Mono-(2-ethylhexyl) Phthalate Inhibits Expression of Antioxidant Enzymes in Mouse Antral Follicles.

Abstract

At birth, the ovaries contain a finite number of primordial follicles. Only a limited number of these primordial follicles will mature into antral follicles. Antral follicles are essential for the proper functioning of the ovary because they synthesize and secrete sex steroid hormones important for the maintenance and regulation of estrous cyclicity and fertility. Di-(2-ethylhexyl) phthalate (DEHP) is a plasticizer found in many commercially used products such as plastic bottles, IV tubes, toys, fragrances and cosmetics. DEHP, a toxic parent compound, can be metabolized by esterases in the body to form mono-(2-ethylhexyl) phthalate (MEHP). MEHP is known to restrict growth, reduce estradiol levels and cause death of antral follicles via apoptosis. Many pathways can lead to cell death via apoptosis, but oxidative stress is a pathway specifically known to cause apoptosis in both non-reproductive and reproductive tissues. Balance between reactive oxygen species production and detoxification by antioxidant enzymes such as Cu/Zn superoxide dismutase (SOD1), glutathione peroxidase (GPX), and catalase (CAT) keep the cell in a healthy and reduced state. Any environmental toxicant exposure that disrupts this balance results in oxidative stress in the cell. Since it was not known if MEHP induces oxidative stress in antral follicles, the goal of this study was to test the hypothesis that MEHP induces the oxidative stress pathway by suppressing the expression of SOD1, GPX and CAT in antral follicles. To test this hypothesis, antral follicles were mechanically isolated from adult cycling CD-1 mice and cultured for 96 hours in alpha-minimal essential media containing either MEHP (0.1-10 µg/ml) or the vehicle control, dimethylsulfoxide (DMSO). Following culture, follicles were subjected to quantitative real time PCR to measure mRNA levels of the Sod1, Gpx, and Cat genes. The results indicate that MEHP significantly decreased mRNA expression of Sod1 compared to controls (DMSO=67.33±8.51 genomic equivalents (GE), MEHP 0.1 µg/ml=24.03±7.73 GE, MEHP 1 µg/ml=19.03 ±4.25 GE, MEHP 10µg/ml=18.73 ±9.41 GE; n=3; p≤0.05). In addition, MEHP significantly decreased mRNA levels of the Gpx gene compared to controls (DMSO=29.16±3.99 GE, MEHP 0.1 µg/ml=3.69±0.45 GE, MEHP 1 µg/ml=4.90±2.04 GE, MEHP 10 µg/ml=2.47±0.60 GE; n=3; p≤0.05). However, the expression levels of Cat were not significantly affected by MEHP exposure compared to controls (DMSO=9.78±0.81 GE, MEHP 0.1 µg/ml=3.85±2.13 GE, MEHP 1 µg/ml=3.47±1.21 GE, MEHP 10µg/ml=4.47±1.77 GE; n=3). Therefore, it is possible that MEHP represses the expression of specific antioxidant enzymes, allowing apoptosis of antral follicles via the oxidative stress pathway. Support: R01ES012893; T32ES07326. (poster)