(111) The Effects of Long-Term Dietary Exposure to Diisononyl Phthalate on Markers of Female Reproductive Aging

Ramsés Santacruz-Márquez, Adira M. Safar, Mary J. Laws, Jodi A. Flaws

Department of Comparative Biosciences, University of Illinois Urbana-Champaign, Urbana, United States of America

 

Abstract Text:

The ovary is essential for producing healthy oocytes through the process of folliculogenesis.  Female mammals are born with a finite number of primordial follicles, which contain an immature oocyte surrounded by a single layer of squamous granulosa cells.  During folliculogenesis, immature primordial follicles develop into primary, preantral, antral, and pre-ovulatory follicles. The antral follicles either undergo atresia (death) or ovulate an egg for fertilization. Thus, depletion of follicles over time leads to the loss of ovarian function, infertility, and the onset of reproductive senescence.  Female reproductive aging is a serious public health concern because it is associated with early onset of infertility and increased risk of cardiovascular disease, depression, osteoporosis, and even early death.  Despite the profound impact of early reproductive aging on women’s health, little is known about the mechanisms underlying early reproductive aging. Reproductive aging is characterized by decreased follicle number, increased ovarian fibrosis, altered expression of telomere-associated genes (TAGs), and increased inflammation/oxidative stress. One likely major contributor to female reproductive aging is exposure to environmental endocrine-disrupting chemicals (EDCs), which are compounds that interfere with hormone synthesis, binding, and/or functions. Further, EDCs can accelerate ovarian follicle loss, leading to early ovarian failure and thus, early reproductive aging.  Phthalates are a class of EDCs widely used in polyvinyl chloride plastics, flooring, food packaging, personal care products, medical tubing, plastic toys, and paints. The use of diisononyl phthalate (DiNP), a member of the phthalate family, has been rising over the past several years. Thus, humans are continuously exposed to increasing levels of DiNP as evidenced by increasing DiNP metabolite levels in human samples.  Previous studies in our lab suggested that exposure to DiNP alters markers of reproductive aging in mice. However, limited data exist regarding the effects of exposure to DiNP on markers of ovarian aging and the mechanisms by which DiNP exposure accelerates the onset of reproductive senescence. We hypothesized that long-term exposure to DiNP affects markers of reproductive aging and induces inflammation in the ovaries of female mice. Adult female mice were exposed to DiNP (0.15 ppm, 1.5 ppm, or 1500 ppm) via the chow for 6 months. After dosing, the mice were euthanized, and some ovaries were collected and processed for picrosirius red staining (PSR) to determine fibrosis levels in the ovary. Other ovaries were used for qPCR analyses to determine changes in the expression of inflammation-, fibrosis-, and TAGs. Sera were collected and processed using a cytokine array to analyze the systemic inflammatory profile. Long-term exposure to DiNP affected both systemic and ovarian inflammatory status mainly by increasing several inflammatory-associated molecules compared to the control. DiNP exposure borderline increased the expression of Il18 (0.15 and 1500 ppm) with no changes in Nlrp3 and Il1b compared to control. Interestingly, DiNP exposure affected extracellular remodeling molecules as evidenced by borderline decreased Ptx3 (1.5 ppm) and increased Sele (1500 ppm) gene expression levels compared to the control. Finally, DiNP long-term exposure borderline increased the expression of the TAGs Trf1 and Trf2 (1.5 ppm) compared to control. Collectively, these data indicate that long-term exposure to DiNP alters markers of reproductive aging, suggesting the exposure can lead to the early onset of female reproductive aging. Supported by R01 ES028661, R01 ES034112, T32 HD 108075.