The Src family kinase Fgr is critical for activation of mast cells and IgE-mediated anaphylaxis in mice.

Mast cells are critical for various allergic disorders. Mast cells express Src family kinases, which relay positive and negative regulatory signals by Ag. Lyn, for example, initiates activating signaling events, but it also induces inhibitory signals. Fyn and Hck are reported to be positive regulators, but little is known about the roles of other Src kinases, including Fgr, in mast cells. In this study, we define the role of Fgr. Endogenous Fgr associates with FcεRI and promotes phosphorylation of Syk, Syk substrates, which include linkers for activation of T cells, SLP76, and Gab2, and downstream targets such as Akt and the MAPKs in Ag-stimulated mast cells. As a consequence, Fgr positively regulates degranulation, production of eicosanoids, and cytokines. Fgr and Fyn appeared to act in concert, as phosphorylation of Syk and degranulation are enhanced by overexpression of Fgr and further augmented by overexpression of Fyn but are suppressed by overexpression of Lyn. Moreover, knockdown of Fgr by small interfering RNAs (siRNAs) further suppressed degranulation in Fyn-deficient bone marrow-derived mast cells. Overexpression of Fyn or Fgr restored phosphorylation of Syk and partially restored degranulation in Fyn-deficient cells. Additionally, knockdown of Fgr by siRNAs inhibited association of Syk with FcεRIγ as well as the tyrosine phosphorylation of FcεRIγ. Of note, the injection of Fgr siRNAs diminished the protein level of Fgr in mice and simultaneously inhibited IgE-mediated anaphylaxis. In conclusion, Fgr positively regulates mast cell through activation of Syk. These findings help clarify the interplay among Src family kinases and identify Fgr as a potential therapeutic target for allergic diseases.

Embryotoxicity assessment of developmental neurotoxicants using a neuronal endpoint in the embryonic stem cell test.

The embryonic stem cell test (EST) is a validated in vitro embryotoxicity test; however, as the inhibition of cardiac differentiation alone is used as a differentiation endpoint in the EST, it may not be a useful test to screen embryotoxic chemicals that affect the differentiation of noncardiac tissues. Previously, methylmercury (MeHg), cadmium and arsenic compounds, which are heavy metals that induce developmental neurotoxicity in vivo, were misclassified as nonembryotoxic with the EST. The aim of this study was to improve the EST to correctly screen such developmental neurotoxicants. We developed a neuronal endpoint (Tuj-1 ID50) using flow cytometry analysis of Tuj-1-positive cells to screen developmental neurotoxicants (MeHg, valproic acid, sodium arsenate and sodium arsenite) correctly using an adherent monoculture differentiation method. Using Tuj-1 ID50 in the EST instead of cardiac ID50, all of the tested chemicals were classified as embryotoxic, while the negative controls were correctly classified as nonembryotoxic. To support the validity of Tuj-1 ID50) , we compared the results from two experimenters who independently tested MeHg using our modified EST. An additional neuronal endpoint (MAP2 ID50), obtained by analyzing the relative quantity of MAP2 mRNA, was used to classify the same chemicals. There were no significant differences in the three endpoint values of the two experimenters or in the classification results, except for isoniazid. In conclusion, our results indicate that Tuj-1 ID50 can be used as a surrogate endpoint of the traditional EST to screen developmental neurotoxicants correctly and it can also be applied to other chemicals.

4-Chlorotetrazolo[1,5-a]quinoxaline inhibits activation of Syk kinase to suppress mast cells in vitro and mast cell-mediated passive cutaneous anaphylaxis in mice.

