Mono-2-ethylhexyl phthalate drives progression of PINK1-parkin-mediated mitophagy via increasing mitochondrial ROS to exacerbate cytotoxicity

Abstract

Phthalate ester plasticizers are used to improve the plasticity and strength of plastics. One of the most widely used and studied, di-2-ethylhexyl phthalate (DEHP), has been labeled as an endocrine disruptor. The major and toxic metabolic derivative of DEHP, mono-2-ethylhexyl phthalate (MEHP), is capable of interfering with mitochondrial function, but its mechanism of action on mitophagy remains elusive. Here, we report that MEHP exacerbates cytotoxicity by amplifying the PINK1-Parkin-mediated mitophagy pathway. First, MEHP exacerbated mitochondrial damage induced by low-dose CCCP via increased reactive oxygen species (ROS) production, decreased mitochondrial membrane potential (MMP), and enhanced fragmentation in mitochondria. Second, co-exposure to MEHP and CCCP ("MEHP-CCCP") induced robust mitophagy. Mechanistically, MEHP-CCCP stabilized PINK1, increased the level of phosphorylated ubiquitin (pSer 65-Ub), and led to Parkin mitochondrial translocation and activation. Third, MEHP-CCCP synergistically caused more cell death, while inhibition of mitophagy, either through chemical or gene silencing, reduced cell death. Finally and importantly, co-treatment with N-acetyl cysteine (NAC) completely counteracted the effects of MEHP-CCCP, suggesting that mitochondrial ROS played a vital role in this process. Our results link mitophagy and MEHP cytotoxicity, providing an insight into the potential roles of endocrine disrupting chemicals (EDCs) in human diseases such as Parkinson's disease.

Keywords: Cell death; Cytotoxicity; MEHP; Mitochondrial ROS; PINK1-Parkin-mediated mitophagy.

Graphical abstract

Fig. 1

MEHP-CCCP induces mitochondrial damage.(A) HeLa cells stably expressing YFP-Parkin were exposed to MEHP (200 μM, 24 h) or CCCP (2.5 μM, 2 h) respectively, or pre-exposed to MEHP (200 μM) for 24 h, followed by CCCP (2.5 μM) for 2 h in the presence or absence of NAC (5 mM). After incubation with MitoSOX™ (2.5 μM) for 30 min, cells were examined and representative cells were photographed using a fluorescence microscope. Scale bar, 20 μm. (B) HeLa-YFP-Parkin cells were treated as in (A). Cell pellets were subsequently collected and subjected to flow cytometry to detect the fluorescence intensity. Data (means ± SD) were representative of three independent experiments (**, P < 0.01, Student's t-test). (C) HeLa-YFP-Parkin cells were exposed to MEHP (200 μM, 24 h) or CCCP (2.5 μM, 6 h) respectively, or pre-exposed to MEHP (200 μM) for 24 h, followed by CCCP (2.5 μM) for 6 h. Cells were collected and incubated with DiIC₁(5) (25 nM) for 15 min, the fluorescence intensity was analyzed by flow cytometry. (D) Statistical analysis of the fluorescence intensity of HeLa-YFP-Parkin cells as treated in (C). Data (means ± SD) were representative of three independent experiments (**, P < 0.01, Student's t-test). (E) HeLa-YFP-Parkin cells were treated as in (A), and cell lysate was harvested for Western blot detection of DRP1 phosphorylation status. (F) HeLa-YFP-Parkin cells were either exposed to MEHP (200 μM, 24 h) or CCCP (2.5 μM, 2 h) respectively, or pre-exposed to MEHP (200 μM) for 24 h, followed by CCCP (2.5 μM) for 2 h or 4 h. Cells were subjected to immunostaining of Tom20 (red) and observed by FV3000 confocal laser scanning microscope. Scale bar, 10 μm. (G) Cells stably expressing YFP-Parkin (green) were treated as in (A) and subjected to immunostaining of HSP60 (red). DNA was stained by DAPI (blue). Cells were examined and photographed using a fluorescence microscope. Scale bar, 20 μm. (H) Statistical analysis of the fluorescence intensity of HSP60 as treated in (G). Data (means ± SD) were representative of three independent experiments (**, P < 0.01; N·S, P > 0.05; Student's t-test). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

