The Effects Of StAR Gene On DEHP Toxicity

Plasticizers are widely used in the industry as polymer materials.A wide variety of plasticizers include phthalate acid esters(PAEs),Di(2-ethylhexyl)phthalate(DEHP)is one of the environmental endocrine disruptors.In general,reproductive toxicity is currently the most obvious effect of DEHP.During the transport of cholesterol into the mitochondria,the StAR protein precursor cleaves off part of its mitochondrial-targeted fragment and becomes the mature StAR protein that eventually transports cholesterol to P450 scc enzyme located on the mitochondrial inner membrane.Cholesterol is converted to progesterone by StAR.Therefore,StAR gene plays an important role in the biosynthesis of testosterone.In order to investigate the changes of StAR gene expression and its effect on the toxicity of DEHP,we conducted a cell experiment on DEHP.We selected human breast cancer MCF-7 cells for experiments.Method: 1.We conducted the experiment with a DEHP exposure concentration of 0-0.80 mmol/L for 24 hours,using Real-time quantitative PCR to detect the expression of StAR gene,observing the change of StAR protein’s expressions by Western blot.Then we observed the dose-effect relationship and time-effect relationship of DEHP exposure.2.We constructed a StAR gene-overexpressing cell line and a StAR-silenced cell line by using lentivirus and identified them with Real-time quantitative PCR and Western blot.3.Different doses of DEHP were used to infect normal MCF-7 cells,high StAR gene-expressing cells,and StAR-silenced cells.The expression levels of the related genes and their protein expression were detected by real-time quantitative PCR and Western Blot.Meanwhile,compared StAR gene-overexpressing cells,StAR-silenced cells and normal MCF-7 cells in order to study the effect of DEHP toxicity on StAR gene.Results: 1.After 24 h and 48 h exposure to DEHP,a significant change of the expression level of StAR gene in the cells was detected by Real-time quantitative PCR,and a dose-effect relationship was found.Additionally,the cells were treated by a single dose of DEHP at 0.8 mmol/L for 3 hours,to 48 hours,the dose-effect relationship was observed on the expression of StAR gene.The cells were treated by DEHP at 0.10-0.80 mmol/L for 24 hoursand the expression of StAR protein was detected by western blot.It turns out that after exposure to DEHP for 24 hours,the expression level of StAR gene in 0.10-0.80 mmol/L DEHP groups was 30%-185% higher than that in the control group,and the differences were statistically significant(P<0.01).2.After the identification by Real-time quantitative PCR and Western blot,two cell lines were successfully constructed.The results of real-time PCR showed that the StAR gene expression in the high-expressing cells was 591.9-fold higher than that in normal MCF-7 cells while Western blot experiments showed that StAR protein expression was 190% higher in StAR than in normal MCF-7 cells.3.After DEHP exposure to StAR gene-expressing cells,its results of real-time quantitative PCR and Western blot showed that the gene expression levels and the protein expression levels of Bax,Caspase-3,Caspase-8,MTH1,hOGG1,ERα,ERβin StAR gene-expressing cells decreased to various degrees compared with the MCF-7 cells in the same dose group(p<0.05 or p<0.01).Conclusion: These experimental results from above indicate that there is a close relationship between the reproductive toxicity of DEHP and the changes in the expression levels of related genes.Therefore,our cell experiments focusing on the changes of StAR gene expression and its effects on the toxicity of DEHP,and we found that after the expression of StAR gene changes,DEHP has a significant effect on the expression level of apoptotic gene,part of estrogen gene,DNA oxidative damage and repair-related genes and protein expression,indicating that to a certain extent,StAR genes participate in the apoptosis of MCF-7 cells,regulate the oxidative damage and repair of estrogen receptor and DNA,and thus affect the toxicity of DEHP.This article not only provides a scientific basis for studying the toxic mechanism of DEHP,but also has an important theoretical and practical application value for further research on the health hazards of plasticizers to the population.