4-Chlorotetrazolo[1,5-a]quinoxaline is a quinoxaline derivative. We aimed to study the effects of 4-chlorotetrazolo[1,5-a]quinoxaline on activation of mast cells in vitro and in mice. 4-Chlorotetrazolo[1,5-a]quinoxaline reversibly inhibited degranulation of mast cells in a dose-dependent manner, and also suppressed the expression and secretion of TNF-α and IL-4 in mast cells. Mechanistically, 4-chlorotetrazolo[1,5-a]quinoxaline inhibited activating phosphorylation of Syk and LAT, which are crucial for early FcεRI-mediated signaling events, as well as Akt and MAP kinases, which play essential roles in the production of various pro-inflammatory cytokines in mast cells. Notably, although 4-chlorotetrazolo[1,5-a]quinoxaline inhibited the activation of Fyn and Syk, minimal inhibition was observed in mast cells in the case of Lyn. Furthermore, consistent with its in vitro activity, 4-chlorotetrazolo[1,5-a]quinoxaline significantly suppressed mast cell-mediated passive cutaneous anaphylaxis in mice. In summary, the results from this study demonstrate that 4-chlorotetrazolo[1,5-a]quinoxaline shows an inhibitory effect on mast cells in vitro and in vivo, and that this is mediated by inhibiting the activation of Syk in mast cells. Therefore, 4-chlorotetrazolo[1,5-a]quinoxaline could be useful in the treatment of mast cell-mediated allergic diseases.

Assessment of penetration of quantum dots through in vitro and in vivo human skin using the human skin equivalent model and the tape stripping method.

Quantum dots (QDs) are rapidly emerging as an important class of nanoparticles (NPs) with potential applications in medicine. However, little is known about penetration of QDs through human skin. This study investigated skin penetration of QDs in both in vivo and in vitro human skin. Using the tape stripping method, this study demonstrates for the first time that QDs can actually penetrate through the stratum corneum (SC) of human skin. Transmission electron microscope (TEM) and energy diverse X-ray (EDX) analysis showed accumulation of QDs in the SC of a human skin equivalent model (HSEM) after dermal exposure to QDs. These findings suggest possible transdermal absorption of QDs after dermal exposure over a relatively long period of time.

Toxicometabolomics of urinary biomarkers for human gastric cancer in a mouse model.

Toxicometabolomics of urinary biomarkers for human gastric cancer in a mouse model was investigated using (1)H-nuclear magnetic resonance (NMR) spectroscopy. A human gastric adenocarcinoma cell line (1 × 10(7) cells/ml) was grafted onto the skin of the back of intact male BALB/c-nu/nu mice. After the xenografted tumors developed, urine was collected and analyzed for endogenous metabolites. Global profiling combined with principal components analysis (PCA), partial least squares-discriminant analysis (PLS-DA), and orthogonal projections to latent squares-discriminant analysis (OPLS-DA) showed distinct separation of clusters between control and tumor-bearing mice. Targeted profiling revealed significant changes in trimethylamine oxide (TMAO), 3-indoxylsulfate, hippurate, and citrate levels in mice carrying human gastric cancer cells compared to normal mice. The levels of TMAO (0.41-fold) and hippurate (0.26-fold) in tumor-bearing mice were significantly decreased, whereas the levels of 3-indoxylsulfate (3.39-fold), 2-oxoglutarate (2.32-fold), and citrate (1.9-fold) were significantly increased in urine samples of tumor-bearing mice. Data suggest that TMAO, hippurate, 3-indoxylsulfate, 2-oxoglutarate, and citrate may serve as useful urinary biomarkers for gastric tumorigenesis in a mouse model.

Functional role of phospholipase D (PLD) in di(2-ethylhexyl) phthalate-induced hepatotoxicity in Sprague-Dawley rats.

Phospholipase D (PLD) is an enzyme that catalyzes the hydrolysis of phosphatidyl choline (PC) to generate phosphatidic acid (PA) and choline. PLD is believed to play an important role in cell proliferation, survival signaling, cell transformation, and tumor progression. However, it remains to be determined whether enhanced expression of PLD in liver is sufficient to induce hepatotoxicity. The aim of this study was to investigate the possible role of PLD in di(2-ethylhexyl) phthalate (DEHP)-induced hepatotoxicity in Sprague-Dawley rats. The phthalate, DEHP (500 mg/kg/d), was administered orally, daily to prepubertal rats (4 wk of age, weighing approximately 70-90 g) for 1, 7, or 28 d. In this study, protein expression levels of PLD1/2, peroxisome proliferator-activated receptor (PPAR), and cytochrome P-450 (CYP) were determined by Western blot analysis using specific antibodies. Liver weight was significantly increased in the DEHP treatment groups. Immunohistochemical analysis demonstrated that DEHP produced strong staining of proliferating cell nuclear antigen (PCNA) at 28 d of exposure, suggestive of hepatocyte proliferation. A significant rise in PLD1/2 expression was observed in liver of DEHP-exposed rats after 7 d. Further, PPARα, constitutive androstane receptor (CAR), pregnane X receptor (PXR), and CYP2B1 protein expression levels were markedly elevated in DEHP-treated groups. Our results suggest that DEHP significantly enhanced the expression of PLD, which may be correlated with PPARα-induced hepatotoxicity through a complex interaction with nuclear receptors including CAR and PXR.

