Effect of di-ethylhexyl phthalate (DEHP) on androgen related proteins in JEG-3 placental cells.

The placenta is a critical fetal endocrine organ that plays an important role in fetal development, xenobiotic detoxification, and sex steroid homeostasis. Although the importance of the placenta in endocrine homeostasis is known, the effect of endocrine disruptors, like phthalates, on placental endocrine function needs further exploration. A common phthalate that expectant mothers can be exposed to is di-ethylhexyl phthalate (DEHP) which is used to make flexible plastics. We aim to determine the effect of DEHP on the androgen related proteome in JEG-3 placental cell model. JEG-3 cells were seeded in 6-well plates at 100,000 cells per well and treated with 0 (vehicle control), 1, 10, and 100 mM DEHP for 72 hours. The cells were harvested, homogenized, and protein sample was digested using an optimized protocol. Global proteomics data were acquired using a Q-Exactive HF Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA). The data were processed with MaxQuant (version 1.6.8.0) using the Andromeda search engine to search the high-resolution MS/MS spectra against the human proteome database curated from Uniprot. Label-free quantification (LFQ) intensities of androgen related proteins were compared across control and treatment groups. Out of the total 2368- 2530 proteins detected, 2085 proteins were common in control and all treatment groups. The control group had 49 unique proteins and DEHP treatment showed 63, 42, and 119 unique proteins to 1, 10 and 100 mM, respectively. ABCG2 was uniquely identified in the higher DEHP treatment group. DEHP treatment elevated the levels of aromatase (CYP19A1) and aldo-keto reductase 1B1 (AKR1B1) in a dose-dependent manner with a 291% and 59% increase in the 100 mM treatment, respectively. Steryl-sulfatase (STS), estradiol 17-beta-dehydrogenase 11, and aldehyde dehydrogenase 3A2levels were unaltered by the DEHP treatments. Determining how environmental endocrine disruptors, like DEHP, affects the placental endocrine pathways is vital to mitigating the toxicity. The elevation of aromatase with DEHP treatment may lead to estrogenic effects as CYP19A1 converts androstenedione and testosterone to estrogens, resulting in hormone imbalance which can cause adverse fetal outcomes both physically and neurodevelopmentally.