Cellular And Molecular Mechanisms Of Mono-(2-Ethylhexyl) Phthalate In Human Vascular Endothelial Cells

Objective:Mono-(2-ethylhexyl)phthalate(MEHP)is an active metabolite of di-(2-ethylhexyl)phthalate(DEHP).DEHP is one of the most abundantly used in PAEs,which can make plastic products become more flexible.Humans may be exposed to high levels DEHP through plastic medical devices.Once DEHP gets into the human body,it is quickly hydrolyzed into MEHP,in the liver and intestine.The present studies mostly focused on potential adverse effects of MEHP on the liver,kidney,reproductive system and developmental toxicities.MEHP also has toxic effects on cardiovascular system,but the possible molecular mechanisms are not completely elucidated.In this study,we concentrated on investigating whether MEHP induces autophagy through the reactive oxygen species(ROS)pathway in EA.hy926 cells.Methods:To investigated whether MEHP induces autophagy through the oxidative stress pathway,we utilized MTT assay to observe the cell viability of 0-1600 ?M MEHP-treated EA.hy926 cells;cells were pretreated with an autophagosome formation inhibitor 3-methyladenine(3-MA),an autophagosome formation stimulator rapamycin,a ROS inhibitor NAC,knockdown of Akt1 with Akt1 siRNA and an Akt1 activator insulin to assess the changes in cell viability under treatment with 0-400 ?M MEHP;we used transmission electron microscope assay to detect cytoplasmic autophagosomes induced by MEHP;the expression of P62 and microtubule-associated protein light chain 3(LC3)in EA.hy926 using Western blot assay was shown;DCFH(2 ',7 ' – dihydro-dichloro within fluorescein)was used to detect ROS levels in 0-200 ?M MEHP-treated or NAC-pretreated EA.hy926 cells;under a fluorescence microscope,the JC-1 staining method was used to evaluate the loss of ??m;pretreatment with NAC,siRNA Akt1,insulin or not,and treatment with MEHP,p-Akt1 level was detected by immunofluorescence staining;pretreatment with siRNA Akt1 or insulin,the expression of LC3 was detected by Western blot assay.Results: MEHP caused a dose-dependent decrease in cell viability in EA.hy926 cells;the 3-MA,NAC and insulin increased the cell viability of MEHP-treated EA.hy926 cells;the rapamycin and siRNA Akt1 decreased the cell viability of MEHP-treated EA.hy926 cells,transmission electron microscope images clearly showed the presence of a large number of autophagosomes in MEHP-treated cells,MEHP increased AVs number significantly;the expression of LC3-II and P62 in EA.hy926 cells increased,these data indicated that accumulation of autophagosomes was because of impaired degradation,pretreatment with NAC the expression level of LC3-II was increased in MEHP-treated cells;the treatment of EA.hy926 cells with MEHP resulted in significant increased ROS level,the pretreatment with NAC caused the ROS level in the MEHP-treated cells to decrease;the ??m of the cells was significantly decreased in MEHP-treated cells,the pretreatment with NAC reversed the increased ??m;p-Akt1 levels were significantly down-regulated in the MEHP-treated cells;after pretreated with NAC or insulin,p-Akt1 level was significantly up-regulated,after pretreated with siRNA Akt1,p-Akt1 level was significantly deceased;the protein level of LC3-II was enhanced in siRNA Akt1-pretreated cells,insulin treatment inhibited the accumulation of LC3-II protein.Conclusion: MEHP increased autophagosome formation in EA.hy926 cells.The ROS-dependent autophagic cell death through Akt1 pathway of EA.hy926 cells.