Combined Effects Of Sterigmatocystin And Ethanol In Increasing DNA Damage In Human Liver Hep G2Cells

Objective: The research found that sterigmatocystin（ST） as thesecond largest mycotoxin following aflatoxin which occurs in a wide rangecrop has several toxic effects. Ethano（l E） also has several toxic effects andits consumption increases these years. Both ST and E were reported to beresponsible for naturally occurring human and animal liver injures andcarcinoma. In daily life, it is very likely that humans are always exposed tothe mixture rather than to individual compounds. Therefore, the aim ofpresent study was to observe whether the combination treatment of ethanoland sterigmatocystin could enhance DNA damage in human liver (Hep G2)cells compared to ethanol or ST effect alone and explore its foundamentalmechanism with oxidative stress and lysosomal membrane destabilizationMethods: human liver Hep G2cells were selected as text system.1.Hep G2cells survival was measured as by MTT assay and determined thetext dose; Using Zhengjun Jin’method to calculate the correlationcoefficient q and determine the interaction between the ST and ethanol.2.DNA damage was evaluated by single cell gel electrophoresis(SCGE) assays.Apoptosis was quantified using Hoechst33258.3.Intracellular ROS productionwas quantified using2,7-dichlorofluorescein diacetate (DCFH-DA).4. Lysosomalpermeabilization was monitored using acridine orange(AO) relocationtechniques.5. Data were statistically analyzed by SPSS v11.5software.Results: MTT assay results show that ST IC50was10.1μg/mL and EIC50was1190.4μg/mL. We selected test doses were ST0.5,1and2μg/mL, E60,120and240μg/mL and combined treatment dose E60μg/mL+ST1μg/mL,E120μg/mL+ST1μg/mL and E240μg/mL+ST1μg/mL. The interaction of240μg/mL E plus1μg/mLST was a simple combined effect.2.In the cometassay, Hep G2cells were cultured with different doses of toxicants for0.5h、 1h and2h individually. Hep G2cells with ST or E for1h induced DNAdamage, but their high combination occurred slight damage after treatmentfor0.5h. And there was a significant dose-dependent and time-dependentincrease of DNA strand breaks founded after treatment for1h. With theDNA damage accelerated, apoptosis cell increased (>10%) after treatmentwith combinations for2h.3.The level of intracellular ROS was significantincreased after treatment with ST or ethanol for0.5h and their combinationscould produce much more ROS than ethanol alone.4.The effect onlysosomal observed in HepG2cells was a statistically significant increaseafter cultured with ST or E and their combination has a significant effect onlysosomal membrane stability than ethanol alone after treatment for1h. Butthis effect disappeared in2h treantment.Conclusion: The combination of ST and E could enhance DNA strandbreaks in Hep G2cells, probably through oxidative stress and lysosomalsignaling pathway at the same time. These treatments could increase ROSproduction and AO fluorescence intensity. Meanwhile their combination hada significant effect on lysosomal membrane stability and ROS productionthan ethanol alone. But after treatment for2h, AO fluorescence intensitydecreased in combination may be due to cells apoptosis...