Efficient synthesis of dichlorodenafil, an unapproved sildenafil analogue appearing in non-prescription supplements.

We have developed an efficient synthesis of dichlorodenafil (4), an unapproved sildenafil analogue isolated from dietary supplements. Our sequence employs POCl(3)-mediated chlorination of readily available chloroacetyl compound 7 followed by selective hydrolysis of the chloro-heterocycle function. Our synthesis confirms the structure of the illegal additive, and will provide regulatory agencies with ready access to authentic standard samples of dichlorodenafil (4) to aid in their mission to protect the public from unapproved and potentially harmful erectile dysfunction (ED) drug analogues that are added to herbal and dietary supplements without providing users with appropriate toxicological or pharmacological information.

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 Cowper's 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.

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.

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.

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 1 microg/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.

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 17beta-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 x 10(-8) M. Both fenvalerate (1 x 10(-6) M) and permethrin (1 x 10(-6) M) did not stimulate the AlkP activity, but progesterone (1 x 10(-8) M)-induced AlkP activity was significantly inhibited at 1 x 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 x 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.

Evaluation of the 20-day pubertal female assay in Sprague-Dawley rats treated with DES, tamoxifen, testosterone, and flutamide.

The Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) has recommended the rodent pubertal female assay as a Tier I test to detect potential endocrine disrupters (EDs). This assay is designed to screen estrogenic activity in immature rats exposed to chemicals during sexual maturation. The aim of this study was to evaluate whether this assay can detect the EDs with effects brought about through various mechanisms. Immature Sprague-Dawley female rats (21 days of age) were dosed daily for 20 days by oral gavage (DES, tamoxifen, and flutamide) or sc injection (testosterone). The mean age at vaginal opening (VO) was 32.3 +/- 0.5 days in control rats. Although VO was unaffected by DES at doses of 0.2 and 1.0 microg/kg, a high dose of DES (5.0 microg/kg) significantly advanced the age at VO to 24 days. Both tamoxifen (50 and 200 microg/kg) and flutamide (25 mg/kg) also significantly accelerated VO to 27.8 +/- 0.5, 25.1 +/- 0.1, and 26.1 +/- 0.1, respectively. However, testosterone dose-dependently delayed VO (exposure to 1.0 mg/kg extended VO to 37.3 +/- 0.8 days, and VO did not occur in 2 of 10 animals by the time of necropsy at 41 days of age). Estrous cyclicity was monitored in rats from VO to necropsy. Irregular cycles were observed in the groups treated with DES (5.0 microg/kg), tamoxifen (200 microg/kg), testosterone (1.0 mg/kg), and flutamide (25 mg/kg). High dose of DES showed a persistent estrus state throughout the entire observation period. In addition, the number of days in diestrus was increased by tamoxifen (200 microg/kg) and flutamide (25 mg/kg) treatments. Significant decreases in ovarian weight were observed in 5.0 microg/kg DES (64% of control), 25 mg/kg flutamide (76% of control), and 200 microg/kg tamoxifen (47% of control). Testosterone also significantly decreased the ovarian weights in all treatment groups. Uterine weights were also decreased significantly at high doses of tamoxifen (200 microg/kg, 39% of control) or testosterone (1.0 mg/kg, 47% of control). In hormone analysis, tamoxifen significantly increased serum E(2) levels at 50 microg/kg. The mean serum levels of TSH were significantly increased in tamoxifen (10 and 50 microg/kg), testosterone (0.2 mg/kg), and flutamide (1.0 and 25 mg/kg) treatment groups compared with the control. However, serum T(4) levels were significantly reduced by testosterone. Furthermore, serum T(3) levels were significantly increased in DES, tamoxifen (10 and 50 microg/kg), testosterone (1.0 mg/kg), and flutamide (1.0 and 5 mg/kg). Our data demonstrate that the rodent pubertal female assay is useful for identifying potential EDs having not only estrogenic/antiestrogenic but also androgenic/antiandrogenic activities. However, further validation study is necessary to identify chemicals that operate through other action mechanisms, including steroid biosynthesis inhibitors and thyroid inhibitors. Moreover, additional data on other compounds with weak endocrine disrupting activity will be required to further characterize the sensitivity of the female pubertal assay.

Comparison of anti-androgenic activity of flutamide, vinclozolin, procymidone, linuron, and p, p'-DDE in rodent 10-day Hershberger assay.

