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  • 1
  • 2
    Keywords: OPTIMIZATION ; RECEPTOR ; CELLS ; IN-VITRO ; CELL ; COMBINATION ; VITRO ; GENE ; GENES ; BIOLOGY ; ASSAY ; CELL-LINE ; LINE ; NETHERLANDS ; RECEPTORS ; ER ; signaling ; SCIENCE ; methods ; ANDROGEN RECEPTOR ; CHEMICALS ; ANTAGONISTS ; ANDROGEN ; androgens ; in vitro ; reporter gene assay ; RANGE ; ANDROGEN-RECEPTOR ; PANEL ; Prevalidation ; ENDOCRINE DISRUPTORS ; REPORTER ; Hershberger assay ; Androgen reporter gene assay ; BIOASSAYS ; HUMAN CELL-LINE
    Abstract: To date there are no validated methods available to test androgenicity or antiandrogenicity in vitro. A problem with testing androgenicity using reporter genes is the possibility by other steroid receptors than androgen receptors to activate the same reporter gene, thereby lowering selectivity. To avoid this we have established a robust and very selective method, the AR CALUX reporter gene assay, to test androgenic and antiandrogenic activity of compounds in vitro. This assay uses a human U2-OS cell line stably transfected with the human androgen receptor and an androgen receptor responsive reporter gene. We optimized protocols to be used in combination with AR CALUX cells and carried out an in house prevalidation. In addition we successfully transferred this assay to another laboratory, leading to comparable test results with a panel of androgen receptor agonists and antagonists. The assay was able to readily rank a range of chemicals on the basis of their EC50 values. The CALUX assay was found to be selective for androgens and seemed not influenced by signaling through other steroid receptors.
    Type of Publication: Journal article published
    PubMed ID: 20438827
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  • 3
    Abstract: Despite more than a decade of research in the field of endocrine active compounds targeting the androgen receptor (AR), and although suitable cell lines can be obtained, no validated human stably transfected androgen sensitive transactivation assay is available. Bayer Schering Pharma (BSP) and the Flemish Institute for Technological Research (VITO), partners within the EU-sponsored 6th framework project ReProTect, made first steps towards such a validation. A standard operation protocol (SOP) developed at BSP based on the androgen sensitive PALM cell line was transferred to VITO and its performance and transferability were thoroughly studied. The investigation followed a generic protocol prepared for all reporter gene assays evaluated within ReProTect, and in both laboratories at least three independent experiments were performed. The highest concentration to be tested was limited to 10 microM, if needed. A few compounds, 17alpha-methyltestosterone (17alpha-MT), vinclozolin and linuron, were studied using a real world scenario, i.e., assuming that their interaction with the AR was not known: A prescreening for agonism and true, competitive antagonism was used to select conditions such as the appropriate mode of action, and the working range excluding cytotoxicity for the final screening. All other compounds were tested according to the generic protocol: Compounds screened for agonism were the reference androgen 17alpha-methyldihydrotestosterone (MDHT), levonorgestrel, norethynodrel, progesterone, o,p'-DDT, and dibutylphthalate (DBP), while compounds screened for antagonism were the reference anti-androgen flutamide, prochloraz, o,p'-DDT, progesterone, norethynodrel, and DBP. Cytotoxicity was assessed in parallel as lactate dehydrogenase release. The prescreen classified 17alpha-MT as androgenic, vinclozolin and linuron as anti-androgenic and compounds were tested accordingly. In the absence of cytotoxicity, appropriate androgenic properties of reference and test compounds were detected by both laboratories, o,p'-DDT and DBP had no androgenic activity. Across the two laboratories EC(50)-values for MDHT, 17alpha-MT, and levonorgestrel varied by not more than a factor of 3.4, for norethynodrel by a factor of 9.7. Progesterone effects could not fully be evaluated, as frequently concentration response curves were incomplete. In the absence of cytotoxicity anti-androgenic properties of reference and test compounds were also detected in both laboratories. DBP, the putative negative reference compound, was inactive, norethynodrel rather showed agonistic properties. Progesterone was an antagonist at low concentrations, but agonistic properties were observed in one laboratory at high concentrations. Since the highest test concentration was limited to 10 microM, for some compounds no complete concentration response curves were obtained and estimation of EC(50)-values was less robust. Our data demonstrated that the SOP was transferable, and that the assay was able to rank compounds with strong, weak, and without affinity for the AR and to discriminate agonists and antagonists. The sensitivity of the assay could be improved further, if the limit of solubility or beginning cytotoxicity was chosen as the highest test concentration. The assay avoids the use of tissues from laboratory animals, and thus contributes to the 3R concept. Furthermore, it could be adjusted to an intermediate/high throughput format. On the whole, this PALM assay is a promising candidate for further validation.
