Long-Term Phthalate Exposure Exacerbates the Genotoxic Estrogen Metabolic Pathway, and Oxidative Stress and Induces Malignant Transformation in Normal Human Prostate Epithelial Cells

Abstract Epidemiological studies suggest a strong association between phthalate exposure and proliferative diseases of the prostate. However, experimental evidence for phthalate-induced malignant transformation in normal prostate epithelial cells is lacking. Therefore, the objective of this study was to evaluate the effects of both the parent di-2-ethylhexyl phthalate (DEHP) and its primary metabolite mono-2-ethylhexyl phthalate (MEHP) on the proliferation and malignant transformation of RWPE-1 normal prostate epithelial cells. Cells were exposed to DEHP and MEHP separately at concentrations of 1 μM and 100 μM for 72 h to evaluate acute effects and for a long term (8 weeks) at 1 μM to evaluate carcinogenic potential. Cell viability and oxidative stress were determined by MTT and DCFH assays, respectively. Cell cycle analysis was performed using flow cytometry, whereas gene expression changes were analyzed at the transcript level by qRT-PCR and at the protein level by Western blot. Phthalate-induced malignant transformation of cells was evaluated by soft agar assay. Results revealed that both DEHP and MEHP caused a decrease in cell viability and increased the intracellular levels of reactive oxygen species (ROS) at 72 h. However, long-term exposure to DEHP and MEHP resulted in increased proliferation and malignant transformation of RWPE-1 cells. Significant changes in the expression of selected genes not only confirmed increased proliferation and oxidative stress but also revealed the activation of a genotoxic estrogen metabolic pathway as an underlying mechanism for phthalate-induced malignant transformation. Decreased expression of the tumor suppressor PTEN and the increased expression of oncogenic c-Myc as well as its upstream regulator β-Catenin further suggest that phthalates cause malignant transformation through the activation of the c-Myc-β-Catenin pathway. This study reveals for the first time that long-term exposure to both DEHP and its metabolite can induce malignant transformation in prostate epithelial cells through activation of the genotoxic estrogen metabolic pathway and oncogenic c-Myc-β-Catenin signaling.