Il Hyun Kang

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.

Effects of in Utero Exposure to DI(n-Butyl) Phthalate on Development of Male Reproductive Tracts in Sprague-Dawley Rats

The purpose of this study was to determine the effects of di(n-butyl) phthalate (DBP) administration on male reproductive organ development in F1 Sprague-Dawley rats following in utero exposure. During gestation days (GD) 10–19, pregnant rats were administered daily, orally, DBP at 250, 500, or 700 mg/kg or flutamide (1, 12.5, or 25 mg/kg/d) as a positive control. The male offspring were sacrificed at 31 d of age. DBP and flutamide dose-dependently significantly increased the incidence of hypospadias and cryptorchidism in F1 male offspring. The weights of testes and accessory sex organs (epididymides, seminal vesicles, ventral prostate, levator ani plus bulbocavernosus muscles (LABC), and Cowpers glands) were significantly reduced in DBP-treated animals. Furthermore, cauda agenesis of epididymides and ventral prostate atrophy were observed in high-dose 700-mg/kg DBP males. Anogenital distance (AGD) and levels of dihydrotestosterone (DHT) and testosterone were significantly decreased in the DBP (700 mg/kg/d)-treated groups. In particular, the expression of androgen receptor (AR) and 5α-reductase type 2 in the proximal penis was markedly depressed following administration of DBP (700 mg/kg/d) or flutamide (25 mg/kg/d). The expression of sonic hedgehog (Shh) in the urethral epithelium of the proximal penis was significantly less in the DBP (700 mg/kg/d)- or flutamide (25 mg/kg/d)-treated groups. In addition, DBP dose-dependently significantly increased the expression of estrogen receptor (ER α) in the undescended testis. Data demonstrated that in utero exposure to DBP produced several abnormal responses in male reproductive organs, and these effects may be due to disruption of the stage-specific expression of genes related to androgen-dependent organs development.

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.

Estrogenic Activity of Persistent Organic Pollutants and Parabens Based on the Stably Transfected Human Estrogen Receptor-α Transcriptional Activation Assay (OECD TG 455)

Screening of estrogenic activity on dichloro diphenyl trichloroethane (DDT), dichloro diphenyl dichloro ethylene (DDE), dieldrin, heptachlor, aldrin, chlordane, lindane, polybrominated diphenyl ethers (PBDE) and parabens was compared using Organization for Economic Cooperation and Development (OECD) test guideline 455 (TG455). The estrogenic activity of DDT was 58,000-fold (PC50, 1.67 × 10−6 M) less than 17β-estradiol(E2) (PC50, 2.88 × 10-11 M) but DDE, dieldrin, heptachlor, aldrin, chlordane, lindane and PBDE did not show any estrogenic activity in this assay system. In the case of paraben compounds, the rank of relative transcriptional activation (logRTA) was butyl paraben −1.63752 (PC50, 1.25 × 10−7M) > isobutyl paraben −2.34008 (PC50, 6.3 × 10−7M) > ethyl paraben −2.64016 (PC50, 1.26 × 10−6 M) > isopropyl paraben −2.73993 (PC50, 1.58 × 10−6M) > propyl paraben −2.84164 (PC50, 2.0 × 10−6 M). Our data suggest that OECD test guideline TG455 may be useful as a screening tool for potential endocrine disruptors.

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.

OECD validation of phase 3 Hershberger assay in Korea using surgically castrated male rats with coded chemicals.

As a participating laboratory for the OECD Hershberger validation program, we conducted a phase 3 trial to test the reliability of the Hershberger assay using coded substances. Male Sprague–Dawley rats were castrated at 6 weeks of age and allowed to recover for 8 days. All the coded substances were administered orally once daily for 10 consecutive days. In the antagonist version of the assay, 0.4 mg kg−1 of testosterone propionate (TP), a reference androgen, was co‐administered with the coded compounds C, D, H, I or K, by a subcutaneous injection. As anticipated, TP alone produced statistically significant increases in the five mandatory accessory sex organ weights. The coded substance L (trenbolone 40 mg kg−1), the test agonist, caused significant increases in the weights of the androgen‐dependent tissues. The five coded compounds, p,p′‐DDE at two doses (codes C and I), linuron at two doses (codes D and K) and flutamide (code H), all significantly decreased the weights of the TP‐stimulated sex organs. These results suggest the OECD Hershberger assay to be a reliable screening method for detecting androgen agonists and antagonists. Copyright

OECD validation of phase-3 Hershberger assay using the stimulated weanling male rat in Korea.

The OECD has proposed a new, validated test guideline with the stimulated weanling male Hershberger assay to avoid the surgical castration step. In the present study, we assessed the relevance and reliability of the stimulated weanling Hershberger assay in four stages. All chemicals except for testosterone propionate (TP) were orally administered to sexually immature male rats of 22 days old for 10 days. The weights of four mandatory accessory sex organs, two additional reproductive tissues and optional systemic organs were evaluated. At the first two stages, TP, as reference androgen, significantly increased the weights of epididymides and accessory sex organs (ASO) at 1.0 mg kg−1 and flutamide (FLU), as a positive anti‐androgen control, decreased the TP‐stimulated organ weights at 3.0 mg kg−1. At stage 3, trenbolone (40 mg kg−1), an anabolic steroid, significantly increased ASO weights, and weak anti‐androgens (DDE and linuron) decreased the TP‐stimulated ASO weights at each high dose. The above results were confirmed in a blind test with coded substances provided by OECD. Compared with results from our previous castrated male assay, the intact weanling version is less sensitive than the castrated male version, in terms of a smaller response at the reference dose of TP or FLU. However, this study suggests that the stimulated weanling Hershberger assay can detect the effects of both potent and weak anti‐androgens on androgen‐producing and androgen‐dependent tissues. Copyright

Alterations of di( n -butyl)phthalate-induced oxidative stress in the testis of hypothyroid rats

The aim of the present study was to investigate the effects of di(n-butyl)phthalate (DBP) on oxidative damage in the testes of hypothyroid rats. Hypothyroidism was induced by administering 0.1% 6-N-propyl-2-thiouracil (PTU) in drinking water for 30 days. DBP was dissolved in corn oil and administered daily for 30 days by oral gavage. Significant decreases in testes weight were observed both in normal (DBP) and hypothyroid (PTU + DBP) groups. Serum testosterone concentrations were significantly reduced in the DBP groups, but no significant change occurred in hypothyroid rats. Di(n-butyl)phthalate significantly increased malondialdehyde (MDA) and 8-hydroxy-2-deoxyguanosine (8-OHdG) level in the testes, whereas the DBP-induced oxidative lipid (MDA) and DNA (8-OHdG) damage were less in hypothyroid rats. PTU-induced hypothyroid rats decreased testicular catalase activity, whereas superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities did not show any significant changes. However, the DBP and PTU + DBP groups significantly increased catalase and SOD activities in testis. The testicular expression of thyroid hormone receptor α-1 (TRα-1) was significantly increased in the DBP and PTU + DBP groups. In contrast, androgen receptor (AR) protein levels were not detected in the DBP and PTU + DBP groups. Di(n-butyl)phthalate significantly increased the peroxisome proliferator-activated receptor-r (PPAR-r) and retinoid X receptor-α (RXR-α) levels. Peroxisome proliferators activated-receptors-α and RXR-r protein levels were markedly decreased in the DBP groups, but these protein levels increased in the PTU + DBP group, as compared to DBP alone. These results suggest that PTU-induced hypothyroidism may protect against oxidative damage in the testis, probably due to the regulation of the PPAR and RXR expression, which is associated with decreased metabolic activation of DBP.

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.