Epidemiologically relevant phthalate mixture and mono(2-ethyl-5-hydroxyhexyl) phthalate exposure alter cell energy metabolism in primary mouse granulosa cells

Abstract

Many products including plastic food containers, medical tubing, and personal care products contain phthalate diesters. Phthalates have been shown to negatively affect the female reproductive system. However, the ovarian cellular impacts of monoester phthalates, the metabolites of phthalate diesters, are largely unknown. This study tested the hypothesis that a monophthalate metabolite mixture or single monophthalate affects cell energy metabolism in granulosa cells. To test this hypothesis, granulosa cells were exposed to vehicle control, the mean urinary level and 100-fold higher monophthalate mixture (2.0 µM and 200.0 µM), or mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) (0.22 µM and 22.0 µM) for 24 or 72 h. Gene expression was assessed for antioxidant enzymes, glycolytic enzymes, and glucose transporters. Cellular metabolism was assessed by Agilent Seahorse assays. MEHHP did not alter expression of the antioxidant enzymes, glycolytic enzymes, or glucose transporters compared to control. However, the monophthalate mixture significantly increased expression of Ldha (200.0 µM, 72-hour exposure) and Glut1 (2.0 µM, 24-hour exposure) compared to control. MEHHP (22.0 µM) increased total ATP production rate at 24 h compared to control, but did not change total ATP production after 72 h. Further, the mixture (2.0 µM and 200.0 µM) altered total ATP production rates compared to control at the 72-hour and 24-hour time points. Short-term phthalate exposure led to significant effects on ATP production rates, making this endpoint a logical indicator of early phthalate toxicity. These data indicate that a monophthalate mixture as well as single monophthalate exposure alter cellular metabolism in granulosa cells.

Keywords: ATP production; Chemical mixtures; Endocrine disrupting chemicals; Female reproduction; Ovaries; Phthalates; Reproduction.