MEHP-CCCP induces mitophagy. (A) SH-SY5Y cells were incubated with MEHP (200 μM) for 24 h or CCCP (5 μM) for 8 h respectively, or pre-incubated with MEHP (200 μM) for 24 h and then incubated with CCCP (5 μM) for 8 h. Western blot was performed to detect proteins expression. (B) Proteins expression from (A) was evaluated by ImageJ, means ± SD from 3 independent experiments were presented (**, P < 0.01; Student's t-test). (C) HeLa-YFP-Parkin cells were exposed to MEHP (200 μM, 24 h) or CCCP (2.5 μM, 8 h) respectively, or pre-exposed to MEHP (200 μM) for 24 h, followed by CCCP (2.5 μM) for 8 h. Mitochondrial proteins were evaluated by Western blot. (D) Proteins expression from (C) was evaluated by ImageJ, means ± SD from 3 independent experiments were presented (**, P < 0.01; Student's t-test). (E) HeLa cells were treated as in (C). Cell lysate was collected and mitochondrial proteins were detected by Western blot. (F) Proteins expression from (E) was evaluated by ImageJ, means ± SD from 3 independent experiments were presented (*, P < 0.05; **, P < 0.01; N·S, P > 0.05; Student's t-test). (G) HeLa-YFP-Parkin cells were exposed to MEHP (200 μM, 4 h) or CCCP (2.5 μM, 4 h) respectively, or pre-exposed to MEHP (200 μM) for 4 h, followed by CCCP (2.5 μM) for 4 h. Cells were treated with DEHP (200 μM) the same as above, and then harvested and subjected to Western blot for the evaluation of mitochondrial proteins. (H) Proteins expression from (G) was evaluated by ImageJ, means ± SD from 3 independent experiments were presented (*, P < 0.05; **, P < 0.01; Student's t-test). MEHP promotes Parkin translocation to damaged mitochondria.

Fig. 3

MEHP promotes Parkin translocation to damaged mitochondria.(A) HeLa-YFP-Parkin cells were either exposed to MEHP (200 μM) for 24 h or CCCP (2.5 μM) for 2 h, or the cells were pre-exposed to MEHP for 24 h, followed by CCCP exposure for 2 h. The localization of YFP-Parkin was observed under a fluorescence microscope. Scale bar, 20 μm. (B) The percentage of cells with YFP-Parkin in mitochondria was quantified by ImageJ, means ± SD were presented of three independent experiments (**, P < 0.01, Student's t-test). (C) HeLa-YFP-Parkin cells were treated as in (A), cells were collected and lysed to detect Parkin expression by Western blot. (D) SH-SY5Y cells were exposed to MEHP (200 μM, 24 h) or CCCP (5 μM, 8 h) respectively, or pre-exposed to MEHP (200 μM) for 24 h, followed by CCCP (5 μM) for 8 h. Protein level of Parkin was evaluated by Western blot. MEHP-CCCP induces mitophagy in a PINK1-Parkin-dependent manner. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

