The Role Of Oxidative Stress In Di-(2-ethylhexyl) Phthalate-induced DNA Damage In HEK293

Background＆Obiective: Di-(2-ethylhexyl) phthalate (DEHP) is commonlyemployed as such a plasticizer. Human exposure to DEHP can occur via the dermal,inhalation, oral, and intravenous routes from medical equipment, and it has beenreported to have cytotoxic, immunotoxic, genotoxic, and reproductive toxic properties.This study by observing the DEHP induced HEK293cells DNA strand breaks, thegeneration of ROS, the level of GSH, lysosomal membrane, mitochondrial membrane,in order to further evaluate the DEHP on human health effects to provide experimentaland theoretical basis. And the aim of present study was to observe whether thecombination treatment of DEHP and ethanol could enhance DNAdamage.Methods: In HEK293cells as the test sample. Experiment using single cell gelelectrophoresis (SCGE) is discussed to detect DNA damage caused by DEHP and verifythe DEHP toxicity of DNA damage. To discuss the possible mechanism of DNAdamage, with2’,7’-dihydro dichlorofluorescein (DCFH), Ophenaldehyde (OPT),acridine orange (AO) and rhodamine123were determined DEHP on intracellularreactive oxygen species (ROS), intracellular tri-peptide glutathione (GSH), the effect onthe stability of the lysosome membrane and the effect on the potential of mitochondrialmembrane. Through n-acetylcysteine (NAC) and DL-buthionine sulphoximine (BSO)intervention trial observe the role of oxidative damage in DNA damage. Thecombination treatment of DEHP and ethanol toxicity of DNA damage, to discuss thepossible mechanism of DNA damage, with2’,7’-dihydro dichlorofluorescein (DCFH), Ophenaldehyde (OPT) were determined on intracellular reactive oxygen species (ROS),intracellular tri-peptide glutathione (GSH). The test results with SPSS v17.0statisticalsoftware for statistical analysis.Results: when DEHP in HEK293cells after1h, the comet experiments showdouble-stranded DNA rupture, compared with blank control group (DMSO) underfluorescence microscope, cell into a comet-like tail, with its concentration increasing,the long tail, tail DNA%and DNA%content increased obviously; meanwhile, DEHPcan result in the expression of ROS levels, the expression of GSH levels, lysosomemembrane stability and mitochondrial membrane potential in HEK293cells. Afterpretreatment with NAC intervention agent, DEHP induced in HEK293cells trailingphenomenon, intracellular ROS, lysosome membrane permeability and mitochondrialmembrane potential have significantly diminished. After pretreatment with BSOintervention agent, DEHP induced in HEK293cells trailing phenomenon, intracellularROS, lysosome membrane permeability and mitochondrial membrane potential enhancesignificantly. In the combination treatment of DEHP and ethanol, the DNA damageaccelerated, and there was a dose-dependent increase of DNA strand breaks.Conclusion: DEHP can cause the DNA damage of HEK293cells, explain it’s genetictoxicity. Meanwhile, DEHP can result in HEK293cells ROS generation increases, GSHgeneration decreases, make the cell in the oxidative stress state, reduce the lysosomemembrane stability and mitochondrial membrane potential. Antioxidantn-acetylcysteine in reducing the intracellular ROS level at the same time, the stability oflysosome membrane showed a trend of protection, DNA strand break is weakenedunder the same conditions caused by DEHP. The result suggests that DEHP cause DNAdamage in relation to oxidative stress. The combination treatment of DEHP and ethanolcould enhance ROS generation increases, GSH generation decreases, make the cell inthe oxidative stress state.