The rodent Hershberger assay proposed by the Organization for Economic Co-operation and Development (OECD) is in the process of the validating a test method to detecting the androgenic or anti-androgenic compounds. The aim of this study was to compare the anti-androgenic properties of flutamide, vinclozolin, procymidone, linuron, and p,p'-DDE in a 10-day Hershberger assay. In the present study, we used immature Sprague-Dawley male rats castrated at 6 weeks of age. Testosterone propionate (TP) was subcutaneously injected for 10 consecutive days at doses of 0.1, 0.2, 0.4, 0.8, or 1.6 mg/kg per day. To compare the anti-androgenic activity of test compounds, flutamide (1, 5, 10, or 20 mg/kg per day), a pure androgen antagonist was used as a positive control, and administered by oral gavage after TP (0.4 mg/kg per day) treatment. In addition, vinclozolin (25, 50, or 100 mg/kg per day), procymidone (25, 50, or 100 mg/kg per day), linuron (25, 50, or 100 mg/kg per day), and p,p '-DDE (25, 50, or 100 mg/kg per day) were also administered by oral gavage after TP (0.4 mg/kg per day) treatment. As expected, TP dose-dependently increased accessory sex organ weights, and statistically significant effects were observed at doses of 0.1 (only seminal vesicles) or 0.2mg/kg per day and above. Serum testosterone levels increased significantly at 0.4 mg/kg per day and above, while serum LH levels were decreased in a dose-dependent manner. Flutamide significantly inhibited the TP-induced re-growth of seminal vesicles, ventral prostate, and Levator ani plus bulbocavernosus muscles (LABC) at 1mg/kg per day and above, and Cowper's glands and glans penis at 5mg/kg per day and above. In contrast to accessory sex organ weights, flutamide did not affect the serum testosterone levels compared to the control at any concentration, but serum LH levels were significantly increased at doses of 10 and 20 mg/kg per day. Similar to flutamide, vinclozolin caused a statistically significant decrease in the weights of seminal vesicles (to 65 and 40% of the control), ventral prostate (to 66 and 51% of the control), LABC (to 81 and 66% of the control), and Cowper's glands (to 81 and 65% of the control) at 50 and 100 mg/kg per day, respectively. Glans penis weight was also significantly reduced (to 79% of the control), but only at 100 mg/kg per day. The most pronounced effects were observed in the procymidone treatment groups. Procymidone significantly inhibited TP-induced re-growth of accessory sex organs at 25mg/kg per day and above, whereas glans penis weight significantly decreased (to 69% of the control), but only at 100 mg/kg per day. Linuron also inhibited TP-induced re-growth of the seminal vesicles (to 72 and 53% of the control), ventral prostate (to 75 and 62% of the control), Cowper's glands (to 74 and 61% of the control) at 50 and 100 mg/kg per day, respectively. LABC (to 65% of the control) and glans penis (to 80% of the control) weights were significantly reduced, but only at 100 mg/kg per day. In case of p,p'-DDE, seminal vesicle weights were significantly decreased at 50 (to 66% of the control) and 100 mg/kg per day (to 58% of the control). In addition, ventral prostate (to 79% of the control), LABC (to 75% of the control), and Cowper's gland (to 82% of the control) weights were reduced, but only at 100 mg/kg per day. On the contrary, no statistically significant differences in serum testosterone or LH levels were observed versus the control. p,p'-DDE significantly increased liver weight in a dose-dependent manner, without affecting on body weights. Our results indicate that procymidone may act as a stronger androgen receptor (AR) antagonist than vinclozolin, linuron, or p,p'-DDE. We conclude that the 10-day Hershberger assay is a sensitive method for detecting potential anti-androgenic compounds.

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.

Biphasic effects of the flavonoids quercetin and naringenin on the metabolic activation of 2-amino-3,5-dimethylimidazo[4,5-f]quinoline by Salmonella typhimurium TA1538 co-expressing human cytochrome P450 1A2, NADPH-cytochrome P450 reductase, and cytochrome b5.