MEHP-CCCP induces mitophagy in a PINK1-Parkin dependent pathway. (A) SH-SY5Y cells were incubated in MEHP (200 μM, 24 h) or CCCP (5 μM, 8 h) respectively, or pre-incubated in MEHP (200 μM) for 24 h, followed by CCCP (5 μM) for 8 h. PINK1 and Parkin levels were detected by Western blot. (B) HeLa-YFP-Parkin cells were exposed to MEHP (200 μM) for 24 h or CCCP (2.5 μM) for 0.5, 1, 2, 4 h respectively, or pre-exposed to MEHP (200 μM) for 24 h, followed by CCCP (2.5 μM) exposure for the indicated time periods. Cell lysate was collected and subjected to Western blot to detect PINK1 and Parkin levels. β-actin was used as a loading control. (C) HeLa-YFP-Parkin cells were exposed to MEHP (200 μM) for 24 h or CCCP (2.5 μM) for 2 h respectively, or pre-exposed to MEHP (200 μM) for 24 h, followed by CCCP (2.5 μM) for 2 h. The phosphorylation of ubiquitin (Ser65) was analyzed by Western blot. (D) HeLa-YFP-Parkin cells were firstly transfected with a non-targeting siRNA or PINK1 specific siRNA. 48 h after transfection, cells were treated as in (C), Western blot was performed to evaluate the phosphorylation status of ubiquitin (Ser65). (E) HeLa-YFP-Parkin cells were treated as in (D). After permeabilization, mitochondria were labeled by HSP60 antibody (1:100) and DNA by DAPI (1:10,000). Cells were observed under a fluorescence microscope. Scale bar, 10 μm. (F) Cells were treated as in (D), cell lysate was collected to evaluate the protein levels of PINK1 and Parkin. (G) PINK1 was knocked down by siRNA in HeLa-YFP-Parkin cells as in (D). 48 h later, cells were incubated with MEHP (200 μM, 24 h) or CCCP (2.5 μM, 8 h) respectively, or pre-incubated with MEHP (200 μM) for 24 h, followed by CCCP (2.5 μM) for 8 h. Western blot was performed to detect the expression of mitochondrial proteins. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Mitophagy promotes MEHP-CCCP-induced cell death. (A) HeLa-YFP-Parkin cells were either exposed to MEHP (200 μM) and CCCP (5 μM) respectively or in combination in presence or absence of zVAD (20 μM) for 24 h. Cell morphology was observed under a microscope. Scale bar, 50 μm. (B) Cells were treated as in (A), cell pellets were subsequently collected and cell death was quantified using propidium iodide (PI, 5 μg/mL) live exclusion staining coupled with flow cytometry. Statistical significance (*, P < 0.05; **, P < 0.01; ns, P > 0.05; Student's t-test) of three independent experiments was indicated in the bar chart. (C) After treated as in (A), cells were collected and stained by DiIC_l(5) (50 nM) and PI (1 μg/mL). Flow cytometry was applied to detect the fluorescence intensity of different wavelength lasers. Shown were representative dot-plots of flow cytometry data. (D) Quantification of the cell death data from panel C (including apoptotic and dead cells) was shown. Data were presented as means ± SD of three independent experiments (*, P < 0.05; **, P < 0.01; ns, P > 0.05; Student's t-test). (E) HeLa-YFP-Parkin cells were transfected with a non-targeting siRNA or PINK1 siRNA for 24 h and then co-treated with MEHP (200 μM) and CCCP (5 μM) for another 24 h. Cell morphology was examined by an inverted microscope. Scale bar, 50 μm. (F) Cells were treated as in (E) and PI live exclusion assay combined with flow cytometry was performed to analyze the percentage of cell death. Shown were the representative dot-plots of flow cytometry data. (G) Data from panel F were presented as means ± SD of three independent experiments (*, P < 0.05; Student's t-test). (H) HeLa-YFP-Parkin cells were either incubated with MEHP (200 μM) and CCCP (5 μM) respectively or in combination with or without of BafA1 (25 nM) for 24 h. Cell morphology was observed under a microscope. Scale bar, 50 μm. (I) PI live cell exclusion assay was used to detect the cell death percentage of panel H and shown were the representative dot-plots. (J) Percentages of cell death were statistically analyzed and means ± SD were presented (**, P < 0.01, Student's t-test). MEHP-CCCP-induced mitophagy and cytotoxicity are mediated by ROS. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Mitophagy promotes MEHP-CCCP-induced cell death. (A) HeLa-YFP-Parkin cells were either exposed to MEHP (200 μM) and CCCP (5 μM) respectively or in combination in presence or absence of zVAD (20 μM) for 24 h. Cell morphology was observed under a microscope. Scale bar, 50 μm. (B) Cells were treated as in (A), cell pellets were subsequently collected and cell death was quantified using propidium iodide (PI, 5 μg/mL) live exclusion staining coupled with flow cytometry. Statistical significance (*, P < 0.05; **, P < 0.01; ns, P > 0.05; Student's t-test) of three independent experiments was indicated in the bar chart. (C) After treated as in (A), cells were collected and stained by DiIC_l(5) (50 nM) and PI (1 μg/mL). Flow cytometry was applied to detect the fluorescence intensity of different wavelength lasers. Shown were representative dot-plots of flow cytometry data. (D) Quantification of the cell death data from panel C (including apoptotic and dead cells) was shown. Data were presented as means ± SD of three independent experiments (*, P < 0.05; **, P < 0.01; ns, P > 0.05; Student's t-test). (E) HeLa-YFP-Parkin cells were transfected with a non-targeting siRNA or PINK1 siRNA for 24 h and then co-treated with MEHP (200 μM) and CCCP (5 μM) for another 24 h. Cell morphology was examined by an inverted microscope. Scale bar, 50 μm. (F) Cells were treated as in (E) and PI live exclusion assay combined with flow cytometry was performed to analyze the percentage of cell death. Shown were the representative dot-plots of flow cytometry data. (G) Data from panel F were presented as means ± SD of three independent experiments (*, P < 0.05; Student's t-test). (H) HeLa-YFP-Parkin cells were either incubated with MEHP (200 μM) and CCCP (5 μM) respectively or in combination with or without of BafA1 (25 nM) for 24 h. Cell morphology was observed under a microscope. Scale bar, 50 μm. (I) PI live cell exclusion assay was used to detect the cell death percentage of panel H and shown were the representative dot-plots. (J) Percentages of cell death were statistically analyzed and means ± SD were presented (**, P < 0.01, Student's t-test). MEHP-CCCP-induced mitophagy and cytotoxicity are mediated by ROS. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

