Di-2-ethylhexyl phthalate antagonizes the action of retinoic acid during neonatal development

Zulema Garcia

Humans are constantly exposed to phthalates, which is a serious concern since human phthalate exposure has been linked to various developmental diseases and reproductive abnormalities. Di-(2-ethylhexyl) phthalate (DEHP) is the highest produced phthalate shown to cause testicular toxicity, manifesting as decreased testosterone levels and sperm counts, and abnormal testicular morphology. Retinoic acid (RA), a metabolite of vitamin A, is essential for the progression of spermatogenesis, especially in the transition of progenitor spermatogonia to differentiated spermatogonia and induction of meiosis. RA action is mediated through retinoic acid receptors (RARs), while DEHP acts through peroxisome proliferator-activated receptors (PPARs). Both receptor families work in the nucleus as transcription factors, where each heterodimerizes to a retinoid X receptor (RXR). It has been shown previously that DEHP antagonizes RA action in cultured somatic Sertoli cells. DEHP causes the translocation of RARA, an RAR subtype, out of the nucleus and into the cytoplasm, while increasing the nuclear localization of PPARA and PPARG, two PPAR subtypes. However, a similar DEHP mechanism of action has not been established in v germ cells. In this work, the hypothesis to be tested is that DEHP inhibits RA action in male germ cells, mediated through PPARs inhibiting RAR signaling activity during neonatal development. To test this hypothesis, a transgenic RARE-hsp68/lacZ mouse model was utilized, with lacZ gene expression ([Beta]-galactosidase) driven by the hsp68 promoter containing a retinoic acid response element (RARE). This mouse model reports [Beta]-galactosidase ([Beta]-gal) activity, directly related to RAR activity, when a RAR/RXR heterodimer is allowed to bind to RARE. Lactational DEHP exposure to transgenic mouse pups caused a significant reduction in [Beta]-gal reporter expression in germ cells during neonatal development. Immunohistochemistry studies identified the DEHP-affected germ cells to primarily be progenitor and differentiated spermatogonia. Similarly, cultured testicular tissue exposed to WY-14643, a potent peroxisome proliferator and PPARA agonist, showed a decrease in [Beta]-gal activity in spermatogonial germ cells. WY-14643 was shown to act through PPARA to inhibit RAR signaling activity, as RNAi-induced inhibition of Ppara rescued [Beta]-gal reporter expression following WY-14643 treatment. These results suggest that PPARA is involved in the DEHP-induced inhibition of RAR signaling.

Di-2-ethylhexyl phthalate antagonizes the action of retinoic acid during neonatal development

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