Mono(2-ethylhexyl) Phthalate Induces Autophagy-dependent Apoptosis In Human Endothelial Cells

Objective: Di(2-ethylhexyl)phthalate(DEHP)is a widely used plasticizer contained within the food containers and medical devices.Additionally,the global production volume of DEHP is of large quantity,approximately 3 million tonnes a year.Phthalate-containing plastics can leach phthalates readily into bodily fluids,blood bags and intravenous tubing can pose a significant route of human exposure to DEHP.Mono(2-ethylhexyl)phthalate(MEHP)is the bioactive monoester metabolite of DEHP.It has been proved that MEHP have adverse effects on the reproductive system,urologic systems,hepatic,developmental toxicities and carcinogenicity.However,reports about the cardiovascular toxicity of MEHP are rare,remains largely unknown.Since the intensive study of autophagy,it has been found that autophagy is a cellular survival mechanism for the cell to degrade and recycle the cell organelles and long-half-life proteins,called type II programmed cell death,which is different from the type I programmed cell death,apoptosis.Although there are differences between apoptosis and autophagy in morphologic and metabolic pathways,it has been shown that many signal transduction pathways exits in both autophagy and apoptosis.It is indicated that MEHP can lead to apoptosis.Overall,our study offers comprehensive insights into the effects of MEHP treatment on LMP(Lysosomal membrane permeabilization),the role of LMP in MEHP-induced apoptosis,and the relationship between autophagy and apoptosis in MEHP-treated EA.hy 926 cells.Methods: In this study,EA.hy 926 cells were treated with MEHP(0-800 μM),and then the cytotoxicity of MEHP was detected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay;we used western blot to detect the expression of LC3,cathepsin B,cytochrome c and caspase 3 after EA.hy 926 cells were treated with 200 μM MEHP at different time(0 h,(3 h),6 h,12 h,24 h);The stability of the lysosomal membrane was evaluated by the acridine orange(AO)relocation test,Samples were immediately observed and photographed using a fluorescence microscope(Olympus BX-63);the mitochondrial membrane potential of MEHP-treated EA.hy 926 cells was determined using the potentiometric fluorescent dye JC-1 under the fluorescence microscope;Apoptosis was observed by TUNEL assay.After pretreatment with 1 mM 3MA for 2 h,we investigated the relationship of autophagy with LMP,MOMP(Mitochondrial outer membrane permeabilization)and apoptosis in MEHP-treated cells.After pretreatment with 10 μM CA-074 Me for 2 h,we investigated the role of cathepsin B in MEHP-induced MOMP and apoptosis.Results: The cell viability of MEHP-treated EA.hy 926 cells was decrease and in a dose-dependent manner.Our result showed that the LC3-Ⅱexpression was upregulated after treatment with 200 m M MEHP for 6 h,12 h and 24 h,While the changewas not significant in 3 h compared with the control.LMP and the lysosomal release of cathepsin B were significantly increased with 200 mM MEHP for 12 h;and these were attenuated by pretreatment with 1 mM 3MA for 2 h.The mitochondrial membrane potential collapsed and the release of cytochrome c was increased in 12 h after treated with 200 mM MEHP,and these were relieved by pretreated with 1 m M 3MA or 10 μM CA-074 Me for 2 h.Elevated apoptosis was found by TUNEL staining in 12 h and 24 h after treated with 200 mM MEHP,at the same time,the level of the cleaved caspase 3 was robustly increased when cells were exposed to 200 m M MEHP for 12 h and 24 h,consistently,3MA and CA-074 Me significantly reduced apoptosis from MEHP.Conclusion: We suggest that MEHP could induced autophagy-dependent apoptosis in EA.hy 926 cells.It is an early MEHP-induced autophagy and a relatively later LMP in cells,and then LMP causes lysosomal release of cathepsin B and leads to MOMP,the release of cytochrome c,ultimately,triggering mitochondrial apoptosis.