Effects of in vitro exposure to dibutyl phthalate, mono-butyl phthalate, and acetyl tributyl citrate on ovarian antral follicle growth and viability

Abstract

Dibutyl phthalate (DBP) is present in consumer products and the coating of some oral medications. Acetyl tributyl citrate (ATBC) has been proposed as an alternative to DBP because DBP causes endocrine disruption in animal models. Following ingestion, DBP is converted to its main metabolite mono-butyl phthalate (MBP) which has been detected in >90% of human follicular fluid samples. Previous studies show that DBP reduces the number of antral follicles present in the ovaries of mice. Thus, this study was designed to evaluate the effects of DBP, MBP, and ATBC on in vitro growth and viability of mouse ovarian antral follicles. Antral follicles were isolated from CD-1 females (PND32-37) and treated with vehicle, DBP, MBP, or ATBC (starting at 0.001 and up to 1000 μg/ml for DBP; 24-72 h). Follicle diameter, ATP production, qPCR, and TUNEL were used to measure follicle growth, viability, cell cycle and apoptosis gene expression, and cell death-associated DNA fragmentation, respectively. While MBP did not cause toxicity, DBP exposure at ≥10 μg/ml resulted in growth inhibition followed by cytoxicity at ≥500 μg/ml. ATBC increased the number of nongrowing follicles at 0.01 μg/ml and did not affect ATP production, but increased TUNEL positive area in treated follicles. Gene expression results suggest that cytotoxicity in DBP-treated follicles occurs via activation of cell cycle arrest prior to follicular death. These findings suggest that concentrations of DBP ≥10 μg/ml are detrimental to antral follicles and that ATBC should be examined further as it may disrupt antral follicle function at low concentrations.

Keywords: acetyl tributyl citrate; antral follicle; apoptosis; cell cycle; dibutyl phthalate; endocrine disruptor; mono-butyl phthalate; ovary; phthalate substitute; toxicology.

Figure 1.

Effect of DBP, MBP, and ATBC exposure on the growth rate of antral follicles in vitro. Early antral follicles (n = 6–12 follicles per treatment) were obtained from the ovaries of untreated CD-1 mice and cultured as described in Materials and Methods section. Follicle diameter was measured every 24-h period and normalized to baseline diameter to obtain percent change. Data are presented as mean percent change ± SEM. Asterisks (*) indicate significantly different from control.

Figure 2.

Effect of DBP, MBP, and ATBC exposure on the growth pattern of antral follicles in vitro. Early antral follicles (n = 6–12 follicles per treatment) were obtained from the ovaries of untreated CD-1 mice and cultured as described in Materials and Methods section. Based on their growth rate, follicles were further classified into “growing,” “slow-growing,” and “nongrowing.” Data are presented as mean percent of follicles ± SEM with asterisks (*) indicating significantly different from control. Gray asterisks indicate statistically significant differences in the slow-growing follicles, while the black asterisks indicate so in nongrowing follicles.

Figure 3.

Effect of DBP, MBP, and ATBC exposure on the viability of antral follicle follicles in vitro. Early antral follicles (n = 6–12 follicles per treatment) were obtained from the ovaries of untreated CD-1 mice and cultured as described in Materials and Methods section. At the end of each culture, the ATP concentration within each follicle was determined using a luminescence assay. Data were normalized to the DMSO vehicle control and are presented as mean normalized luminescence ± SEM with asterisks (*) indicating significantly different from control.

Figure 4.

Effect of DBP exposure on cell cycle progression gene expression in cultured antral follicles. Early antral follicles (n = 6–12 follicles per treatment) were obtained from the ovaries of untreated CD-1 mice and cultured as described in Materials and Methods section. At the end of each culture, RNA was extracted and processed for qPCR analysis of Ccnd2 (A) and Ccne1 (B). Data were normalized to housekeeping genes and are presented as mean relative expression ± SEM with asterisks (*) indicating significantly different from control.

Figure 5.

Effect of DBP exposure on cell cycle arrest gene expression in cultured antral follicles. Early antral follicles (n = 6–12 follicles per treatment) were obtained from the ovaries of untreated CD-1 mice and cultured as described in Materials and Methods section. At the end of each culture, RNA was extracted and processed for qPCR analysis of Cdkn1a (A) and Cdkn2a (B). Data were normalized to housekeeping genes and are presented as mean relative expression ± SEM with asterisks (*) indicating significantly different from control.

Figure 6.

Effect of DBP exposure on apoptosis gene expression in cultured antral follicles. Early antral follicles (n = 6–12 follicles per treatment) were obtained from the ovaries of untreated CD-1 mice and cultured as described in Materials and Methods section. At the end of each culture, RNA was extracted and processed for qPCR analysis of Bax (A), Bid (B), and Bcl2 (C). Data were normalized to housekeeping genes and are presented as mean relative expression ± SEM with asterisks (*) indicating significantly different from control.

Figure 7.

Representative images of antral follicles subjected to TUNEL. Early antral follicles (n = 6–12 follicles per treatment) were obtained from the ovaries of untreated CD-1 mice, cultured, and processed for TUNEL as described in Materials and Methods section. Representative merged images show DAPI (blue) and TUNEL (green) staining in follicles treated in vitro with vehicle control (DMSO, A), apoptosis positive control (HU, B), DBP 10 μg/ml (C), 100 μg/ml (D), 500 μg/ml (E), 1000 μg/ml (F), and ATBC 0.01 μg/ml (G) for 48 h. Panels H and I show representative images of the assay negative (no enzyme added) and positive (DNAse digestion of tissue) controls, respectively. Images were taken at ×200 magnification. Scale bar represents 100 μm.

Figure 8.

Effect of DBP and ATBC exposure on TUNEL. Early antral follicles (n = 6–12 follicles per treatment) were obtained from the ovaries of untreated CD-1 mice, cultured, subjected to TUNEL, and quantified as described in Materials and Methods section. Data are presented as the mean percentage of TUNEL/DAPI area ± SEM with asterisks (*) indicating significantly different from control at the P < 0.05 level. Actual P-values are provided for groups showing visual trends.