Hyun Ju Moon

Neonatal exposure to di(n-butyl) phthalate (DBP) alters male reproductive-tract development.

The purpose of this study was to evaluate male reproductive-organ development in early postnatal male rats following neonatal exposure to di(n-butyl) phthalate (DBP) and identify a mechanism of action. Neonatal male rats were injected subcutaneously from d 5 to 14 after birth with corn oil (control) and DBP (5, 10, or 20 mg/animal). Animals were killed at postnatal day (PND) 31 and PND 42, respectively, and testes, epididymis, seminal vesicles, ventral prostate, levator ani plus bulbocavernosus muscles (LABC), and Cowpers glands were weighed. In addition, the expressions of androgen receptor (AR), estrogen receptors (ERs), and steroidogenic factor-1 (SF-1) were also examined in the testes. Total body weights gains were significantly reduced at PND 29–31, but gradually recovered on PND 42. However, DBP (20 mg/animal) significantly reduced the weights of testes and accessory sex organs (seminal vesicles, LABC, and Cowpers glands), but not of the epididymis. These adverse effects persisted through puberty at PND 42. Serum testosterone levels did not show any significant changes in the control and DBP treatment groups. Histomorphological examination showed mild diffuse Leydig-cell hyperplasia in the interstitium of severely affected tubules on PND 31. Only a few multinuclear germ cells were observed. DBP (20 mg/animal) significantly decreased the expression of AR, whereas ERβ expression and SF-1 expression were increased in a dose-dependent manner on PND 31 in the rat testes. On PND 42, DBP (20 mg/animal) significantly inhibited ERβ expression in the testes, but not AR, ERα, and SF-1. These results demonstrate that neonatal exposure to DBP produces permanent changes in the endocrine system and leads to abnormal male reproductive-tract development until puberty. Thus our data suggest that DBP is likely to exert its antiandrogenic actions through disruption of AR or ERβ expression during the early neonatal stage.

Comparative estrogenic effects of p-nonylphenol by 3-day uterotrophic assay and female pubertal onset assay.

Nonylphenol (NP) is widely used as a component of detergents, paints, pesticides, and many other formulated products. Several studies have demonstrated that NP is estrogenic in fish, avian, and mammalian cells. NP also competitively inhibits the binding of 17 beta-estradiol (E2) to the estrogen receptor (ER). However, there are relatively few in vivo data related to this issue in mammals. The aim of this study was to investigate the estrogenic activity of NP in animal models. We performed a 3-day uterotrophic assay using immature female rats for comparison with other endpoints of Tier I screening including vaginal opening (VO) in prepubertal intact female rats. For the uterotrophic assay, diethylstilbestrol (DES) (0.2 and 1.0 microg/kg) and p-NP (10, 25, 50, 100, and 200 mg/kg) were administered subcutaneously to immature Sprague-Dawley female rats for 3 consecutive days (postnatal days (PND) 20, 21, and 22). For the female pubertal onset assay, DES (0.2, 1.0, and 5.0 microg/kg) and p-NP (10, 50, and 100 mg/kg) were administered daily by oral gavage from 21 days of age for 20 days. In the uterotrophic assay, statistically significant increases in uterine wet weight were observed at doses of 100 and 200 mg/kg p-NP. DES (0.2 and 1.0 microg/kg) also significantly increased uterine weight compared to the vehicle control. In the female pubertal onset assay, the age of VO was advanced following oral exposure to DES (1.0 and 5.0 microg/kg) and p-NP (50 and 100 mg/kg). Estrous cyclicity was monitored in prepubertal rats from the day of VO to the day of necropsy. Irregular estrous cycles were observed in the groups treated with DES (5.0 microg/kg) and p-NP (50 and 100 mg/kg). High-dose DES (5.0 microg/kg) produced a persistent estrus state, whereas p-NP (50 and 100 mg/kg) increased the number of days in diestrus. Serum thyroxine (T(4)) concentrations were decreased in a dose-dependent manner by DES and p-NP treatment. A significant decrease in serum T(4) level was observed at high-dose DES (5.0 microg/kg) and p-NP (100 mg/kg). Serum TSH level was significantly increased by DES (5.0 microg/kg) treatment. Statistically significant decreases in ovarian weight were observed in female rats treated with DES (5.0 microg/kg) and p-NP (100 mg/kg). Our data demonstrate that p-NP can accelerate the onset of puberty and alter estrous cyclicity in prepubertal female rats at oral doses lower than the subcutaneous doses typically used in the uterotrophic assay. We therefore suggest that the female pubertal onset assay may be used as a sensitive testing method to detect environmental agents with weak estrogenic activity, but requires further research.