Heterocyclic amines (HCAs) produced by cooking meat products at high temperatures are promutagens that are activated by cytochrome P450 (CYP) lA2. Using a newly developed Salmonella typhimurium TA1538/1A2bc-b5 strain, we tested the effect of quercetin and naringenin on the mutagenicity of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ). TA1538/1A2bc-b5 bears two plasmids, one expressing human CYP1A2 and NADPH-P450 reductase (NPR), and the other plasmid which expresses human cytochrome b5 (cyp b5). TA1538/1A2bc-b5 cells showed high activities of 7-ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) associated with CYP1A2 and are very sensitive to mutagenesis induced by several HCAs. MeIQ was found to be the strongest mutagen among the HCAs tested in this system. Mutagenicity of MeIQ was enhanced 50 and 42% by quercetin at 0.1 and 1 microM, respectively, but suppressed 82 and 96% at 50 and 100 microM. Naringenin also increased the MeIQ-induced mutation about 37 and 22% at 0.1 and 1 microM, but suppressed it 32 and 63% at 50 and 100 microM concentrations, respectively, in TA 1538/1A2bc-b5 cells. Thus, they stimulated the MeIQ induced mutation at low concentrations, but strongly suppressed it at high concentrations. This biphasic effect of flavonoids was due to the stimulation or the inhibition of CYP1A2 activity in a dose-dependent manner judging by the activities of EROD or MROD in the Salmonella cells. These results indicate that quercetin and naringenin can exhibit inhibitory or stimulating effects on CYP1A2 mediated mutagenesis by MeIQ, depending on their concentrations.

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 Cowper's 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 Cowper's 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 ER 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 ER expression in the testes, but not AR, ER, 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 ER expression during the early neonatal stage.

Evaluation of in vitro screening system for estrogenicity: comparison of stably transfected human estrogen receptor-α transcriptional activation (OECD TG455) assay and estrogen receptor (ER) binding assay.

The estrogenic activity of industrial chemicals, di(2-ethylhexyl) phthalate (DEHP), di(n-butyl) phthalate (DBP), benzylbutyl phthalate (BBP), diethyl phthalate (DEP), tetrabromobisphenol A (TBBPA), bisphenol A (BPA), and nonylphenol (NP), was compared using OECD test guideline 455(TG455), stably transfected transcriptional activation (STTA) and estrogen receptor (ER) binding assays. The estrogenic activity of BBP, BPA and NP were approximately 180,000-fold (PC(50), 4.32 x 10(-6 )M), 5,000-fold (PC(50), 1.26 x 10(-7) M) and 120,000-fold (PC(50), 2.92 x 10(-6 )M) less than 17ß-estradiol (PC(50), 2.43 x 10(-11)M), whereas DEHP, DBP and DEP did not show any estrogenicity activity in the STTA assay. Moreover, binding affinities to human ERα of BBP, BPA, and NP were approximately 200,000-fold (IC(50), 4.91 x 10(-4) M), 8000-fold (IC(50), 1.92 x 10(-5) M) and 1400-fold (IC(50), 3.34 x 10(-6) M) less than 17ß-estradiol (IC(50), 2.45 x 10(-9) M) in competitive human ERα binding assay. The relative potencies of STTA assay were very similar to ER binding, E-screen, and Yeast screening assays. Therefore, our results suggested that OECD test guideline TG455 may be useful as a screening test for potential endocrine disruptors.

Aroclor1254 interferes with estrogen receptor-mediated neuroprotection against beta-amyloid toxicity in cholinergic SN56 cells.

Because estrogen plays important neurotrophic and neuroprotective roles in the brain by activating estrogen receptors (ERs), disruption of normal estrogen signaling can leave neurons vulnerable to a variety of insults, including ß-amyloid peptide (Aß). Aroclor1254 (A1254) belongs to the endocrine-disrupting chemical (EDC) polychlorinated biphenyls and has anti-estrogenic properties. In the present study, we evaluated the effect of A1254 on the protective activity of estrogen against Aß toxicity in differentiated cholinergic SN56 cells. Aged Aß25-35 causes apoptotic cell death in differentiated SN56 cells, and the cytotoxic evidences are effectively rescued by estrogen. We found that A1254 abolishes the neuroprotective activity of estrogen against Aß toxicity, and attenuates the suppressive effect of estrogen on Aß-induced tau phosphorylation and JNK activation. The effects of A1254 on the neuroprotective effects of estrogen in Aß toxicity are very similar to the effects of the estrogen receptor antagonist ICI182,780. Thus, exposure to EDCs that have anti-estrogenic activity might interfere with normal estrogen-activated neuroprotective signaling events and leave neurons more vulnerable to dangerous stimuli. Our present results provide new understanding of the mechanisms contributing to the harmful effects of EDCs on the function and viability of neurons, and the possible relevance of EDCs in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease.

