Di Lorenzo, Mariana (2017) Phthalates effects on male reproductive tract. [Tesi di dottorato]

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Phthalates are an interesting group of endocrine disruptors widely used in the manufacture of PVC. Dibutylphthalate (DBP) and di-2-ethylhexyl phthalate (DEHP) are the two most commonly used phthalates and toxicological studies showed their toxic effect for the reproduction. Thus, in the present research project, the effects of DBP and DEHP on male reproductive system have been evaluated using in vitro and in vivo studies. In vitro studies were used to evaluate DBP and 17-β-estradiol (E2) effects on human prostate adenocarcinoma epithelial cells (LNCaP). First we assessed the effects of DBP and E2 on the cell viability after 24h of exposure. DBP induced a cell proliferation decrease at 10-8M, instead E2 at 10-9M stimulated cell viability. RT-qPCR and western blot analysis were used to evaluate the expression of genes and proteins involved in the regulation of cell cycle such as MCT4, Cyclin D1, Ki-67. Then, to evaluate through which pathway DBP induced a decreased cell viability, we performed western blot for Bax an Bak, two pro-apoptotic proteins involved in intrinsic apoptosis pathway. DBP treatment strongly enhanced both Bax and Bak expression, suggesting its involvement in programmed cell death processes. Moreover, in order to study estrogen (ER) and androgen (AR) receptors involvement, we evaluated their expression with western blot after 24 h of exposure and their cellular localization with immunofluorescence after three different times of exposure (30’, 2h, 4h). DBP induced a minor expression of ERα and its cytoplasm-nucleus translocation after 4h of treatment; whereas DBP had no effects on ERβ and AR expression and cell localization. Results confirm that DBP may be involved in the deregulation of prostate cell cycle and it may interfere with estrogen hormonal receptor pathway. In vivo studies were used to evaluate the effects of different doses of DEHP on testis histopathology in neonatal rats after in utero and lactation exposure. Pregnant Wistar rats were gavaged from gestation day (GD) 7 to GD 21 and from postnatal day (PND) 1 to 6 with vehicle, 30, 300, 900 mg/kg bw/day DEHP. Gonocytes appeared to be enlarged and multinucleated only after treatment with high DEHP doses and the treatment did not affect neonatal Sertoli cells. Immunofluorescence for 3β hydroxysteroid dehydrogenase (3β-HSD) revealed that Leydig cells tended to group together in clusters dose dependently from DEHP 30 mg/kg/bw-d. Moreover, in rats treated with DEHP 900 mg/kg/bw-d, it was possible to note malformed cords with positive 3βHSD Leydig cells inside the tubules. Furthermore, DEHP treatment reduced cord diameters after exposure to all DEHP doses. DEHP did not induce gonocytes proliferation or Leydig cells hyperplasia and did not cause apoptosis. To highlight a mechanism for DEHP antiandrogenic effects, immunohistochemistry for AR and PPARγ has been performed. Treatment did not interfere with AR expression, instead it induced a reduced expression of PPARγ in Leydig cells of rats treated with DEHP 900 mg/kg/bw-d. In conclusion, DEHP impairs testis development during neonatal period; in particular, the most evident effects are registered on Leydig cells through PPARγ involvement.

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