tested with western-blot test when CHL cells exposing to NaNO₂ of different doses.Results:1.MTT test reveals that NaNO₂ can enhance proliferation of cells when the doses are between 1.52mg/L and 40mg/L.The survival rates of CHL cells are higher than that of the normal control group.And the hormesis effect can be observed.NaNO₂ can induce the adaptive response of CHL cells to the cytotoxicity of 1g/L NaNO₂ when the cells were pretreated with the doses between 0.0005mg/L and 0.01mg/L for 6 hours.But the pretreatment with low dose NaNO₂ cannot induce the adaptive response of CHL cells to much higher dose NaNO₂(such as 5g/L).2.Chromosomal aberration test reveals that NaNO₂ can increase the rate of chromosome aberration in CHL cells when the exposing doses are higher than 128mg/L.The types of chromosome aberration include break,chromatid exchange,dicentric chromosome,ring chromosome and polyploid.When CHL cells are pretreated with doses between 0.5mg/L and 2.5mg/L for 6 hours,NaNO₂ can induce the adaptive response to chromosome or chromatid aberration caused by 1.5g/L NaNO₂.And 3AB can inhibit the adaptive response induce by low dose NaNO₂.3.NaNO₂ can cause DNA damage in CHL cells when the doses are higher than 200mg/L and the treatment time is 18 hours.After CHL cells are treated with low dose(0.01mg/L～0.1mg/L) NaNO₂ for 6 hours,NaNO₂ can induce the adaptive response to DNA damage caused by 1g/L NaNO₂.3AB can block the adaptive response of DNA damage caused by low dose NaNO₂ before the pretreatment of the cells.But after 6 hours of low dose NaNO₂ pretreatment,3AB cannot block the adaptive response induced by NaNO₂.4.NaNO₂ can enhance apoptosis of CHL cells,cause the blockage of the cells in G₁ stage,and decrease the percentage of cell numbers in S and G₂ stages,and make the DNA related concentrations(DNA_RC) and the cell proliferation index(PI) decreasing.That indicates the cells suffer from severe DNA damage when they are exposed to 1.5g/L NaNO₂ for 18 hours.The adaptive response of apoptosis can be induced by pretreatment cells for 6 hours with low dose NaNO₂(0.01mg/L～0.1mg/L).When the activity of PARP-1 is inhibited by 3AB,the function of DNA repair is blocked,but the actions related with apoptosis is not affected.5.Western-blot test reveals that the strap of PARP-1 protein with 116KD molecular weight become more pale when CHL cells exposing to NaNO₂ of 0.1mg/L group for 6 hours than that of other groups.It can be known that the NaNO₂ dose of 0.1mg/L is the optimal dose to induce adaptive response from the above other tests results.This also indicates that PARP-1 has a close relationship with adaptive response induced by low dose NaNO₂.Because low dose NaNO₂ can activate PARP-1 through the phosphorylated ERK₂ route,and change PARP-1 itself to polyADP-ribose(PAR) polymers,the molecular weight of PARP-1 protein is also changed.The strap of 116KD PARP-1 protein of 1g/L group is also more pale,but this can only be related with severe DNA damage caused by 1g/L NaNO₂.Conclusion:1.NaNO₂ pretreatment of low dose between 0.01mg/L and 0.1mg/L.can induce adaptive response of cultured CHL cells to oxidative damage caused by NaNO₂ of 1.0g/L or 1.5g/L.But to much higher dose NaNO₂(5g/L),low dose NaNO₂ pretreatment cannot induce the adaptive response. 2.Low dose NaNO₂ can trigger hormesis effect in CHL cells.But the hormetic zone is much higher than the dose range that can provoke adaptive response.It indicates that hormosis may be a precaution signal of DNA damage.And only the much lower dose under the lowest limit of hormetic zone that can induce adaptive response is the real safe dose.3.PARP-1 is a critical factor in the formation of adaptive response induced by low dose NaNO₂. PARP-1 can probably be activated through the phosphorylated ERK₂ route when CHL cells are treated with low dose NaNO₂.Then the function of DNA repair is enhanced,and meanwhile,the apoptosis procedure is also provoked in the cells with their DNA damaged severely.The DNA repair function of PARP-1 is crucial to the adaptive response.But the apoptosis mediated by PARP-1 is not the only way.PARP-1 only is one of initiated factors of apoptosis procedure probably.The function of PARP-1 may be related with the activation of its downstream relative genes.4.In this research,the receptive biosystem is cultured Chinese hamster lung cells.The above conclusions cannot be extrapolated to human conditions because of the differences between the species.Such problem should be further studied in animal vivo or in other human somatic cells.