ROS mediate MEHP-CCCP-induced mitophagy and cytotoxicity. (A) HeLa-YFP-Parkin cells were firstly exposed to MEHP (200 μM) for 24 h and then exposed to CCCP (2.5 μM) for 2 h. NAC (5 mM) was added at the same time of MEHP exposure. YFP-Parkin localization was examined and photographed with a fluorescence microscope. Scale bar, 20 μm. (B) HeLa cells stably expressing YFP-Parkin were exposed to MEHP (200 μM, 24 h) or CCCP (2.5 μM, 2 h) respectively, or pre-exposed to MEHP (200 μM) for 24 h, followed by CCCP (2.5 μM) for 2 h in the presence or absence of NAC (5 mM). Cell lysate was harvested and subjected to Western blot to detect the protein levels of PINK1 and Parkin. (C) HeLa-YFP-Parkin cells were exposed to MEHP (200 μM, 24 h) or CCCP (2.5 μM, 8 h) respectively, or pre-exposed to MEHP (200 μM) for 24 h, followed by CCCP (2.5 μM) for 8 h in the presence or absence of NAC (5 mM). The degradation of mitochondrial proteins was evaluated by Western blot. (D) HeLa-YFP-Parkin cells were either exposed to MEHP (200 μM) and CCCP (5 μM) respectively or in combination in presence or absence of NAC (5 mM) for 24 h. Cell morphology was observed under a microscope. Scale bar, 50 μm. (E) Cells were treated as in (D). Cell pellets were then harvested and subjected to PI live cell exclusion assay combined with flow cytometry to detect the cell viability. Shown were the representative dot-plots. (F) Statistical analysis of three independent experiments as in (E) was presented as means ± SD (**, P < 0.01; Student's t-test). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)