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Mono-(2-ethylhexyl) phthalate induced ROS-dependent autophagic cell death in human vascular endothelial cells
Nairong Liu1, Liping Jiang2, Xiance Sun2
1Department of Food Nutrition and Safety, Dalian Medical University, No. 9W. Lushun South Road, Dalian 116044, China.
Abstract:
Mono-(2-ethylhexyl) phthalate (MEHP) is an active metabolite of di-(2-ethylhexyl) phthalate (DEHP). MEHP has toxic effects on cardiovascular system, but the possible molecular mechanisms are not completely elucidated. In our study, 3-methyladenine (3-MA), an autophagosome formation inhibitor, protected the EA.hy926 cells against MEHP cytotoxicity, and rapamycin, an autophagosome formation stimulator, further decreased the cell viability in the MEHP-treated EA.hy926 cells. Thus, autophagy may play an important role in MEHP-induced toxicity. MEHP increased the autophagosome number in EA.hy926 cells detected under transmission electron microscope. Collapses of ΔΨm and reactive oxygen species (ROS) level were increased in a dose-dependent manner under treatment with 0-200μM MEHP for 24h. N-acetyl-l-cysteine (NAC), a ROS inhibitor, protected against MEHP-induced cytotoxicity and decreased the protein expression of LC3-II. These findings suggested that MEHP-induced autophagic cell death was ROS-dependent in EA.hy926 cells. Knockdown of Akt1 with Akt1 siRNA aggravated MEHP-induced cell death, and insulin, an Akt1 activator, alleviated MEHP-induced cell death. These results were consistent with the expression of LC3-II using western blot. The phospho-Akt1(Ser473) (p-Akt1) level was enhanced after pretreatment with NAC. In conclusion, it is possible that ROS elicited autophagy through Akt1 pathway in the MEHP-treated EA.hy926 cells.
Insights
Mono-(2-ethylhexyl) phthalate (MEHP) triggers cell death by inducing autophagy, a process dependent on reactive oxygen species (ROS) and the Akt1 pathway in EA.hy926 cells.
Area of Science:
- Toxicology
- Cell Biology
- Molecular Mechanisms
Background:
- Mono-(2-ethylhexyl) phthalate (MEHP), a metabolite of di-(2-ethylhexyl) phthalate (DEHP), exhibits cardiovascular toxicity.
- The precise molecular mechanisms underlying MEHP-induced toxicity remain incompletely understood.
Purpose of the Study:
- To investigate the role of autophagy in MEHP-induced cytotoxicity in EA.hy926 cells.
- To elucidate the involvement of reactive oxygen species (ROS) and the Akt1 signaling pathway in MEHP toxicity.
Main Methods:
- EA.hy926 cells were treated with varying concentrations of MEHP.
- Autophagy was modulated using 3-methyladenine (autophagy inhibitor) and rapamycin (autophagy stimulator).
- Cell viability, mitochondrial membrane potential (ΔΨm), ROS levels, LC3-II expression, and Akt1 phosphorylation were assessed. N-acetyl-l-cysteine (NAC) and Akt1 siRNA/insulin were used to investigate specific pathways.
Main Results:
- MEHP treatment increased autophagosome formation and decreased cell viability in EA.hy926 cells.
- MEHP induced mitochondrial dysfunction and elevated ROS levels in a dose-dependent manner.
- Inhibition of ROS by NAC protected cells and reduced LC3-II levels, indicating ROS-dependent autophagy.
- Akt1 knockdown exacerbated MEHP toxicity, while Akt1 activation by insulin alleviated it. NAC treatment enhanced Akt1 phosphorylation.
Conclusions:
- Autophagy plays a significant role in MEHP-induced cytotoxicity in EA.hy926 cells.
- The observed autophagic cell death is dependent on ROS generation.
- ROS may elicit autophagy via the Akt1 pathway in MEHP-exposed cells.
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