Assessment of heavy metal exposure via the intake of oriental medicines in Korea.

Oriental medical herbs are mainly natural products that are generated by simple processes, and therefore there is the possibility of contamination with various pollutants, including heavy metals. Heavy metals produce adverse effects in humans, and the toxicities of lead (Pb) and cadmium (Cd) are well established. This study evaluated the effects of exposure to Pb and Cd via the intake of the frequent prescriptions of oriental medicines, and assessed the risk to the Korean population based on domestic data. The average daily exposures to Pb and Cd were estimated. This is the first study to evaluate exposure and risk of heavy metal intoxication through intake of oriental medicines in Korea. Despite the uncertainties and limits of the data, these results simulate realistic exposure levels.

Expression of surface markers on the human monocytic leukaemia cell line, THP-1, as indicators for the sensitizing potential of chemicals.

BACKGROUND: Evaluation of skin sensitization potential is an important part of the safety assessment of cosmetic ingredients and topical drugs. Recently, evaluation of changes in surface marker expression induced in dendritic cells (DC) or DC surrogate cell lines following exposure to chemicals represents one approach for in vitro test methods. OBJECTIVE: The study aimed to test the change of expression patterns of surface markers on THP-1 cells by chemicals as a predictive in vitro method for contact sensitization. METHODS: We investigated the expression of CD54, CD86, CD83, CD80, and CD40 after a 1-day exposure to sensitizers (1-chloro-2,4-dinitrobenzene; 2,4-dinitrofluorobenzene; benzocaine; 5-chloro-2-methyl-4-isothiazolin-3-one; hexyl cinnamic aldehyde; eugenol; nickel sulfate hexahydrate; potassium dichromate; cobalt sulfate; 2-mercaptobenzothiazole; and ammonium tetrachloroplatinate) and non-sensitizers (sodium lauryl sulfate, benzalkonium chloride, lactic acid, salicylic acid, isopropanol, and dimethyl sulphoxide). The test concentrations were 0.1x, 0.5x, and 1x of the 50% inhibitory concentration, and the relative fluorescence intensity was used as an expression indicator. RESULT AND CONCLUSION: By evaluating the expression patterns of CD54, CD86, and CD40, we could classify the chemicals as sensitizers or non-sensitizers, but CD80 and CD83 showed non-specific patterns of expression. These data suggest that the THP-1 cells are good model for screening contact sensitizers and CD40 could be a useful marker complementary to CD54 and CD86.

Suitability of macrophage inflammatory protein-1beta production by THP-1 cells in differentiating skin sensitizers from irritant chemicals.

BACKGROUND: Worldwide restrictions in animal use for research have driven efforts to develop alternative methods. OBJECTIVE: The study aimed to test the efficacy of the macrophage inflammatory protein-1beta (MIP-1beta) assay for testing chemicals' skin-sensitizing capacity. METHODS: The assay was performed using 9 chemicals judged to be sensitizing and 7 non-sensitizing by the standard in vivo assays. THP-1 cells were cultured in the presence or absence of 4 doses, 0.01x, 0.1x, 0.5x, or 1x IC(50) (50% inhibitory concentration for THP-1 cell proliferation) of these chemicals for 24 hr, and the MIP-1beta level in the supernatants was determined. Skin sensitization by the test chemicals was determined by MIP-1beta production rates. The MIP-1beta production rate was expressed as the relative increase in MIP-1beta production in response to chemical treatment compared with vehicle treatment. RESULTS AND CONCLUSION: When the threshold MIP-1beta production rate used was 100% or 105% of dimethyl sulfoxide, all the sensitizing chemicals tested (dinitrochlorobenzene, hexyl cinnamic aldehyde, eugenol, hydroquinone, dinitrofluorobenzene, benzocaine, nickel, chromium, and 5-chloro-2-methyl-4-isothiazolin-3-one) were positive, and all the non-sensitizing chemicals (methyl salicylate, benzalkonium chloride, lactic acid, isopropanol, and salicylic acid), with the exception of sodium lauryl sulfate, were negative for MIP-1beta production. These results indicate that MIP-1beta could be a biomarker for classification of chemicals as sensitizers or non-sensitizers.

