The Role Of Oxidative Stress In Plasticizer Di-(2-ethylhexyl) Phthalate-induced DNA Damage In Pancreatic β Cell

Objective: Di-(2-ethylhexyl)Phthalate(DEHP)is the most widely used plasticizer in toys,medical equipment,building materials,food packaging and so on.DEHP leach out easily since its structure is loose.The route of human exposure is extensive.More and more evidences showed that,DEHP exposure can affect the nutritional metabolic state.Studies have found that phthalates associated with insulin resistance,and confirmed that DEHP leads to insulin resistance by oxidative stress.Alcohol can damage the digestive,nervous,endocrine,circulatory system,and the toxicity is associated with oxidative stress.Pyrroloquinoline quinone(PQQ)play a role of antioxidant by scavenging active oxygen and mitochondria-associated cell signaling pathways effectively.This study we selected rat pancreatic β cells Ins-1 as our objects to investigate the toxic effects of DEHP,and explore the molecular mechanisms associated with this toxicity.Meanwhile,the effects of alcohol and PQQ on this toxicity were explore.Method: In this study Ins-1 cells were selected as research objects.We assess the effect of different concentrations of DEHP on Ins-1 cells viability by MTT;single cell gel electrophoresis assay(SCGE)was observed,to detecte DNA damage DEHP-induced in Ins-1 cells;RT-PCR test was used to detect the expression levels of p53 and ATM gene,which wre related to DNA damage.To explore the mechanism of DNA damage,Rhodamine 123(rhodamine 123),phthalaldehyde(OPT),2’,7’-dihydro-dichlorofluorescein(DCFH-DA)and acridine orange(AO)wre used as fluorescent probes.And fluorescence spectrophotometer was used to measure mitochondrial membrane stability,intracellular GSH levels,intracellular reactive oxygen species(ROS)and cell lysosomal membrane stability.Based on the test methods above,we detect the effects of PQQ and alcohol in DEHP-induced cell injury respectively.SPSS 17.0 was selected to analysis the test results statistically.Results: After treated with different concentrations of DEHP 1 h,Ins-1 cells showed comet-like tails,and the higher the dose,the more obvious the phenomenon.Compared with the control group,the tail,tail DNA / head DNA(%)and tail moment of groups treated with DEHP were significantly increased,and the difference was statistically significant.At the same time,DEHP can cause to decreasing intracellular GSH levels,lysosomal and mitochondrial membrane stability.While,ROS levels were increased.Compared with the same concentrations of DEHP treated cells,the cells treated with DEHP and PQQ showed results as follow: the DNA strand was alleviated;the level of intracellular GSH level was increased;the stability of lysosomal and mitochondrial membrane were increased;ROS levels were decreased.In the group of cells treated with DEHP and alcohol the trends were opposite.It also means that: the DNA strand was aggravated;the level of intracellular GSH level was decreased;the stability of lysosomal and mitochondrial membrane were decreased;ROS levels were increased.Conclusion: Oxidative stress is the mechanism of the genotoxicity DEHP-induce in Ins-1.Meanwhile,lysosomes-mitochondrial pathway plays an important role in the toxicity.PQQ protected Ins-1 from the injury DEHP-induce by clearing intracellular ROS,increasing levels of intracellular GSH,increasing the instability of lysosomal membrane and mitochondrial membrane.Ethanol exacerbated DEHP-induced cell damage by enhancing cell oxidation stress state,such as increasing of intracellular ROS levels,lowering levels of intracellular GSH,breaking the instability of lysosomal membrane and mitochondrial membrane.