Systemic and Reproductive Effects of Persistent DBP Exposure in Superovulated Mice

Abstract

Phthalates are endocrine disrupting chemicals commonly used in plastics to make them flexible. Due to their prevalence in our environment and their negative associations to female reproductive health outcomes, it is imperative to study the effects of environmentally relevant levels of phthalates on female reproduction. We have previously modeled human relevant exposures to di-n-butyl phthalate (DBP) using a mouse model and confirmed its metabolite in the ovary and short-term effects on natural ovulation following in vivo dosing. The aim of this project was to investigate whether persistent oral dosing with DBP beyond an entire follicle developmental cycle (~53 days in the rodent), causes systemic and/or reproductive perturbances in mice. We hypothesized that DBP would only negatively affect reproductive parameters (oocyte health and yield) and reproductive organ weights (ovaries, uterus, pituitary) with minimal systemic effects. CD-1 female mice, upon their first proestrus (~30days old), were weighed and pipet-fed daily for 60-62 days with one of three treatments: tocopherol-stripped corn oil control (CON, n=13), DBP at 10ug/kg/day (10DBP; n=13) or 100ug/kg/day (100DBP, n=12) and superovulated for oocyte collection. Mice treated with 100DBP spent significantly less time in Metestrus (p=0.0482), significantly more time in Diestrus (p=0.0394) and had significantly increased estrous cycle lengths (p=0.0314) compared to the CON group. Mice in the 100DBP group, after 4 weeks of dosing and until the end of the study, on average, weighed more than the CON group (p≤0.05). No differences were observed between treatments in terms of reproductive and most non-reproductive organ weights (uteri, ovaries, pituitaries, adrenals, spleen, kidneys), except for liver weights which was significantly increased in the 100DBP (p=0.0177). However, when the liver and spleen weights were normalized relative to body weight, there was no difference amongst groups. There was a decreased trend of ova retrieved in the 100DBP group compared to CON (p=0.1023). There was no significant nor trending difference between percent of ‘healthy’ and ‘unhealthy’ oocytes. There was no difference in total antral follicle counts, nor in the subclassifications of small or large antral follicles. However, the DBP treated groups had higher mean large antral follicle counts than the CON groups (0.4±0.2449 vs 1.6±0.6782 vs 1.4±0.7483). There was a trend of fewer CL numbers in the 10DBP group compared to the CON group (p=0.0778), though 100DBP was unaffected. There was a significant decrease in LUF numbers in both 10 & 100DBP groups compared to the control (p=0.0083 and p=0.0107, respectively). Lastly, the sera hormone analyses showed significant differences in progesterone and 17B-estradiol levels. There was a strong trend in lower progesterone levels in the 10DBP when compared to the 100DBP group (p=0.0536), though neither DBP treatment group was significantly different from the CON group. However, both 10 & 100DBP treatment groups had significantly increased serum estradiol levels compared to the CON group (p=0.0190 and p=0.0009). Altogether, our findings provide new knowledge regarding the impact of persistent DBP exposure on systemic and reproductive parameters in newly pubertal mice during the entire length of a cycle of female gamete production, a vital process for female fertility. This work was supported by NIH Grant R01ES026998 (ZRC).