Effects of Endocrine Disruptors on the Reproductive Function in Rodents

Abstract

Endocrine disrupters (EDs) are chemicals with the potential to interfere with the function of the endocrine system. In this study, we demonstrate the reproductive toxicity of the EDs butyl-paraben (BP), propyl-paraben (PP), and di-(2-ethylhexyl) phalate (DEHP) using the international toxicity test protocol proposed by the Organization for Economic Cooperation and Development (OECD). We also analyze the effect of these chemicals on mice offspring following prenatal and lactational exposure. In Chapter 1, we discuss the use of the Hershberger assay and the Enhanced TG 407 assay to investigate the short- and long-term toxicity of BP and PP. For the Hershberger assay, male Sprague-Dawley (SD) rats were administrated BP and PP (0, 10, 100, and 1000 mg/kg/day) orally for 10 d together with testosterone propionate (TP, 0.4 mg/kg per day). They were then castrated on PND 42. For the Enhanced TG 407, male SD rats were administrated BP and PP (0, 10, 100, and 1000 mg/kg/day) orally for 28 days starting on PND 49. In the short-term toxicity test (the Hershberger assay), the weight of the liver, adrenal glands, and preputial gland significantly decreased, together with body weight at BP 1000 mg/kg + TP, suggesting that BP at 1000 mg/kg + TP may have systemic toxicity. In the histological analysis, liver tissues showed vacuolization at BP 100 and 1000 mg/kg + TP. Serum T3 levels were significantly decreased at PP 10, 100, and 1000 mg/kg + TP. Serum T4 levels were significantly decreased at PP 1000 mg/kg + TP and BP 1000 mg/kg + TP. Together, this suggests that PP induces thyroid toxicity. In the long-term toxicity test (Enhanced TG 407), body weight significantly decreased at BP 1000 mg/kg and PP 1000 mg/kg. The thyroid, heart, and liver weights significantly decreased in the BP 1000 mg/kg and PP 1000 mg/kg groups, suggesting that a high dose of paraben induces systemic toxicity. Serum testosterone levels significantly decreased at BP or PP 1000 mg/kg. Serum T3 levels significantly decreased at BP 100 mg/kg, suggesting BP induces thyroid toxicity at a dose of 100 mg/kg. T4 levels significantly decreased at BP 1000 mg/kg and PP 1000 mg/kg. This may be attributable to systemic toxicity. In Chapter 2, we examine the effects of long-term exposure to BP and PP on the reproductive organs based on Enhanced TG 407. The height of parathyroid epithelial cells significantly decreased in the BP 100 and BP 1000 mg/kg groups. This suggests that long-term exposure to BP may result in thyroid toxicity. Daily sperm production, the number of offspring, and the expression of StAR and 3?-HSD6 mRNA were significantly reduced in the BP 1000 mg/kg group. The number of Leydig cells was significantly reduced in the PP 1000 mg/kg group. Together, this suggests that BP and PP dosing at 100 mg/kg may not induce male reproductive toxicity. However, random amplified polymorphic DNA (RAPD) analysis of DNA methylation of epdidymal sperm was increased following BP or PP dosing at 10 mg/kg. This suggests that long-term exposure to low-dose BP and PP may cause DNA methylation in male germ cells. In Chapter 3, we examine the reproductive toxicity of DEHP through the prenatal and lactational periods in male and female mice offspring. Pregnant imprinting control region (ICR) mice were administrated DEHP orally (0.015, 0.15, 1.5, 15, and 150 mg/kg/day) from gestation day (GD) 18 to postnatal day (PND) 20. The reproductive toxicity of male offspring was determined at PND 35 and PND 180 and that of female offspring was determined at PND 49. Preputial separation in F1 male offspring was significantly shortened at DEHP 0.15, 1.5, and 15 mg/kg. This suggests prenatal and lactational exposure to low-dose DEHP may cause the early onset of puberty in male offspring. However, at PND 35, body weight and the weight of the reproductive organs was significantly reduced at DEHP 150 mg/kg in male offspring. The number of Leydig cells at PND 35 was significantly reduced at DEHP 1.5, 15, and 150 mg/kg, but serum testosterone levels did not differ among the groups. In the testes, bax mRNA levels and the number of TUNEL-positive germ cells were significantly increased at a DEHP dose of 150 mg/kg. At PND 180, body weight and testes weight did not differ among the experimental groups, but epididymis and prostate and pituitary weights were significantly decreased at DEHP 150 mg/kg. Seminal vesicle weight was significantly decreased at DEHP doses of 0.15, 1.5, 15, and 150 mg/kg. The serum testosterone level and daily sperm production were significantly decreased at DEHP 150 mg/kg. The steroidogenic pathway gene StAR, 3?-HSD6, and 17?-HSD3 mRNA expression were significantly decreased at DEHP doses above 0.15 mg/kg. Testes bax mRNA levels were significantly increased at DEHP 0.15, 1.5, 15, and 150 mg/kg. The number of TUNEL-positive germ cells was significantly increased at a DEHP dose of 150 mg/kg. Together, prenatal and lactational exposure to DEHP may disrupt normal development of Leydig cells and steroidogenesis and the development of male accessory sex organs in male offspring. In female offspring, body weight at PND 49 was significantly decreased at DEHP 0.015 and 150 mg/kg. Ovary weight in the diestrus stage was significantly decreased at DEHP doses of 15 and 150 mg/kg. Uterus weight in the diestrus stage was significantly decreased at DEHP doses of 1.5, 15, and 150 mg/kg. Adrenal, liver, spleen, thymus, and pituitary weights were significantly decreased at DEHP 150 mg/kg. The day of vaginal opening (VO) was significantly advanced at DEHP doses of 1.5, 15, and 150 mg/kg. The corpus luteum number and Cyp11a1 mRNA levels in the diestrous ovary were significantly decreased at DEHP 1.5, 15, and 150 mg/kg. At the estrus stage, the oviduct weight was significantly decreased at DEHP 1.5, 15, and 150 mg/kg. Spleen weight was significantly decreased at DEHP 0.015, 0.15, 1.5, 15, and 150 mg/kg. Thymus and brain weights were significantly decreased at DEHP 150 mg/kg. Serum estrogen levels were significantly decreased at DEHP 1.5, 15, and 150 mg/kg. In cyclic female offspring, the duration of the estrous cycle was significantly increased at DEHP 1.5, 15, and 150 mg/kg. The ovulated oocyte number and fertilization rate were significantly decreased, together with an increase in the abnormality of oocytes, at DEHP 1.5, 15, and 150 mg/kg. When mated with normal males, the number of live births (F2) for females was significantly decreased at DEHP 150 mg/kg. Ovarian steroidogenic genes (StAR, SF-1, 3?-HSD6, 17?-HSD3, and Cyp19a1) and ?-SMA mRNA levels were decreased by DEHP, but the sensitivity of these genes to DEHP was different during the estrous cycle. This suggests that prenatal and lactational exposure to DEHP inhibits ovarian follicle development and steroidogenesis in female offspring. Taken together, prenatal and lactational exposure to DEHP may cause the early onset of puberty in female offspring, but their fertility during adulthood was compromised.