(92) Acute Exposure to Di(2-ethylhexyl) Phthalate or Diisononyl Phthalate Leads to Increased Inflammation and Oxidative Stress in the Uterus of Mice

Adriana R. Andrus¹, Lyda Y. Parra Forero¹, Coba N. Sexton^1, Kalysta C. Liu¹, Sarah M. Rush¹, Mary J. Laws², Jodi A. Flaws², Romana A. Nowak¹

¹Department of Animal Sciences, College of Agricultural, Consumer and Environmental Sciences, University of Illinois, Urbana, IL, USA; ²Department of Comparative Biosciences, College of Veterinary Medicine at the University of Illinois Urbana-Champaign (UIUC), Urbana, USA

Abstract Text:

Phthalates are a class of synthetic compounds known as endocrine-disrupting chemicals (EDCs), meaning they are exogenous substances that alter the functions of the endocrine system. They are frequently used in consumer products such as personal care items, medical equipment, and food products. Two of the most common phthalates, diisononyl phthalate (DiNP) and di(2-ethylhexyl) phthalate (DEHP), are plasticizers used to improve the flexibility and durability of the numerous plastics they are found in.  Both DEHP and DiNP exposure have been shown to have negative effects on female reproductive health, likely due to their EDC properties. The goal of this study was to investigate the impacts of acute DEHP and DiNP exposure on the female reproductive tract of mice with a focus on inflammation and oxidative stress responses. Over a 10-day period, adult female CD-1 mice were orally dosed with DEHP (0, 20 μg/kg/day, 200 μg/kg/day, or 200 mg/kg/day) or DiNP (0, 20 μg/kg/day, 100 μg/kg/day, or 200 mg/kg/day). The mice were euthanized when they were in diestrus, and the uteri were collected for histology and RNA analysis. Fixed tissue sections were stained with hematoxylin and eosin to measure morphological changes. Immunohistochemistry was performed for the proliferation marker Ki67, for the smooth muscle cell and pericyte marker α-smooth muscle actin, and the macrophage marker CD68. Quantitative PCR (qPCR) was used to investigate changes in the levels of gene expression of genes associated with the inflammasome (IL18, IL1β, Nlrp3), with inflammation (IL6 and IL10), and with regulation of responses to oxidative stress (Sod1, Cat, Gpx1, and Prdx2). Data were normalized to the housekeeping genes GAPDH and Rplp0 (n=6/treatment group). Mice treated with DEHP at 200ug/kg/day and DiNP at 200mg/kg/day showed an upregulation in the expression of the inflammasome genes IL18, IL1β, and Nlrp3, while those treated with DEHP at 20ug/kg/day showed upregulation for IL1β and Nlrp3. DEHP treatment did not increase either Il10 or Il6 gene expression while DiNP at 200 mg/kg/day increased Il10.  Genes involved in the oxidative stress response showed different patterns for the two phthalate chemicals.  DEHP treatment did not cause any changes in Sod1, Cat, or Gpx1 but all three doses markedly increased levels of Prdx2.  DiNP treatment at 20 ug/kg/day caused an increase in Prdx2.  Interestingly, DiNP treatment at the higher doses decreased levels of Sod1, Cat, and Gpx1.  Histological analysis showed that acute exposure to DiNP 200mg/kg/day resulted in a decreased thickness of the outer myometrium. All doses of DiNP caused a thinning of the epithelial cells lining the lumen with a decreased height, while DEHP only at 200mg/kg/day caused the same effect. Analysis of CD68 staining showed a significant increase in macrophage influx when dosed with DEHP at 20 μg/kg/day and 200 μg/kg/day DEHP as well as DiNP at 200mg/kg/day. Staining with Ki67 revealed no changes in cell proliferation. These results indicate that acute exposure to DiNP and DEHP leads to increased inflammation and oxidative stress in the uterus. This study also provides insight into the fact that the two phthalates do not always act in a similar manner and the dose with the most significant impact is not the same for both chemicals. (Funded by NIH ES034112 to RAN and JAF).