ALTERATION OF MITOCHONDRIAL MEMBRANE POTENTIAL (Delta psi M): POTENTIAL ROLE IN THE MECHANISM OF MEHP-INDUCED INHIBITION OF STEROID BIOSYNTHESIS IN MA-10 LEYDIG CELLS

INTRODUCTION AND OBJECTIVES Mono‐(2‐ethylhexyl) phthalate (MEHP) is the active metabolite of di‐(2‐ethylhexyl) phthalate (DEHP), a plasticizer with endocrine disruptor activity that is widely used in the manufacturing industry. MEHP inhibits luteinizing hormone (LH)‐stimulated steroid biosynthesis in Leydig cells by molecular mechanisms that remain unclear. In the present study, using MA‐10 mouse tumor Leydig cells, we examined changes in mitochondrial function caused by MEHP exposures in relationship to possible link to alteration of progesterone biosynthesis. METHODS The effect of MEHP on LH‐stimulated progesterone production was assessed after treating MA‐10 cells with (0–300 μM) MEHP for 24 hrs. Progesterone production was quantified by enzyme‐linked immunosorbent assay (ELISA). To measure oxygen consumption, cells were treated with MEHP (0–300 μM) for 24 hrs, and oxygen consumption was monitored using a Clarke‐type oxygen electrode. Mitochondrial membrane potential (ΔΨm) was assessed using the JC‐1 dye mitochondrial membrane potential indicator. Mitochondria‐derived superoxide was measured using the MitoSOX™ red mitochondrial superoxide indicator. RESULTS Exposures of MA‐10 cells to 0–300 μM MEHP caused inhibition LH‐stimulated progesterone biosynthesis in a dose‐dependent manner. The decrease in the Leydig ability to produce progesterone correlated with a loss in the Leydig cell mitochondrial membrane potential (ΔΨm), and was linked to perturbation of the capacity to energize the mitochondrial membrane. MEHP exposure was suggested to cause a disruption in the electron flow through the electron transport chain, evidenced by a decrease in the ability to consumption molecular oxygen and an increase in the generation of superoxide in the mitochondria. CONCLUSIONS Together, these observations suggest that MEHP inhibits steroid biosynthesis in MA‐10 cells via a mechanism involving a loss of the mitochondrial membrane potential (ΔΨm) and an increase in the oxidative stress level, two crucial changes known to affect mitochondrial function and cause inhibition of STAR‐mediated cholesterol transport and steroid biosynthesis. Support or Funding Information This material is based upon work supported by NIH grant SC2GM099578 and by the North Carolina Biotechnology Center, NCBC grant #2019‐IDG‐1017.