Sterigmatocystin Induced DNA Damage Related To Oxidative Stress And Lysosomal Membrane Destabilization In Human HepG2Cells

Objective: The object of present study is to investigate whether ST causes DNAdamage in HepG2cells and to elucidate the foundamental mechanism of ST-inducedDNA damage.As a result it may provide us some information for safety assessment tohumans on ST.Methods: HepG2cells were selected as test system.First,DNA damage inducedby ST was assessed by single cell gel electrophoresis(SCGE) assays to evaluate thegenotoxicity of ST.To further study the genetic mechanism of toxicity,the2,7-dichlorofluorescein diacetate (DCFH-DA) was used to monitor the levels of reactiveoxygen species(ROS).the Acridine orange(AO) was used to measure the changes oflysosomal membrane stability,the levels of8-hydroxy-deoxyguanosine (8-OhdG)generated in DNA were measures with immunohistochemistry.Meanwhile we usedN-Acetylcysteine(NAC) and NH4CL to protect DNA from damage induced by ST.Thedata were statistically analyzed by SPSS v11.5software.Results:In the comet assay, a significant dose-dependent increase of DNA strandbreaks was found after treatment with ST(0,0.5,1,and2μg/ml) for1h. After beingpretreated with NH4CL(10mM) and NAC(10mM) for1h,the DNA strand breaksinduced by ST were almost completely prevented.After being treated withST(0,0.5,1,and2μg/ml) for1h,the effect on lysosomes observed in HepG2cells was astatistically significant increase and levels of ROS and the expression of8-OhdG inDNA both increased after the treating.It has been proved that lysosomal leakageinduced by ST in a response that required an acidic lysosomal enviorment.Cellspretreated with weak base ammonium chloride to increase lysosomal pH,showeddecreased lysosomal injury.Besides,NAC is an effective antioxidant,the increasing levels of ROS in HepG2cells was significanty reduced with the pretreatment of NACfor1h.Conclusion:ST induced DNA strand breaks in Hep G2cells,probably throughlysosomal signaling pathway and oxidative stress at the same time.This researchsuggests that lysosomal leakage can activate the lysosomal enzyme and the lysosomalacidic compartment that contribute to ST-induced DNA strand beaks.Also,ST increasedthe levels of ROS and the expression of8-OhdG in DNA,besides,as an effectiveAntioxidants,NAC significantly reduced the DNA strand breaks in Hep G2cellsinduced by ST,and this proved that oxidative stress probably is another pathway in thisresearch.