Environmentally relevant mixtures of phthalates and phthalate metabolites differentially alter the cell cycle and apoptosis in mouse neonatal ovaries†

Phthalates are a group of chemicals used as additives in various consumer products, medical equipment, and personal care products. Phthalates and their metabolites are consistently detected in humans, indicating widespread and continuous exposure to multiple phthalates. Thus, environmentally relevant mixtures of phthalates and phthalate metabolites were investigated to determine the effects of phthalates on the function of the ovary during the neonatal period of development. Neonatal ovaries from CD-1 mice were cultured with dimethyl sulphoxide (DMSO; vehicle control), phthalate mixture (0.1-100 μg/mL), or phthalate metabolite mixture (0.1-100 μg/mL). The phthalate mixture was composed of 35% diethyl phthalate, 21% di(2-ethylhexyl) phthalate, 15% dibutyl phthalate, 15% diisononyl phthalate, 8% diisobutyl phthalate, and 5% benzylbutyl phthalate. The phthalate metabolite mixture was composed of 37% monoethyl phthalate, 19% mono(2-ethylhexyl) phthalate, 15% monobutyl phthalate, 10% monoisononyl phthalate, 10% monoisobutyl phthalate, and 8% monobenzyl phthalate. After 96 h of culture, ovaries were harvested for histological analysis of folliculogenesis, gene expression analysis of cell cycle and apoptosis regulators, and immune staining for cell proliferation and apoptosis. The metabolite mixture significantly decreased the number and percentage of abnormal follicles (100 μg/mL) compared to controls. The metabolite mixture also significantly increased the expression of cell cycle inhibitors (100 μg/mL) and the antiapoptotic factor Bcl2l10 (10 μg/mL) compared to controls. The phthalate mixture did not significantly alter gene expression or follicle counts, but ovaries exposed to the phthalate mixture (0.1 μg/mL) exhibited marginally significantly increased apoptosis as revealed by DNA fragmentation staining. Overall, these data show that parent phthalates and phthalate metabolites differentially impact ovarian function.