Evaluation of immunotoxicity induced by pirimiphos-methyl in male Balb/c mice following exposure to for 28 days.

Pirimiphos-methyl (O-2-diethylamino-6-methylpyrimidin-4-yl O,O-dimethyl phosphorothioate: POM) is widely used organophosphorous (OP) insecticide as a grain protectant to control insects during storage. This study was carried out to assess the immunologic effects of POM in Balb/c mice after 28-day oral exposure. Three dose levels of POM (10, 60, or 120 mg/kg/day) were administered orally to mice for 4 weeks. At autopsy after 28-day exposure, there were significant decreases in relative spleen weight and splenic cellularity found at 120 mg POM, but body weight, relative thymic weight, thymic cellularity, and splenic and thymic subsets were not affected. T cell proliferation response induced by Con A was significantly decreased at all dosages though no statistical differences were observed in splenic B cell proliferation. Significant increases in the production of cytokines (IL-2, IL-4, IL-6, IFN-gamma, and IL-10) were evident on the whole, but the increase in production of inflammatory cytokines overwhelmed that of the T(H)1 cell suppressive cytokine (IL-10). The relative levels of three types of autoantibodies, anti-dsDNA, anti-histone, and antinuclear antibody (ANA) were dose-dependently decreased in serum. Oral exposure to POM induced a significant decrease in Immunoglobulin M production capability in Balb/c mice. This decrease in antibody production capability may result from disturbances in cytokine balance produced by splenic immune cells. These results show that POM may induce allergic responses by relatively enhancing T(H)2 development and additionally contribute to chronic inflammation by attracting macrophage by IFN-gamma.

Di(2-ethylhexyl) phthalate induces apoptosis through peroxisome proliferators-activated receptor-gamma and ERK 1/2 activation in testis of Sprague-Dawley rats.

Di(2-ethylhexyl) phthalate (DEHP) is a well-known hepatic and reproductive toxicant whose toxicity may be mediated by peroxisome proliferators-activated receptor (PPAR). This study examined the effects of DEHP on the expression of PPAR-regulated genes involved in testicular cells apoptosis. Sprague-Dawley male rats were treated orally with 250, 500, or 750 mg/kg/d DEHP for 28 d, while control rats were given corn oil. The levels of cell cycle regulators (pRb, cyclins, CDKs, and p21) and apoptosis-related proteins were analyzed by Western blot analysis. The role of PPAR-gamma (PPAR-gamma), class B scavenger receptor type 1 (SR-B1), and ERK1/2 was further studied to examine the signaling pathway for DEHP-induced apoptosis. Results showed that the levels of pRB, cyclin D, CDK2, cyclin E, and CDK4 were significantly lower in rats given 500 and 750 mg/kg/d DEHP, while levels of p21 were significantly higher in rat testes. Dose-dependent increases in PPAR-gamma and RXRalpha proteins were observed in testes after DEHP exposure, while there was a significant decrease in RXRgamma protein levels. In addition to PPAR-gamma, DEHP also significantly increased SR-B1 mRNA and phosphorylated ERK1/2 protein levels. Furthermore, DEHP treatment induced pro-caspase-3 and cleavage of its substrate protein, poly(ADP-ribose) polymerase (PARP), in a dose-dependent manner. Data suggest that DEHP exposure may induce the expression of apoptosis-related genes in testes through induction of PPAR-gamma and activation of the ERK1/2 pathway.