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 17ß-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(-7)M) > isobutyl paraben -2.34008 (PC50, 6.3 × 10(-7)M) > ethyl paraben -2.64016 (PC50, 1.26 × 10(-6) M) > isopropyl paraben -2.73993 (PC50, 1.58 × 10(-6)M) > 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.

In vitro study of Organization for Economic Co-operation and Development (OECD) endocrine disruptor screening and testing methods- establishment of a recombinant rat androgen receptor (rrAR) binding assay.

The androgen receptor (AR) binding assay can be used to determine the ability of probable endocrine disruptors (EDs) to compete with synthetic androgen methyltrienolone (R1881) for binding to recombinant rat AR (rrAR). In this study, we assessed AR binding of various chemicals using Lexius Freyberger's method. The rank of relative binding affinity (RBA, IC(50)) on the tested chemicals was trenbolone 1.3 x 10(-8) M (RBA 138) > dihydrotesterone (DHT) 1.8 x 10(-8) M (RBA 100) > methyl testosterone 5.7 x 10(-8) M (RBA 31.6) > nonylphenol (NP) 1.3 x 10(-5) M (RBA 0.14) > bisphenol A (BPA) 1.1 x 10(-4) M (RBA 0.016) > isobutyl paraben 3.1 x 10(-4) M (RBA 0.0058) > butyl paraben 6.2 x 10(-4) M (RBA 0.0029) > propyl paraben 9.7 x 10(-4) M (RBA 0.0019). However, di(n-butyl) phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP), known anti-androgenic chemicals, did not show any significant AR binding activity. Our data suggests that in vitro AR binding assay may be useful as a screening tool for potential EDs.

Evaluation of liver and thyroid toxicity in Sprague-Dawley rats after exposure to polybrominated diphenyl ether BDE-209.

Our goal in the present study was to evaluate whether decabromodiphenyl ether (BDE-209), which is the most abundant polybrominated diphenyl ether (PBDE) found in human samples, affects against target organs. Sprague-Dawley male rats were exposed to vehicle or BDE-209 (100, 300, or 600 mg/kg body weight, daily) from postnatal day (PND) 10 to PND 42. There was no significant difference in body and male reproductive organ weight changes compared with controls. However, liver, thyroid and adrenal gland weights were significantly increased in the high-dose of BDE-209 group. BDE-209 significantly induced the expression of cytochrome P450 (CYP1A2, CYP3A1, and CYP2B1) enzymes in the liver. Furthermore, constitutive androstane receptor (CAR) and pregnane xenobiotic receptor (PXR) expression levels were also increased in a dose-dependent manner. Total serum triiodothyronine (T3) concentration was significantly reduced in a dose-dependent manner, whereas the level of thyroid-stimulating hormone was significantly increased with BDE-209 treatment. In the histological findings, multiple areas of degenerated follicular epithelium and slight attenuation of the follicular epithelium were observed in the thyroid glands by high doses (300 and 600 mg/kg) of BDE-209 treatment. The presence of hepatocytic fatty degeneration and inflammatory foci were also observed in the 300 and 600 mg/kg of BDE-209 group. These findings demonstrate that BDE-209 induces hyperthyroidism and hepatotoxicity. In the future, further research is needed to determine the relationship between target organ toxicity and blood concentrations of BDE-209.

Effects of postnatal administration of diethylstilbestrol on puberty and thyroid function in male rats.

To examine the effects of diethylstilbestrol (DES) on male pubertal development and thyroid function, juvenile male Sprague-Dawley rats were given DES daily by oral intubation at doses of 10, 20 and 40 microg/kg/day from postnatal day 33 for 20 days. Prepuce separation was significantly delayed at the dose of 20 microg/kg/day and above in the DES-treated rats. DES treatment induced a significant reduction in the weights of testes, epididymides, the ventral prostate, seminal vesicles plus coagulating glands and fluid, levator ani bulbocavernosus muscles, Cowper's glands and the glans penis. The weights of the liver and adrenals increased in the DES-treated animals. DES caused a dose-dependent reduction in germ cells; in particular the spermatids were mainly affected. The serum levels of testosterone and luteinizing hormone were significantly reduced in the DES-treated groups, but that of estradiol decreased. No differences were observed in the serum thyroxine levels of the control and DES-treated groups. In microscopic observation of the DES-treated animals, degeneration of germ cells and tubular atrophy in the testis were noted, but there were no microscopic changes in the thyroid. These results indicate that DES affected the pubertal development of juvenile male rats and that its mode of action may be related to alterations in hormone levels.

