Evaluation for reliability and feasibility of the draft protocol for the enhanced rat 28-day subacute study (OECD Guideline 407) using androgen antagonist flutamide

As part of an international validation project to establish a test protocol for the 'Enhanced OECD Test Guideline no. 407', a 28-day repeated dose study of flutamide was performed (1) to examine which of the current and/or additional parameters can detect endocrine effects of test chemicals most reliably and sensitively, (2) to investigate whether it is actually feasible to routinely include all additional parameters into the testing routine, and (3) to assess intra-laboratory variability by performing two identical studies (experiments A and B) in parallel using groups of five animals each per dose and sex. Groups of five male and five female CD(SD)IGS rats were treated by oral gavage with 0, 1, 10 and 100 mg flutamide/kg body weight for at least 28 days. The dose level considered to be around the MTD (100 mg/kg) exerted the expected antiandrogenic effects on androgen related tissues: significant decrease of the weights of androgen dependent organs and the sperm count and increase in histopathological lesions. At the middle dose (10 mg/kg), significant decrease of prostate weight (ventral and dorso-lateral parts combined) was observed and it was suggested that weight measurement of androgen dependent organs provides the most reliable and sensitive endpoint with this protocol. As for the feasibility, because of many items in this protocol, selection should be based on the sensitivity. From our data, addition of weight measurement of androgen dependent organs to the items of the existing OECD 407 guideline might allow accurate screening for endocrine disruptors. At the dose level considered to be around the MTD, the findings achieving statistical significance in one experiment with five animals/dose/sex could be reproduced in the second experiment, and evaluation with the small groups was consistent with findings using the combined groups of 10 animals/dose/sex. The results demonstrate that the protocol can reliably detect antiandrogenic effects of flutamide.     

Expression of endothelial nitric oxide synthase is induced by estrogen with glycogen synthase 3beta phosphorylation in MCF-7 cells

The endothelial nitric oxide synthase (eNOS) gene is induced by a variety of extracellular signals and NOS plays a key role in many physiological as well as pathological processes, including tumorgenesis. Some studies showed a positive correlation between the level of NOS protein and progression of malignancy in human breast cancer. In this study, we examined eNOS mRNA expression in human breast cancer cell lines. MCF-7 cells, which showed an estrogen receptor positive phenotype, were treated with estradiol or LiCl, a selective inhibitor of glycogen synthase kinase (GSK)-3beta. Both estradiol and LiCl enhanced the expression of eNOS mRNA with the phosphorylation of GSK-3beta, but not Akt. The induction was completely suppressed by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002, but not by PD98059, MEK-1 inhibitor nor rapamycin, p70S6 kinase inhibitor. We conclude that the estradiol-induced eNOS expression is modulated by PI3-kinase-dependent GSK-3beta pathway.     

Effect of prenatal and prepubertal genistein exposure on N-methyl-N-nitrosourea-induced mammary tumorigenesis in female Sprague-Dawley rats

BACKGROUND: The effect of prenatal and prepubertal genistein exposure on the development of chemically-induced mammary carcinomas in rat was investigated. Materials and METHODS: Genistein was subcutaneously (s.c.) injected daily, from gestational days 15 to 19, into pregnant Sprague-Dawley rats at 0, 1.5 or 30 mg/kg/day. Female offspring of mothers not exposed to genistein during pregnancy received daily s.c. injection, from prepubertal days 15 to 19, at a dose of 1.5 or 30 mg/kg/day. At 28 days of age, 6 female offspring from each group were sacrificed to observe the influence of genistein and the remaining rats were injected intraperitoneally with 50 mg/kg N-methyl-N-nitrosourea (MNU). Rats injected with MNU were sacrificed at 26 weeks of age or when their largest mammary tumor was > or = 1 cm in size. RESULTS: At the time when MNU was administered, the different groups had comparable mammary gland development; genistein-treated and -untreated rats had similar numbers of terminal end buds (TEBs) at the periphery of the mammary glandular tree. However, estrogen receptor alpha (ER alpha)- and progesterone receptor (PgR)-positive cells, p63-positive cells and proliferating cell nuclear antigen (PCNA)-labeling index were lower in genistein-exposed TEBs. Although tumor multiplicity and latency were not significant, prenatal or prepubertal genistein exposure, at low or high dosage, tended to suppress the incidence of mammary carcinomas > or = 1 cm; suppression was significant in the prepubertal low-dose group. CONCLUSION: The majority of the mammary carcinomas in the present study were hormone-dependent. The present findings suggest that administration of genistein in the perinatal period has protective effects against MNU-induced mammary carcinoma in Sprague-Dawley rats, via reduction of levels of ER alpha- and/or PgR-positive cells (presumed progenitor cells of mammary carcinomas), p63-positive mammary progenitor/stem cells (involved in cell renewal) and PCNA-positive cells (necessary for cell proliferation).     

