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.

Assessing effects of germline exposure to environmental toxicants by high-throughput screening in C. elegans

“…In vitro studies of the effects of DBP exposure on Sertoli cell culture showed increased apoptosis stemming from inhibiting the PI3K/AKT and mTOR pathways which promote the proliferation and survival of sperm and the maintenance of testicular homeostasis [26]. Antral follicles isolated from female mice and exposed to DBP exhibited increased expression of the cyclin-dependent kinase inhibitors Cdkn1a and Cdkn2a and pro-apoptotic factors Bax and Bid along with down regulation of cyclin Ccnd2 resulting in growth inhibition and follicular death [28]. Permethrin has been shown to cause DNA damage in mitotic cells where exposure of peripheral blood mononuclear cells (PBMCs) induced breaks in the KMT2A and IGH genes which can be driver mutations for lymphoma and leukemia along with increased aneuploidy [36].…”

“…Higher levels have been detected in human urine, follicular fluid, and serum in different occupationally exposed groups [21,24]. In vitro and animal studies have shown that DBP disrupts the reproductive system resulting in inhibition of ovarian antral follicle growth and viability, altered gene expression in ovaries, testicular malformation and dysfunction, inhibition of spermatogenesis, and altered androgen signaling in males [25–28]. In humans, a significant inverse relationship has been observed between levels of DBP metabolites (i.e.…”

Pharmacokinetic Properties of Acetyl Tributyl Citrate, a Pharmaceutical Excipient

“…Rasmussen et al investigated the effects of in vitro exposure to ATBC on ovarian antral follicle growth and viability [17]. At a dose of 0.01 μg/mL, ATBC increased the number of nongrowing follicles and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive area in treated follicles, pointing to the possibility that ATBC may disrupt antral follicle function at low concentrations.…”

Proposed Key Characteristics of Female Reproductive Toxicants as an Approach for Organizing and Evaluating Mechanistic Data in Hazard Assessment

“…DBP decreases growth of cultured mouse antral follicles, the last stage of ovarian follicle development before the preovulatory stage (Craig et al. 2013; Rasmussen et al. 2017).…”

“…DBP causes arrest of follicle cells in the G1 phase of the cell cycle by up-regulating expression of cyclin-dependent kinase (CDK) inhibitors and down-regulating cyclins, resulting in decreased follicle growth (Craig et al. 2013; Rasmussen et al. 2017).…”