IFN-gamma/IRF-1-induced p27kip1 down-regulates telomerase activity and human telomerase reverse transcriptase expression in human cervical cancer.

Telomerase activation is regulated by the expression of human telomerase reverse transcriptase (hTERT) and is a key step in the development of human cancers. Interferon-gamma (IFN-gamma) signaling induces growth arrest in many tumors through multiple regulatory mechanisms. The p27 tumor suppressor protein inhibits the formation of tumors through the induction of cell cycle arrest and/or apoptosis. We demonstrate here that p27Kip1 inhibits hTERT mRNA expression and telomerase activity through post-transcriptional up-regulation by IFN-gamma/IRF-1 signaling. The ectopic expression of p27 suppressed hTERT expression and telomerase activity in human cervical cancer cell lines, HeLa and HT3. Furthermore, hTERT promoter activity of mouse embryonic fibroblasts (MEFs) deficient in p27 (p27-/- MEFs) was significantly higher than that of wild-type MEFs. Overexpression of p27 suppressed hTERT promoter activity and telomerase activity of p27-/- MEFs. In addition p27 down-regulated E7 protein expression and in transiently transfected HeLa cells, E7 increased hTERT promoter activity. In conclusion, we propose that inhibition of the hTERT expression and telomerase activity may be a novel tumor suppressor function of p27.

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.

Neurotoxic effects of alcohol and acetaldehyde during embryonic development.

Alcohol drinking during pregnancy results in abnormal fetal development, including fetal alcohol syndrome (FAS) in humans and experimental animals. FAS is characterized by two major effects, including central nervous system (CNS) dysfunction and multiple anomalies recognizable mainly as a typical face. However, the mechanisms of alcohol-induced embryotoxicity have not been clearly demonstrated. The aim of the present study was to investigate the possible mechanisms underlying ethanol-induced FAS in the developing embryo. First, ethanol-induced developmental abnormalities were investigated in vitro. Postimplantation embryos at gestation day (GD) 9.5 were cultured for 48 h and observed for morphological changes. Ethanol-mediated changes in proteins regulated apoptosis (p53 and bcl-2), antioxidant (vitamin E and catalase) activities, generation of reactive oxygen species (ROS), and oxidative DNA damage shown as 8-hydroxy-2'-deoxyguanosine (8-OHdG) were measured in embryonic midbrain cells. Alcohol or acetaldehyde significantly induced cytotoxicity in cultured rat embryonic midbrain cells. The levels of p53, bcl-2, and 8-OHdG were concomitantly changed by alcohol and acetaldehyde treatment in midbrain cells. Injured cells induced by ROS were increased by alcohol or acetaldehyde treatment in midbrain cells. Cotreatment with alcohol or acetaldehyde and catalase decreased cytotoxicity in midbrain cells. In postimplantation embryo culture, alcohol or acetaldehyde-treated embryos showed retardation of embryonic growth and development in a concentration-dependent manner. These results indicate that alcohol and its metabolite acetaldehyde induce fetal developmental abnormalities by disrupting cellular differentiation and growth. Data demonstrate that some antioxidants can partially protect against the alcohol-induced embryonic developmental toxicity.

Multigeneration reproductive and developmental toxicity study of bar gene inserted into genetically modified potato on rats.

Each specific protein has an individual gene encoding it, and a foreign gene introduced to a plant can be used to synthesize a new protein. The identification of potential reproductive and developmental toxicity from novel proteins produced by genetically modified (GM) crops is a difficult task. A science-based risk assessment is needed in order to use GM crops as a conventional foodstuff. In this study, the specific characteristics of GM food and low-level chronic exposure were examined using a five-generation animal study. In each generation, rats were fed a solid pellet containing 5% GM potato and non-GM potato for 10 wk prior to mating in order to assess the potential reproductive and developmental toxic effects. In the multigeneration animal study, there were no GM potato-related changes in body weight, food consumption, reproductive performance, and organ weight. Polymerase chain reaction (PCR) was carried out using extracted genomic DNA to examine the possibility of gene persistence in the organ tissues after a long-term exposure to low levels of GM feed. In each generation, the gene responsible for bar was not found in any of the reproductive organs of the GM potato-treated male and female rats, and the litter-related indexes did not show any genetically modified organism (GMO)-related changes. The results suggest that genetically modified crops have no adverse effects on the multigeneration reproductive-developmental ability.

