MEHP induces alteration of mitochondrial function and inhibition of steroid biosynthesis in MA-10 mouse tumor Leydig cells

Abstract

Di-(2-ethylhexyl) phthalate (DEHP) is a plasticizer that is widely used in manufacturing. Previous studies have shown that mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of DEHP, has inhibitory effects on luteinizing hormone (LH)-stimulated steroid biosynthesis by Leydig cells. The molecular mechanisms underlying its effects, however, remain unclear. In the present study, we examined the effects of MEHP on changes in mitochondrial function in relationship to reduced progesterone formation by MA-10 mouse tumor Leydig cells. Treatment of MA-10 cells with MEHP (0-300 μM for 24 h) resulted in dose-dependent inhibition of LH-stimulated progesterone biosynthesis. Biochemical analysis data revealed that the levels of the mature steroidogenic acute regulatory protein (STAR), a protein that works at the outer mitochondrial membrane to facilitate the translocation of cholesterol for steroid formation, was significantly reduced in response to MEHP exposures. MEHP also caused reductions in MA-10 cell mitochondrial membrane potential (ΔΨm) and mitochondrial respiration as evidenced by decreases in the ability of the mitochondria to consume molecular oxygen. Additionally, significant increases in the generation of mitochondrial superoxide were observed. Taken together, these results indicate that MEHP inhibits steroid formation in MA-10 cells at least in part by its effects on mitochondrial function.

Keywords: Mitochondria; Phthalates; Steroidogenesis.

Fig. 1.. MEHP inhibits LH-stimulated progesterone production by MA-10 Leydig cells.

MA-10 cells were treated with MEHP (10-300μM) for 24 h and then with LH for 2 h. (A) Effect of MEHP on LH-stimulated progesterone production. (B) Dose-response effects of MEHP on cell viability and cytotoxicity. Data are presented as mean ± SEM of three separate experiments. * P < 0.05; ** P < 0.01.

Fig. 2.. Treatment with 22HC but not dcAMP alleviates the inhibitory effect of MEHP on progesterone production by MA10 cells.

MA10 cells were treated with DMSO-containing culture medium alone (control) or culture medium containing MEHP (300μM) for 24 h. At the end of the incubation time, cells were incubated with LH, dbcAMP or 22HC for 2 h. Culture media were analyzed for progesterone. Data are presented as mean ± SEM of three separate experiments. * P < 0.05; ** P < 0.01.

Fig. 3.. Effect of MEHP on the synthesis of the 30 kDa STAR protein.

(A) MA-10 cells were incubated in the absence or presence of LH (100 ng/mL) for 0.5, 1, 2 or 3 h. Total proteins were analyzed by immunoblot analysis using rabbit anti-mouse STAR antibodies. For loading control, levels of α-tubulin in samples were assessed using rabbit anti-mouse α-tubulin antibodies. (B) STAR protein band density were analyzed by densitometry using ImageJ software, and normalized to that of α-tubulin, and converted to percent control. (C) MA-10 cells were incubated with DMSO or MEHP (10-300μM) for 24 h followed by their stimulation with LH (100 ng/mL) for 2 h. STAR protein levels in samples were examined by immunoblot analysis. (D) Bar graph representation of relative band density. Data are presented as mean ± SEM of three separated experiments. * P < 0.05; ** P < 0.01.

Fig. 4.. MEHP induces ROS generation by the mitochondria of MA-10 cells.

MA10 cells were incubated with DMSO or MEHP (10-300μM) for 24 h and then with DCFH-DA or MitoSox-red. The fluorescence intensity of 10 × 10³ cells was quantified using fluorescence-activated cell sorting-scan analysis. (A) Intracellular ROS (hydrogen peroxide) production (DCF-positive cells) in relationship to increasing MEHP dose. (B) ROS production per cell in response to increasing MEHP concentration (Mean Fluorescence Intensity). (C) Representative dot plots of superoxide production by cells treated with MEHP. Cells in the upper-left quadrant (low fluorescence) migrate toward the upper right quadrant (high fluorescence) in response to MEHP treatment. (D) Percent positive superoxide producing cells. (E) Histogram representation of intracellular superoxide production induced by MEHP. (F) Bar graph representations of average level of superoxide production per cell (Mean Fluorescence Intensity).

Fig. 5.. MEHP exposures cause loss of mitochondria oxygen consumption capacity by MA-10 cells.

MA10 cells were incubated with DMSO or MEHP (10-300μM) for 24 h. The rate of oxygen consumption by 1 × 10⁶ cells was monitored. Data are mean ± SEM from three experiment. * P < 0.05; ** P < 0.01.

Fig. 6.. Evaluation of mitochondria membrane potential, ΔΨm, in MA-10 Leydig cells following MEHP treatment.

MA-10 cells were incubated with DMSO or MEHP (10-300μM) for 24 h and then in PBS containing JC-1. Changes in mitochondrial membrane potential in response to increasing concentrations of MEHP were assessed by measuring JC-1-derived fluorescence. (A) Representative dot plots of mitochondria in MA-10 cells treated with vehicle (DMSO) or MEHP. Gating strategy was set to select cells containing functional mitochondria with normal membrane potential in the upper right quadrant (orange color) and dysfunctional mitochondria with low membrane potential in the low-right quadrant (green color). Background fluorescence was designated as “unstained” (no JC-1 added). As a positive control, JC-1 fluorescence was assessed following exposure of the cells to CCCP. (B) Ratios of cells with normal potential (orange) to lower potential (green). Data are mean ± SEM of three separate experiments. * P < 0.05; ** P < 0.01.

Fig. 7.. Effect of MEHP on the synthesis of HIF-1α, CYP11A1 and MnSOD proteins.

(A) MA-10 cells were incubated for 24 h in the absence or presence of MEHP (10-300μM). Total proteins were extracted and analyzed by immunoblot analysis using rabbit anti-mouse HIF-1α, MnSOD, or CYP11A1. (B) Band densities were analyzed by densitometry using ImageJ software, normalized to that of α-tubulins, and converted to percent control. Data represent mean ± SEM of three separate experiments. * P < 0.05; ** P < 0.01.

Fig. 8.

Proposed mechanism of MEHP-induced inhibition of steroidogenesis by MA10 Leydig cells.