Validation Study of OECD Rodent Uterotrophic Assay for The Assessment of Estrogenic Activity in Sprague-Dawley Immature Female Rats

The Organization for Economic Cooperation and Development (OECD) is developing a screening and testing method to identify estrogenic/antiestrogenic compounds. Based on these demands, phase 1 study for OECD uterotrophic assay was undertaken. The OECD is in the process of validating the assay results from international participating laboratories, which carried out this study with established environmental estrogenic compounds using designed protocols. The aim of this study was to provide data for validating the OECD uterotrophic assay using Sprague-Dawley immature female rats when testing with weak or partial estrogenic compounds. Ethinyl estradiol (EE) at 0.3 or 1μg/kg/d, a positive control used in the present study, significantly increased both uterine wet and blotted weights. In the case of weak estrogenic compounds, the uterine wet weights were significantly increased by bisphenol A (BPA) at 300 mg/kg/d, nonylphenol (NP) at 80 mg/kg/d, genistein (GN) at 35 mg/kg/d, and methoxychlor (MXC) at 500 mg/kg/d. In addition, the increase in uterine blotted weights also showed a similar pattern to that of uterine wet weights. However, both 1,1,1-trichloro-2,2-bis(p-chlorphenyl)ethane (o,p-DDT) and dibutyl phthalate (DBP) did not affect uterus (wet and blotted) weights at doses of 100 and 500 mg/kg/d. These results suggest that the increase in uterine weights should be considered useful as a sensitive endpoint for detecting weak estrogenic compounds in 3-d rodent uterotrophic assay. However, further combination studies using surrogate biomarkers may be needed to improve the sensitivity of this assay for the detection of weak estrogenic compounds, such as o,p-DDT.

Effects of flutamide on puberty in male rats: an evaluation of the protocol for the assessment of pubertal development and thyroid function.

To establish a test protocol for the rodent 20-d thyroid/pubertal assay, flutamide, a nonsteroidal androgen antagonist, was administered to intact male Sprague-Dawley rats from postnatal d 33 for 20 d, and several reproductive endpoints were examined to assess the sensitivity of a number of parameters with respect to the detection of endocrine-related effects. Immature male rats were divided into 4 groups and given flutamide once daily by oral gavage at doses of 0, 1, 5, or 25 mg/kg/d. Prepuce separation was significantly delayed in flutamide-treated rats (5 and 25 mg/kg/d). One day after the last dose, the rats were sacrificed. Flutamide treatment resulted in a significant reduction in the weights of epididymides, ventral prostate, seminal vesicles plus coagulating glands and fluid, levator ani plus bulbocavernosus muscles, Cowpers glands, and glans penis. The weight of adrenal glands decreased at 25 mg/kg/d, while testes and any other organ weights were unaffected. No microscopic changes were observed in the thyroid glands. Serum levels of testosterone were significantly increased in the flutamide-treated groups (5 and 25 mg/kg/d) and serum levels of estradiol were also increased (25 mg/kg/d). No differences were observed in the serum thyroxine levels. These results indicate that flutamide delays puberty in the male rat, and its mode of action appears to be via altered secretion of steroids, which subsequently affect the development of the reproductive tract. Thus, this assay might be used as an alternative for screening antiandrogenic activities of chemicals.

Pyrethroid insecticides, fenvalerate and permethrin, inhibit progesterone-induced alkaline phosphatase activity in T47D human breast cancer cells.

Pyrethroid insecticides exhibited a weak estrogenic activity by stimulation of MCF-7 cell proliferation and induction of alkaline phosphatase (AlkP) enzyme activity in cultured Ishikawa cells. Previously it was reported that fenvalerate and permethrin significantly inhibited the 17β-estradiol-induced MCF-7 BUS cell proliferation. Although certain pyrethroid insecticides exert estrogenic or antiestrogenic activities, it is not clear whether pyrethroid insecticides act as progesterone agonists or antagonists. Therefore, the aim of this study was to evaluate the effects of fenvalerate and permethrin on AlkP activity as a progesterone-specific response in T47D cells. In the present study, the stimulation of AlkP activity was concentration dependent with addition of progesterone, and maximum activity was observed at concentration of 1 × 10−8 M. Both fenvalerate (1 × 10−6 M) and permethrin (1 × 10−6 M) did not stimulate the AlkP activity, but progesterone (1 × 10−8 M)-induced AlkP activity was significantly inhibited at 1 × 10−6 M concentration of fenvalerate and permethrin, respectively. Progesterone receptor (PR) levels in cytosolic protein of T47D cells were studied to determine the relationship between cellular PR expression and AlkP activity. Similar to AlkP activity, progesterone (1 × 10−8 M) significantly increased PR protein levels compared to control. However, PR protein levels were not affected in T47D cells cultured with fenvalerate and permethrin alone, whereas fenvalerate and permethrin significantly decreased progesterone-induced PR protein levels. Our data indicate that fenvalerate and permethrin exhibit antiprogestagenic activity in T47D human breast cancer cells.