Assessment of estrogenic and androgenic activities of tetramethrin in vitro and in vivo assays.

Tetramethrin, a synthetic pyrethroid insecticide, is used globally for agriculture, and thus potential environmental exposure to tetramethrin is a concern. Environmental chemicals that are hormonally active (particularly estrogen or androgen) may adversely affect the reproductive and endocrine systems. However, little is known about the estrogenic and androgenic activities of tetramethrin. In this study, uterine CaBP-9k gene expression assay and a uterotrophic assay were conducted for estrogenic activity assessment of tetramethrin, and a Hershberger assay was conducted for androgenic activity. Estrogen receptor (ERalpha and ERbeta) protein levels were also measured in tetramethrin-treated rat uteri. Northern blot analysis showed reduction in uterine CaBP-9k mRNA levels in response to tetramethrin, as well as when rats were given both tetramethrin and 17beta-estradiol (E2). In the uterotrophic assay using 18-d-old female Sprague-Dawley rats, subcutaneous treatment with tetramethrin (5 to 800 mg/kg/day) for 3 d led to a statistically significant decrease in absolute and relative uterine wet weights at all doses tested. Moreover, tetramethrin blocked the effect of E2 on uterine weights. In addition, tetramethrin reduced absolute and relative vaginal wet weights, and also inhibited the increases of vaginal weights produced by E2. Tetramethrin showed no androgenic on antiandrogenic activities in the Hershberger assay. These results suggest that tetramethrin might exert endocrine-disrupting effects on female rats through antiestrogenic action.

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.

No androgenic/anti-androgenic effects of bisphenol-A in Hershberger assay using immature castrated rats.

Several studies have demonstrated that bisphenol A (BPA) exhibited weak estrogenic activity in the 3-day uterotrophic assay using ovariectomized (OVX) and immature rats (Toxicol. Lett. 115 (2000) 231; Regul. Toxicol. Pharmacol. 32 (2000) 118; J. Toxicol. Sci. 26 (2001) 111) and BPA also possessed anti-androgenic activity in in vitro yeast based assays (J. Endocrinol. 158 (1998) 327). To investigate anti-androgenic effects of BPA. a rodent Hershberger assay was carried out using immature Sprague-Dawley male rats. An androgen agonist, testosterone (0.4 mg/kg per day), was administered for 7 consecutive days by subcutaneous (s.c.) injection as a positive control. Additionally, a pure androgen antagonist, flutamide (1, 5. 10 mg/kg per day. oral) was co-administered with testosterone (0.4 mg/kg per day s.c.). BPA was also administered orally with or without testosterone (0.4 mg/kg per day, s.c.) for 7 consecutive days. In the testosterone treated groups, glans penis, seminal vesicles, ventral prostate, and levator ani plus bulbocavernosus muscles (LABC) weights were significantly increased compared with control. However. flulamide dose-dependently inhibited the testosterone-induced re-growth of seminal vesicles, ventral prostate, and LABC, with a significant decrease at flutamide 1.0 mg/kg and above (P<0.05). Serum LH levels were also significantly increased (5 mg/kg and above, P<0.05), but no changes in serum testosterone levels. In contrast, BPA had no effects on the re-growth of seminal vesicles, ventral prostate and LABC induced by testosterone, and no significant differences were observed in serum LH and testosterone levels. In summary, the Hershberger assay could be a sensitive method for detecting androgenic or anti-androgenic chemicals, but BPA did not exhibit any androgenic or anti-androgenic activities in Hershberger assay.

Comparative evaluation of alkylphenolic compounds on estrogenic activity in vitro and in vivo.

