Early testicular effects in rats perinatally exposed to DEHP in combination with DEHA--apoptosis assessment and immunohistochemical studies.

This study aimed to characterize the effects of di(2-ethylhexyl) phthalate (DEHP) on the fetal rat testes and relate them to the effects seen in adults. Histopathological effects in fetal testes were examined with immunohistochemistry for anti-Mullerian hormone (AMH), 3beta-hydroxysteroid dehydrogenase, smooth muscle actin (SMA), proliferating cell nuclear antigen (PCNA), histone H3 and vimentin. Additionally, testicular apoptosis levels were assessed in fetal, prepubertal and adult rats. As the plasticizer di(2-ethylhexyl) adipate (DEHA) has similarities with DEHP in chemical structure and metabolism, we investigated if the testicular effects of DEHP were modulated by co-administration with DEHA. Wistar rats were gavaged during gestation and lactation with vehicle, DEHP (300 or 750 mg/kg/day), or DEHP (750 mg/kg/day) in combination with DEHA (400mg/kg/day), and male offspring were examined at gestation day (GD) 21, postnatal day (PND) 22, 26 and 190. In fetal testes, Leydig cells were found in large clusters containing AMH positive Sertoli cells. At GD 21, seminiferous chords appeared enlarged with an apparently increased number of gonocytes. However, proliferation of gonocytes did not appear increased. A few animals had a high number of TUNEL positive apoptotic cells in degenerating seminiferous tubules at PND 22 and 190, whereas most exposed animals had low levels of germ cell apoptosis at GD 21, PND 22 or PND 26, as evaluated by DNA laddering, TUNEL staining, Caspase-3 immunohistochemistry and Caspase-3 activity measurement. No differences between DEHP and DEHP+DEHA exposed groups were observed.