The Role Of Oxidative Stress In Gliotoxin-induced DNA Damage In HEK-293

Background＆Obiective:Gliotoxin (GT) widely exists in the Trichoderma reesei,penicillium, aspergillus fumigatus,dida albicans and other pathogenic bacteria orenvironmental microbial secondary metabolites, studies have shown that GTproduced mainly comes from aspergillus fumigatus, and by liquid chromatographytandem mass spectrometry (HPLC) method for the detection of gliotoxin exists inanimal feed (such as pigs, cows, poultry), also produced in the process of the cornstorage. In vitro experiments show that gliotoxin has strong antibacterial, antiviral effect,showing a good medicinal prospects, briefly considered can be used in anti-tumortherapy. But subsequent research has shown that gliotoxin has immunosuppression,mediated apoptosis toxicity and so on. This study by observing the GT induce HEK-293cells genetic toxicity, as well as it cause the generation of ROS and lysosomalmembrane permeability change to explore the toxicity mechanism of the GT on DNAdamage, in order to further evaluate the GT on human health effects to provideexperimental and theoretical basis.Methods: in HEK-293cells as the test sample. Experiment using single cell gelelectrophoresis (SCGE)and the protein imprinting method (western blot) are discussedto detect DNA damage caused by GT and the DNA damage (ATM) and P53proteinof high expression level in HEK-293cells in vitro, and verify the GT toxicity of DNAdamage. To discuss the possible mechanism of DNA damage, with2’,7’-dihydrodichlorofluorescein (DCFH) and acridine orange (AO) were determined gliotoxin onintracellular reactive oxygen species (ROS), and the effect on the stability of thelysosome membrane. through n-acetyl cysteine (NAC) intervention trial to observe therole of oxidative damage in DNA damage, as well as its influence in the lysosomemembrane stability. The test results with SPSS v12.7statistical software for statisticalanalysis.Results: when gliotoxin (GT) in HEK-293cells after1hour, the comet experiments show double-stranded DNA rupture, compared with blank control group (DMSO)under fluorescence microscope, cell into a comet-like tail, with its concentrationincreasing, the long tail, tail DNA%%and DNA content increased obviously; At thesame time, the GT (0.3125μM to1.25μM) can result in the expression of ROS levels,lysosome membrane stability in HEK293cells. After pretreatment with NACintervention agent, GT induce in HEK293cells trailing phenomenon,, intracellularROS and lysosome membrane permeability have significantly diminished. Effect after24hours, GT is not cause HEK293cells apoptosis,but DNA damage protein expressionin dose dependent effect appears high trend, at the same time presents NAC interventioneffect.Conclusion: GT (0.625μM–1.25μM) can cause the DNA damage of HEK293cells,explain it’s genetic toxicity. At the same time, GT can result in HEK-293cells ROSgeneration increases, make the cell in the oxidative stress state and rsduce the lysosomemembrane stability. And antioxidant n-acetyl cysteine in reducing the intracellular ROSlevel at the same time, the stability of lysosome membrane showed a trend of protection,DNA strand break is weakened under the same conditions caused by GT. All resulsesuggests that GT cause lysosomal membrane stability in HEK-293cells and DNAdamage in relation to oxidative stress. DNA damage high expression of protein (ATM,P53) and protective effects of NAC, further illustrate the GT can cause oxidative DNAdamage.