OECD validation of the rodent Hershberger assay using three reference chemicals; 17alpha-methyltestosterone, procymidone, and p,p'-DDE.

The rodent Hershberger assay is being validated as an in vivo test method for detecting androgenic or antiandrogenic compounds by the Organization for Economic Cooperation and Development (OECD). As part of the international validation work, we studied 17alpha-methyltestosterone for evaluating androgenic activity, and procymidone and p,p'-DDE for evaluating antiandrogenic activity. Male Sprague-Dawley rats were castrated at postnatal day 42, and only the rats that showed preputial separation were used in this study. Seven days after castration, chemicals were administered daily by gavages to groups of rats for 10 days, as recommended by OECD phase-2 protocol. Administration of 17alpha-methyltestosterone induced increases of weights of accessory sex tissues and glands in a dose-dependent manner. Administration of procymidone and p,p'-DDE produced a dose-dependent decrease of weights of accessory sex tissues and glands in the rats co-treated with testosterone propionate (0.4 mg/kg/day) subcutaneously. Our data strongly suggested that the current protocol of OECD Hershberger assay (phase-2) should be used as a reliable method for the detection of endocrine related toxicity of other chemicals.

Gestational exposure to nonylphenol causes precocious mammary gland development in female rat offspring.

This study examined whether or not exposure to 4-nonylphenol (NP) during late gestation affects reproductive and mammary development in the offspring of female rats. Time pregnant Long Evans rats were gavaged with NP (10 or 100 mg/kg), atrazine (ATR, 100 mg/kg), or corn oil on gestation days 15-19. The uterus weights of the NP (100 mg/kg/d)-exposed pups were higher than those of the controls but the weights of the other organs were unchanged. Delayed mammary gland (MG) development was detected in the ATR pups on PND 4 and persisted through to PND 66. The high dose NP pups had advanced lobular development of their MG on PND 22, while the glands from the low dose NP pups were no different morphologically from the controls. Immunohistochemical comparisons of the mammary sections from PND 41 demonstrated low levels of estrogen receptor (ER) staining in the control gland stroma and epithelium but higher levels in the tissue of the pups exposed to NP and ATR. ATR also elevated ER in the stroma surrounding the epithelial layer of the terminal end buds. The level of progesterone receptor (PR) staining was markedly lower in the epithelium of the 100 mg/kg NP glands vs. the control glands. However, PR was present at high levels in the epithelium of the 10 mg/kg NP glands and was even more prominent in the ATR-exposed ductal epithelium and fat cell nuclei. The level of prolactin staining was only elevated in glands containing lobule areas (NP-exposed) compared with the control levels. These results suggest that NP and ATR have opposite effects on the development of MG after gestational exposure. Exposure to them during the critical period of epithelial outgrowth altered the receptor levels of mammary progesterone and prolactin and might contribute to the differences in the mammary morphology at PND 41.

Calbindin-D9k mRNA expression in the rat uterus following exposure to methoxychlor: a comparison of oral and subcutaneous exposure.

Calbindin-D(9k) (CaBP-9k) is a cytosolic calcium-binding protein that is induced by estrogenic compounds possibly through estrogen receptors. We compared CaBP-9k mRNA expression in the uterus with uterotrophic response in immature rats exposed to methoxychlor (MC), an environmental chemical with estrogenic activity. MC was orally or subcutaneously administered to 3-week-old female Sprague-Dawley rats for 3 days. The weights of the uterus and vagina significantly increased in the oral treatment group at a dose of 50, 100 and 200 mg/kg, but those of the subcutaneous (SC) treatment group only increased at 200 mg/kg. Northern blot analysis showed that CaBP-9k mRNA expression was significantly induced in a dose-dependent manner at doses of 50, 100 and 200 mg/kg/day in the oral treatment group. SC administration of MC induced significant expression at only a dose of 200 mg/kg/day; this was similar to the uterotrophic response. MC has an estrogenic effect on the uterus as shown by the increase in weight and induction of CaBP-9k mRNA expression, which were much greater following exposure via oral gavage than via the SC route. The strong correlation between the results of in vivo uterotrophic assay and CaBP-9k mRNA expression suggests that CaBP-9k mRNA expression in the rat uterus may be used as an early gene marker for detection of the estrogenic effects of putative environmental chemicals.