Dibutyl Phthalate (DBP) Induces Cell Cycle Arrest and Apoptosis in Ovarian Antral Follicles.

Dibutyl phthalate (DBP) is found in plastics, cosmetics, insecticides and oral medications. We have shown previously that DBP (1000 μg/mL) inhibits growth of mouse antral follicles treated in vitro. The purpose of this study was to investigate the mechanisms by which DBP inhibits follicle growth. We hypothesized that DBP disrupts the expression of genes that regulate the cell cycle and inhibits proliferation of follicular cells. To test our hypothesis, antral follicles were isolated from adult CD-1 mice (32-37 days old) and individually exposed (n= 8-12/culture) to DBP (1-1000 μg/mL) or vehicle (dimethylsulfoxide, DMSO) for 24 h. Following culture, follicles were subjected to qPCR analysis for the expression of cyclins D2 (Ccnd2), E1 (Ccne1), A2 (Ccna2) and B1 (Ccnb1), which are known cell cycle regulators. DBP treatment decreased expression of Ccnd2, Ccne1, Ccna2 and Ccnb1 compared to control follicles (p=0.05). Decreased levels of cyclins are suggestive of cell cycle arrest, thus, we determined the expression of cyclin-dependent kinase inhibitor 1A (Cdkn1a), which is an indicator of stress-activated arrest. Expression of Cdkn1a was greater in DBP-treated follicles than in controls, but this observation was only statistically significant in the 10 and 1000 μg/mL groups. Further, because altered expression of cyclins and increased expression of Cdkn1a were also observed in follicles treated with doses that do not affect follicle growth, we hypothesized that at higher doses DBP also triggers apoptosis. Therefore, we determined the expression of BH3 interacting domain death agonist (Bid), which is activated via the same pathway as Cdkn1a but promotes apoptosis rather than cell cycle arrest. DBP at 1-100 μg/mL did not alter the expression of Bid, but at 1000 μg/mL it increased the expression of Bid compared to controls (p=0.05). These data suggest that DBP inhibits antral follicle growth by altering the expression of important cell cycle regulators and causing cell cycle arrest. The observation that DBP increased the expression of the pro-apoptotic gene Bid only at the highest dose, suggests that DBP may act via different mechanisms depending on dose. Supported by NIH ES019178 (JAF), Billie Field Fellowship (ZRC and WW).