The Cellular Effects of Di(2-ethylhexyl) Phthalate in Non-Malignant Colonic Epithelia Involve Oxidative Stress

Human exposure to Di(2-ethylhexyl) Phthalate (DEHP) occurs through ingestion of contaminated food. Yet, the effects of DEHP on gastrointestinal toxicity at the cellular level are poorly understood and studies conducted to date have used malignant cell lines, limiting our understanding of molecular signaling in intestinal epithelia of otherwise healthy individuals. The objective of our study was to use a non-transformed, colonic epithelial cell line (Young Adult Mouse Colonocytes; YAMCs) to characterize the in vitro effects of DEHP on non-malignant colonic epithelia. A 72 h DEHP exposure significantly reduced cell number and proliferation while short-term exposure increased: cellular apoptosis, <i>BAX</i> expression, Reactive Oxygen Species (ROS) production, gene expression linked to oxidative stress (<i>NRF2</i>, <i>GCLC</i>, <i>HO-1</i>, <i>CHOP</i>). Antioxidant pretreatment prior to DEHP exposure attenuated the phthalate's apoptotic effect, suggesting a link between oxidative stress and apoptosis. Using YAMCs with a CRISPR-deleted Aryl Hydrocarbon Receptor (AhR) we further showed that the apoptotic and pro-oxidative effects of the phthalate are partially mediated through AhR. In conclusion, we have demonstrated that DEHP-induced toxicity in non-malignant colonocytes is due to ROS-induced oxidative stress and subsequently, apoptosis. We have further demonstrated that these effects are partly mediated by the AhR, a mechanism that deserves further investigation. Future studies should build on these findings by (a) characterizing the specific mechanisms linking ROS production to apoptosis demonstrated in our model of exposure, (b) measuring the dynamics of the receptor following DEHP exposure and (c) examining these effects over a longer exposure period.