This study was undertaken to compare the sensitivity of screening test methods and to investigate the structure-activity relationships of the estrogenic activity of alkylphenolic compounds (APs) using in vitro and in vivo assays. Two in vitro systems, MCF-7 cell proliferation (E-screen assay) and competitive binding assay to estrogen receptor (ER), were selected to evaluate the estrogenic effects. Uterotrophic assay and Calbindin-D9K (CaBP9K) mRNA expression were also examined in ovariectomized Sprague-Dawley female rats. A series of APs with various alkyl groups were examined, namely, 4-propylphenol, 4-butylphenol, 4-t-butylphenol, 4-pentylphenol, 4-nonylphenol, 4-octylphenol, 4-t-octylphenol, and 4-phenylphenol, and 17beta-estradiol (E2) was used as a positive control. In the E-screen assay, E2 was found to induce maximum proliferation of MCF-7 cells at 1 nM. Among the APs, 4-t-octylphenol and 4-nonylphenol were found to be considerably more potent than any other compound and estrogenic effects were detectable at 1 and 10 microM, respectively. 4-t-Octylphenol and 4-nonylphenol inhibited the binding of E2 to the ER of MCF-7 cells in a competitive ER binding assay. The uterotrophic effects to APs (10, 50, 200, and 400 mg/kg/d) were compared to E2 (1 microg/kg) in ovariectomized rats after treatment for 3 d. 4-Nonylphenol, 4-t-octylphenol, and 4-phenylphenol produced dose-dependent increases in the uterine weights of ovariectomized rats. In the CaBP-9K mRNA expression test, CaBP-9K mRNA levels were detected in the uteri of ovariectomized rats treated with 4-pentylphenol (400 mg/kg), 4-nonylphenol, 4-phenylphenol (200 and 400 mg/kg), and 4-t-octylphenol (50 mg/kg and above), respectively. In the dot blot assay, CaBP-9K mRNA levels were significantly increased in rats exposed to 4-t-octylphenol (200 and 400 mg/kg), 4-pentylphenol, 4-nonylphenol, and 4-phenylphenol (400 mg/kg), respectively. Among the APs, compounds with bulky alkyl groups or higher carbon numbers possessed higher estrogenic capacity. In addition, the pattern of CaBP-9K expression correlated with that of the 3-d uterotrophic assay. Therefore, our results suggest that the CaBP-9K gene might be used as a potential biomarker for the screening of endocrine disruptors.

Maternal-fetal disposition of bisphenol a in pregnant Sprague-Dawley rats.

This study describes the maternal-fetal disposition of bisphenol A and its distribution into the placenta and amniotic fluid after iv injection (2 mg/kg) to pregnant Sprague-Dawley rats. Bisphenol A was distributed extensively to the placenta and fetus, with their respective AUC values 4.4- and 2.2-fold greater than AUC for the maternal serum. In contrast, the distribution of bisphenol A into the amniotic fluid was low, with the mean amniotic fluid-to-maternal serum AUC ratio of 0.2. The decay curves of bisphenol A in the placenta, fetus, and amniotic fluid paralleled that of the maternal serum during the terminal elimination phase. A five-compartment open model consisting of the maternal central, maternal peripheral, placental, fetal, and amniotic fluid compartments was used to describe the disposition of bisphenol A in pregnant rats, with the elimination occurring from the maternal central and fetal compartments. Based on this model, bisphenol A delivered to the placenta was transferred primarily to the fetus [kpf/(kpf + kpc + kpa) = 65.4 %], with the remaining fraction transported to the maternal central (33.2%) and amniotic fluid (1.4%) compartments. Bisphenol A was eliminated from the amniotic fluid by the fetal (63.9%) and placental (36.1%) routes. On the other hand, bisphenol A was eliminated from the fetus primarily by the placental route back to mother [kfp/(kfp + kfa + kfo) = 100%], with the amniotic route playing an insignificant role in fetal elimination. The percent contribution of the fetal elimination to the total elimination in the maternal-fetal unit was 0.0% [CLfoAUCfetus/(CLcoAUCmaternal serum + CLfoAUCfetus)]. The pharmacokinetic model used in this study provides insights into the routes of elimination of bisphenol A in the maternal-fetal rat upon maternal administration.