Mono-(2-Ethylhexyl) Phthalate Induces Oxidative Stress and Inhibits Growth of Mouse Ovarian Antral Follicles1

Abstract

Mono-(2-ethylhexyl) phthalate (MEHP) is the active metabolite of the most commonly used plasticizer, di-(2-ethylhexyl) phthalate, and is considered to be a reproductive toxicant. However, little is known about the effects of MEHP on ovarian antral follicles. Thus, the present study tested the hypothesis that MEHP inhibits follicle growth via oxidative stress pathways. The data indicate that MEHP increases reactive oxygen species (ROS) levels and inhibits follicle growth in antral follicles, whereas N-acetylcysteine (NAC; an antioxidant) restores ROS levels to control levels and rescues follicles from MEHP-induced inhibition of follicle growth. To further analyze the mechanism by which MEHP induces oxidative stress and inhibits follicle growth, the expression and activities of various key antioxidant enzymes (copper/zinc superoxide dismutase [SOD1], glutathione peroxidase [GPX], and catalase [CAT]) and the expression of key cell-cycle regulators (Ccnd2, Ccne1, and Cdk4) and apoptotic regulators (Bcl-2 and Bax) were compared in control and MEHP-treated follicles. The data indicate that MEHP inhibits the expression and activities of SOD1 and GPX; does not inhibit Cat expression; inhibits the expression of Ccnd2, Ccne1, Cdk4, and Bcl-2; but increases the expression of Bax compared to controls. Furthermore, NAC blocks these toxic effects of MEHP. Collectively, these data suggest that MEHP induces oxidative stress by disrupting the activities of antioxidant enzymes. This may lead to decreased expression of cell-cycle regulators and antiapoptotic regulators and increased expression of proapoptotic factors, which then may lead to inhibition of follicle growth.

Prenatal exposure to di-(2-ethylhexyl) phthalate (DEHP) affects reproductive outcomes in female mice

“…A decrease in ovarian weight, but no change or an increase in preantral follicle numbers could be explained by the follicles being smaller on average than follicles in control ovaries, with fewer granulosa cells per follicle. This possibility is supported by previous studies indicating that DEHP and its metabolite MEHP inhibit follicle growth [43-45]. …”

“…It is likely that DEHP, like other endocrine disrupting chemicals, exhibits non-monotonic dose response curves. Specifically, previous studies have indicated that DEHP has a non-monotonic dose response curve specific to gene expression in cultured mouse antral follicles [44-45]. Further, studies in males have shown non-monotonic responses in terms of testicular and serum testosterone levels and anogenital distance and brain aromatase activity [25, 46].…”

Bisphenol A exposure inhibits germ cell nest breakdown by reducing apoptosis in cultured neonatal mouse ovaries

“…Oxidative stress is a major factor that triggers the apoptosis pathway (reviewed in [14]), and endocrine disrupting chemicals have been shown to cause oxidative stress in ovaries [17,30]. Thus, we examined whether BPA treatment affects expression of major antioxidant enzymes ( Sod1 , Cat , Gpx , and Gsr ).…”

“…Many endocrine disrupting chemicals have been shown to cause oxidative stress in mouse ovaries [17,30], and germ cell nest breakdown is a process that is associated with apoptosis [40]. Thus, the present study also examined whether the ability of BPA to inhibit germ cell nest breakdown was due to effects on the oxidative stress and apoptosis pathways.…”

“…Ovaries were collected after 2, 4, and 8 days of culture (PND 2, 4, and 8), and subjected to ROS/RNS assays for measurement of total free radicals, including reactive oxygen species (ROS) and reactive nitrogen species (RNS) using an OxiSelect In vitro ROS/RNS Assay Kit (Cell Biolabs, Inc., San Diego, CA) according to the manufacturer’s instructions and as previously described by Wang et al [30]. Data were normalized to protein level (measured by BCA Protein Assay Kit, Thermo Scientific, Rockford, IL).…”