Lack of estrogenic or (anti-)androgenic effects of d-phenothrin in the uterotrophic and Hershberger assays

Synthetic pyrethroids are among the most common insecticides and pesticides currently in use worldwide. Recently, d-phenothrin, a synthetic pyrethroid, is suspected to have endocrine activities through the estrogen and androgen receptors. However, no study has been conducted to evaluate its potential for hormonal activity using an in vivo test specifically focused on estrogenic and androgenic activities. In this study, we evaluated the interaction of d-phenothrin (0, 100, 300 or 1000 mg/kg per day, p.o.) with estrogen- or androgen-mediated mechanisms using in vivo short-term assays. While internationally standardized protocols for the uterotrophic and Hershberger assays have not yet been fully developed, both are widely used and are being considered by the OECD as short-term screening assays for hormonal activity. The highest dose level tested for d-phenothrin was a limit dose (1000 mg/kg per day) designated in the current draft protocol by the OECD, and in fact there was no excessive systemic toxicity in both assays; slightly increased liver weight but no change of serum androgen levels in accessing anti-androgenicity. Potential estrogenic effect of d-phenothrin was evaluated by means of 3-day uterotrophic assay using immature Crj:CD(SD)IGS rats (20 days of age). No increase in uterine weight (wet or blotted) was observed following oral exposure to d-phenothrin. Reference control ethynyl estradiol (0.001 mg/kg per day) showed a significant effect in this assay protocol. A 10-day Hershberger assay using castrated peripubertal male rats measures the androgenic or anti-androgenic effects of the test chemicals on several accessory glands/tissues (the ventral prostate, dorso-lateral prostate, seminal vesicles with coagulating glands, levator ani plus bulbocavernosus muscles, glans penis and Cowper's glands). d-Phenothrin was administered by oral gavage for 10 days to castrated male Crj:CD(SD)IGS rats (7 weeks of age, rats were castrated at 6 weeks of age) with or without co-administration of 0.2 mg/kg per day testosterone propionate (subcutaneous injection on the dorsal surface). Reference controls of methyltestosterone and p,p'-DDE (100 mg/kg per day) provided significant effects in this assay protocol, whereas d-phenothrin did not show any androgenic or anti-androgenic effects. It is concluded that, based on the results of these two reliable in vivo assays, d-phenothrin exhibits no potential to cause adverse estrogenic or (anti-)androgenic effects even at dose of 1000 mg/kg per day, the limit dose designated in the current draft protocol by the OECD.     

A member of the Pyrin family, IFI16, is a novel BRCA1-associated protein involved in the p53-mediated apoptosis pathway

We identified IFI16 as a BRCA1-associated protein involved in p53-mediated apoptosis. IFI16 contains the Pyrin/PAAD/DAPIN domain, commonly found in cell death-associated proteins. BRCA1 (aa 502-802) interacted with the IFI16 Pyrin domain (aa 1-130). We found that IFI16 was localized in the nucleoplasm and nucleoli. Clear nucleolar IFI16 localization was not observed in HCC1937 BRCA1 mutant cells, but reintroduction of wild-type BRCA1 restored IFI16 nuclear relocalization following IR (ionizing radiation). Coexpression of IFI16 and BRCA1 enhanced DNA damage-induced apoptosis in mouse embryonic fibroblasts from BRCA1 mutant mice expressing wild-type p53, although mutant IFI16 deficient in binding to BRCA1 did not induce apoptosis. Furthermore, tetracycline-induced IFI16 collaborated in inducing apoptosis when adenovirus p53 was expressed in DNA-damaged p53-deficient EJ cells. These results indicate a BRCA1-IFI16 role in p53-mediated transmission of DNA damage signals and apoptosis.     

