DEHP interferes with pituitary androgenic signaling in vitro

Abstract

DEHP phthalate is an endocrine disruptor that exhibits affinity for the androgen receptor (AR) and interacts with the ligand-binding pocket, suggesting its potential to interfere with AR signaling. The target organ of this study is the pituitary, which is regulated by testosterone through the AR expressed in its different cell populations. Considering this, the objective was to determine whether DEHP affects androgenic signaling in the pituitary using an in vitro system.

Three independent primary pituitary cultures from adult male rats were stimulated with DEHP 0.1 or 1µM for 72 hours. In parallel, co-stimulation with testosterone 0.01µM was performed during the final 15 or 30 minutes. The cultures were processed to determine the subcellular localization of the AR by immunofluorescence and subcellular fractionation followed by western blot (WB) and analysis of MAPK pathway activation by determining ERK phosphorylation (pERK/tERK) by WB. Statistical analysis ANOVA-Tukey, p<0.05 considered statistically different.

Both DEHP doses significantly decreased the percentage of AR+ nuclei by approximately 32% compared to the control. Testosterone increased the percentage of AR+ nuclei, while co-incubation of this hormone with both DEHP doses significantly reduced AR+ nuclei by approximately 60% compared to testosterone alone at both analyzed times. AR protein expression showed a similar pattern to that observed by immunofluorescence, with a reduction in nuclear AR and an increase in cytoplasmic AR in cells exposed to DEHP alone or co-incubated with testosterone. Both DEHP and testosterone alone significantly increased ERK1/2 phosphorylation compared to control. DEHP/testosterone co-stimulation for 30 minutes showed pERK levels higher than those observed with testosterone alone, similar to individual DEHP treatment.

These results demonstrate that DEHP retains AR in the cytoplasm, suggesting that it inhibits testosterone-induced nuclear translocation and alters androgenic signaling by increasing testosterone-induced ERK activation.