Di(2-ethylhexyl) phthalate induces apoptosis through mitochondrial pathway in GC-2spd cells

Abstract

Di(2-ethylhexyl) phthalate (DEHP), a plasticizer of synthetic polymers, is a well-known endocrine disrupting chemical (EDC) and reproductive toxicant. Addressing the unclear mechanism of DEHP-induced reproductive dysfunction, this study used GC-2spd cells to investigate the molecular mechanism involved in the DEHP-induced toxicity in the male reproductive system. The results indicated that the apoptotic cell death was significantly induced by DEHP exposure over 100 μM. Furthermore, DEHP treatment could induce oxidative stress in GC-2spd cells involving in the decrease of superoxide dismutase (SOD) activity (200 μM) and glutathione peroxidase (GSH-Px) activity (50 and 100 μM). In addition, DEHP induction also caused the elevated ratios of Bax/Bcl-2, release of cytochrome c and decomposition of procaspase-3 and procaspase-9 in GC-2spd cells. Taken together, our work provided the evidence that DEHP exposure might induce apoptosis of GC-2spd cells via mitochondria pathway mediated by oxidative stress. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1055-1064, 2017.

Keywords: Di(2-ethylhexyl) phthalate (DEHP); GC-2spd cells; apoptosis; mitochondrial pathway; oxidative stress.

Fig. 1

Cell viability of GC-2spd cells after treatment with 0.1% DMSO, 50, 100, 200, or 400 μM DEHP for 24 h. Data are obtained from MTT assay (mean ± SD) and are normalized to DMSO group. Statistical significance was analyzed by one-way ANOVA. Significant difference: (*) P <0.05. [Color figure can be viewed at wileyonlinelibrary.com]

Fig. 2

Flow cytometric analysis of GC-2spd cells after treatment with DEHP. Cellular apoptosis was tested by apoptosis detection kit. A–D represented as treatment of 0.1% DMSO (A), 50 μM DEHP (B), 100 μM DEHP (C), 200 μM DEHP (D). The comparison of apoptotic rate(%) after treatment with different concentration of DEHP is presented in E. Data were presented as mean ± SD. There are three independent experiments performed in triplicate. Significant difference: (*) P <0.05, compared with the control group. [Color figure can be viewed at wileyonlinelibrary.com]

Fig. 3

A–C represent the oxidative stress levels after treatment with DEHP. Effect of different concentration of DEHP on Malondialdehyde (MDA) Level (A), Superoxide Dismutase (SOD) Activity (B) and Glutathione Peroxidase (GSH-Px) Activity (C) in GC-2spd cells. Significant difference: (*) P <0.05, compared with the control group. [Color figure can be viewed at wileyon-linelibrary.com]

Fig. 4

(A,B): This represents the real time quantitative PCR dissociation curve of Bcl-2(A) and Bax(B). C, D is the effect of different concentration of DEHP on Bcl-2 and Bax relative mRNA expression (C), and the ratio of Bcl-2 and Bax(D), Significant difference: (*) P <0.05, compared with control. [Color figure can be viewed at wileyonlinelibrary.com]

Fig. 5

A–C represent the real time quantitative PCR dissociation curve of cytochrome c (A) caspase-9(B) and caspase-3(C). D is the effect of different concentration of DEHP on cytochrome c, caspase-9 and caspase-3 relative mRNA expression (D). Significant difference: (*) P <0.05, compared with control. [Color figure can be viewed at wileyonlinelibrary.com]

Fig. 6

Effects of DEHP exposure on the expression levels of Bcl-2, Bax, cytochrome c, procaspase-9, and procaspase-3 in GC-2spd cells (A). The level of proteins was measured using immunoblotting (B). Values represent the mean ± SD. Significant difference: (*) P <0.05, compared with control.