Novel Concept to Detect an Optimum Thixoforming Condition of Al-Al3Ni Functionally Graded Material by Wavelet Analysis for Online Operation

A novel technique to characterize the transition phenomenon from solid to melt of Al-Al₃Ni functionally graded material (FGM) through a wavelet analysis for the development of a thixoforming system is investigated. Identification of an optimum semi-solid condition for thixoforming is necessary not only for the construction of a system but also the fabrication of a near-net-shape product with fine microstructure. An online wavelet analysis system using Haar’s wavelet function, which is applied for its simplicity compared with Daubechies’ wavelet function, is developed to find the optimum operating condition. A thixoforming system, which is constructed adapting a threshold value as an index, monitors successfully a discontinuity of deformation of Al-Al₃Ni FGM with the temperature rise. Thus, the timing of an operation is not at pre-fixed temperature but at the time when the index related to a wavelet function is satisfied. The concept is confirmed to be suitable from the micro-structural observation of the Al-Al₃Ni FGM product, because the product under the optimum condition is found to have refined Al₃Ni grains, which change from coarse grains and are expected to improve the mechanical properties.     

Calcification and osteopontin localization in the peritoneum of patients on long-term continuous ambulatory peritoneal dialysis therapy.

BACKGROUND: Peritoneal calcification is an uncommon complication of continuous ambulatory peritoneal dialysis (CAPD), which is mainly observed in patients on long-term therapy. Although some asymptomatic patients must have microscopic calcification in their peritoneum, little information on this topic has been published. Recent studies have revealed active participation of adhesive/chemotactic protein osteopontin (OPN) in dystrophic calcification. METHODS: Peritoneal tissue was obtained by biopsy or at autopsy from 18 CAPD patients (median duration, 122 months), 5 control haemodialysis (HD) patients, and 3 pre-CAPD patients. The distribution of calcium deposits and OPN protein was determined by von Kossa staining and immunohistochemistry, respectively. Smooth muscle cells and macrophages were identified with anti-alpha smooth muscle actin (alpha-SMA) and anti-CD68 antibodies. RESULTS: Calcium deposits with various configurations were observed in specimens from 12 of the 18 CAPD patients. They included massive calcification facing the peritoneal cavity, scattered granular or crystalloid deposits in the submesothelial stroma, and oval-shaped deposits formed within hyalinized vasa. Most were present in highly sclerosed areas and accompanied by extracellular OPN precipitation. Cytoplasmic OPN was detected in infiltrating leukocytes, granulation tissue cells, fibroblast-like cells and mast cells. Computerized tomography examination also detected peritoneal calcification in seven of the CAPD patients. No calcium deposits or OPN staining was detected in control specimens. CONCLUSIONS:The results of our study suggest that microscopic peritoneal calcification is frequent in patients on CAPD for more than 10 years. Myofibroblast infiltration, OPN expression, calcium deposition, and associated OPN precipitation seem to be components of the peritoneal changes in such patients.     

Influence of Age on Diurnal Variability in Measurements of Spirometric Indices and Respiratory Pressures

We evaluated variations in spirometric indices (i.e., forced vital capacity [FVC], peak expiratory flow [PEF], and forced expiratory volume in 1 sec [FEV,₁]) and static respiratory muscle pressures (i.e., maximum static inspiratory pressure [PI_max] and maximum static expiratory pressure [PE_max]) within a span of 12 hr in 60 healthy elderly subjects, 60 young subjects, and 30 Chronic Obstructive Pulmonary disease (COPD) patients. There were no differences among data of FVC, PEF, FEV₁, Pl_max, and PE_max on three separate occasions within a day in the elderly or the young. The mean coefficient of variation (CV) values of PEF and Pl_max on three occasions were 3.0 ± 0.3% and 4.2 ± 0.4% in the elderly, and 2.4 ± 0.2% and 3.7 ± 0.3% in the young, respectively. No subjects had more than 9% CV on each measurement in the study, suggesting that there is no significant daytime variation in measurement of expiratory flow and respiratory pressures in young and elderly people. However, FVC, PEF FEV₁, and Pl_max values in the morning were smaller than those measured at the other two occasions in COPD patients. The results indicate that COPD affects diurnal variation in pulmonary function, but age alone has little impact on diurnal variation.