Oxidative DNA Damage Induced By Plasticizer DNOP In Pancreatic β Cellline

Objective: Di-n-octyl phthalate(di-n-octy1 phthalate,DNOP)is a very common plasticizer,which has good plasticity;it is widely used in plastic products plastic packaging products.The DNOP acute toxicity is relatively low,the body after ingestion guts often has no acute symptoms,but the chronic toxicity for human harm is relatively large,past studies have found that DNOP can cause insulin resistance by oxidative stress pathway,thus initiator type ⅱ diabetes.PQQ(pyrroloquinoline-quinone,PQQ)is essential to the body in physiological regulator;the literature indicates that PQQ by the cellular oxidative stress-induced disease has a therapeutic effect.Alcohol is a common substance in life,which should be a very wide range;there are a large number of alcohols content in liquor,its toxicity and cellular oxidative stress.The recent well-known liquor brands found to contain plasticizer component,causing more people for alcohol and DNOP combined effects of concern.This topic selected rat islet β cell Ins-1 as the research object,DNOP islet β cell Ins-1 mechanism of toxicity,and studied the protective effect of PQQ Ins-1 cells and the combined effects of alcohol and DNOP.Methods: The subjects selected rat islet β cell Ins-1 for the study,the first to use different concentrations of DNOP MTT toxicity test toxicity Ins-1 Cells,and determine the median lethal dose.Single cell gel electrophoresis assay(SCGE)to detect damage of islet β cells of rat DNOP Ins-1DNA and the protection of PQQ for Ins-1 cell DNA,the combining effects of alcohol.To obtain the protective effect of DNOP Ins-1 cell DNA damage mechanisms and PQQ,the combined effects of alcohol,the use of phthalaldehyde(OPT)Detection of intracellular glutathione(GSH)content,using acridine orange within(AO)to detect cell lysosomal membrane stability,7’-dihydro-2’-dichloro-fluorescein(DCFH)to detect intracellular reactive oxygen species(ROS),the use of rhodamine 123 to detect cells mitochondrial membrane potential.So as to arrive glutathione Ins-1 cells(GSH)levels,lysosomal stability level,within the reactive oxygen species(ROS)and mitochondrial membrane potential.PQQ was added as a protective agent in the pre-test,the observed indicators.Then add alcohol and DNOP observed effects of alcohol results.The test results using statistical software SPSS17.0 to test results were statistically analyzed.Results: In the SCGE test,DNOP alone treated rat pancreatic beta cell Ins-1,the Ins-1cells of the DNA chain rupture and a dose-dependent relationship.With the increasing doses of DNOP tailing phenomenon gradually increased,tail length,tail moment and tail DNA/head DNA(%)compared with the control group,the difference was significant;in the PQQ pretreatment group,reduced beta cell Ins-1 tail,tail length,tail DNA/head DNA(%),tail moment is less than the same dose of DNOP alone treatment group in pre alcohol;treatment group,stronger tailing phenomenon of islet beta cell Ins-1,Ins-1 cell tail length,tail DNA/head DNA(%),tail moment relative to DNOP alone treatment group increased significantly.Reduced glutathione in the cell(GSH)trial,DNOP reduced Ins-1 cells increased GSH level;PQQ pretreatment group GSH level;compared with DNOP alone treated group,alcohol pretreatment group GSH levels compared with the same concentration of DNOP alone treatment group decreased.The lysosomal membrane stability test,reduce the lysosomal membrane stability in DNOP treated alone,increased lysosomal membrane stability in PQQ pretreatment group,lysosomal membrane stability in alcohol pretreatment group compared with the same concentration of DNOP alone treatment group decreased.Active oxygen in the cell(ROS)test,the elevated level of ROS DNOP in the cells treated with ROS,the level of PQQ pretreated group cells decreased,while the level of ROS alcohol pretreatment group cells compared to DNOP treatment group with the same concentration increased.In the cell mitochondrial membrane potential assay,mitochondrial membrane potential level of DNOP in the cells treated decreases with increasing concentration,elevated levels of mitochondrial membrane potential in PQQ pretreatment group,while the level of mitochondrial membrane potential alcohol pretreatment group decreased.Conclusion: The DNOP induced oxidative stress damage in DNA of Ins-1 cells,DNOP cells can increase the level of ROS,lower the level of GSH,resulting in oxidative damage to cells,PQQ can reduce the number of ROS,increase the level of GSH,thereby reducing the oxidative stress injury,alcohol can aggravate oxidation stress damage in Ins-1 cells with the combined effects of DNOP.In DNOP the lysosomal mitochondrial pathway Ins-1 cell injury in DNA,DNOP can make the lysosomal membrane stability of Ins-1 cells decreased,and the level of mitochondrial membrane potential decreased,PQQ can make the lysosomal membrane stability of Ins-1 cells increased,mitochondrial membrane potential increased,and alcohol can make cell stability of lysosomal membrane and mitochondrial membrane potential decreased,thereby increasing injury DNOP of the Ins-1 DNA.In summary,DNOP by oxidative stress and lysosomal pathway-mitochondrial pathway injury Ins-1 cells,DNA,PQQ has a protective effect,the combined effects of alcohol.