(95) Effect of Phthalate Exposure on the Aryl Hydrocarbon Receptor Pathway in the Mouse Ovary over Time

Angela E. Dean¹, Ramsés Santacruz-Márquez¹, Mary J. Laws¹, Justin Huff¹, Jodi A. Flaws¹

1. Department of Comparative Biosciences, University of Illinois Urbana-Champaign, Urbana, IL, United States

Abstract Text: Diethyl (2-ethylhexyl) phthalate (DEHP) and one of its replacements, diisononyl phthalate (DiNP), are phthalates that are commonly used in plastics and other consumer products, and both phthalates are known as endocrine-disrupting chemicals (EDCs). Exposure to these chemicals over a person’s lifetime is a concern, especially as women age. Most women reach menopause around the age of 51. However, other factors such as exposure to EDCs can influence the age at menopause. Recently, we have shown that a DEHP metabolite, mono-2-ethylhexyl phthalate (MEHP), alters aryl hydrocarbon receptor (AHR) signaling in ovarian cells in vitro. However, it is not known how these individual phthalates affect AHR signaling in the ovary and throughout reproductive aging in vivo. The AHR is a transcription factor that is a member of the Per/ARNT/Sim- basic helix-loop-helix (bHLH) family, which plays a role in ovarian follicle development, function, and xenobiotic metabolism. Once a ligand is bound, the AHR heterodimerizes in the nucleus with ARNT to promote transcription of its downstream targets such as CYP1A1 and CYP1B1. We hypothesized that DEHP and DiNP exposure alters expression of genes and proteins associated with the AHR signaling pathway in the ovary throughout reproductive aging. We treated adult female CD-1 mice with varying doses (0 ppm, 0.15 ppm, 1.5 ppm, or 1500 ppm) of DEHP or DiNP in the diet for 1 and 6 months. The 0.15 ppm and 1.5 ppm doses correspond to human daily exposure and occupational exposure, respectively. We also included a high dose (1500 ppm) to examine a range of doses. At each time point, ovaries were collected, and we examined the expression of genes and proteins associated with AHR signaling using quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC), respectively. DEHP and DiNP did not alter gene expression of Ahr, Cyp1a1, Cyp1b1, Arnt, or Ahrr (a downstream target of the AHR) in the whole ovary or protein expression of AHR, CYP1A1, or CYP1B1 in the antral follicles and corpora lutea (CL) after one month of exposure to these phthalates. The highest dose (1500 ppm) of DEHP increased the expression of Cyp1a1, but not the other genes examined in the AHR pathway after 6 months of exposure. DiNP had no effect on gene expression of this pathway in the ovary. DEHP (1500 ppm) and DiNP (1.5 and 1500 ppm) increased AHR in the antral follicles, but not in the CL after 6 months of exposure. Neither phthalate altered CYP1A1 or CYP1B1 in the antral follicles or CL after 6 months. Overall, the higher doses of DEHP and DiNP increased AHR expression in the antral follicles, and only DEHP increased ovarian expression of Cyp1a1 after 6 months of exposure. Collectively, these data indicate that longer phthalate exposure and increased age influenced the AHR pathway in the ovary. Supported by NIH T32 ES007326 and NIH R01 ES034112.