    Type of Publication: Journal article published
    PubMed ID: 19836445
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  • 4
    Keywords: IN-VITRO ; Germany ; IN-VIVO ; TOXICITY ; VITRO ; VIVO ; BIOLOGY ; TRIAL ; ASSAY ; PREDICTION ; in vitro tests ; FUTURE ; PROJECT ; SPRAGUE-DAWLEY RATS ; PROGRAM ; FRAMEWORK ; WEIGHT ; SCIENCE ; development ; methods ; PROFILES ; ANDROGEN RECEPTOR ; in vivo ; CHEMICALS ; in vitro assay ; PROFILE ; BREAST-CANCER CELLS ; FERTILITY ; in vitro ; DEVELOPMENTAL TOXICITY ; Alternative methods ; Reproductive toxicology ; ReProTect ; Alternative reproductive toxicity testing ; CONGENITAL DIAPHRAGMATIC-HERNIA ; DIETARY BISPHENOL-A ; Embryotoxicity ; Endocrine disruption ; ENDOCRINE DISRUPTOR VINCLOZOLIN ; ETHYLENE-GLYCOL MONOMETHYL ; GLUFOSINATE-AMMONIUM ; Hershberger assay ; Ring trial
    Abstract: ReProTect is a project within the 6th European Framework Program which has developed alternative methods aimed to reduce or replace animal experimentation in the field of reproductive toxicology. In its final year, a ring trial, named the "Feasibility Study", was conducted, in which 10 blinded chemicals with toxicologically well-documented profiles were analyzed by employing a test battery of 14 in vitro assays. EC(50) (half maximal effective concentration) or equivalent endpoints were determined and the test compounds were ranked relative to chemicals previously assayed in the tests of the battery. This comparative analysis together with a weight of evidence approach allowed a robust prediction of adverse effects on fertility and embryonic development of the 10 test chemicals in vivo. In summary, the vast majority of the predictions made based on the in vitro results turned out to be correct when compared to the whole animal data. The procedure used here, a nearest neighbor analysis coupled with a weight of evidence approach, may guide future activities in the field of alternative toxicity testing.
    Type of Publication: Journal article published
    PubMed ID: 20493943
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  • 5
    Keywords: RECEPTOR ; CELLS ; IN-VITRO ; CELL ; Germany ; MODEL ; MODELS ; VITRO ; screening ; TOOL ; GENE ; validation ; REDUCTION ; DOMAIN ; BIOLOGY ; hormone ; ASSAY ; CELL-LINE ; LINE ; PREDICTION ; RECEPTORS ; PROJECT ; AFFINITY ; ANTAGONIST ; ESTROGEN-RECEPTOR ; CYTOTOXICITY ; FRAMEWORK ; SCIENCE ; development ; ESTROGEN ; estrogen receptor ; CHEMICALS ; in vitro ; reporter gene assay ; RANGE ; ReProTect ; Agonist ; Estrogen activity ; hER transactivation assay ; MELN cell line ; TRANSACTIVATION ASSAY
    Abstract: The need for development and validation of in vitro hormone receptor transactivation assays as important alternative tools to study interactions with sex hormone receptors is outlined by international organisations, as such assays should be included in the OECD conceptual framework for the testing and assessment of endocrine active chemicals. Therefore as part of the European Union (EU)-sponsored 6th framework project ReProTect, the validation study with MELN cells, MCF-7 cells (ER+, estrogen receptor positive) which were stably transfected with the estrogen responsive gene ERE-beta Glob-Luc-SVNeo was set up. Standard operating procedures including a prescreen assay for unknown chemicals, an ER-agonist assay and an ER-antagonist assay were developed at the Flemish Institute for Technological Research, Belgium, and successfully transferred to Bayer Schering Pharma AG, Germany. Test results were obtained for 16 chemicals, and it was demonstrated that the MELN assay is transferable, robust and reproducible which allowed to rank chemical compounds according to their strong to weak affinity for the estrogen-alpha receptor, or identify negative chemicals within the test range up to 10(-5) M. Besides the screening for agonism, we demonstrated the suitability of MELN cells to test for antagonistic activity, which is of added value compared to current validated assays. As the MELN assay successfully passed the first modules of the ECVAM validation procedure, it now should be considered for further steps including the definition of a prediction model and application domain to get it accepted as an alternative screening assay, contributing to the 3R's with a reduction of animal experiments. (C) 2010 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 20362049
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  • 6
    ISSN: 1432-0800
    Source: Springer Online Journal Archives 1860-2000
    Topics: Energy, Environment Protection, Nuclear Power Engineering , Medicine
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1432-0703
    Source: Springer Online Journal Archives 1860-2000
    Topics: Energy, Environment Protection, Nuclear Power Engineering , Medicine
    Notes: Abstract. In this study, the toxicity of cadmium-contaminated clay to the zebrafish Danio rerio was evaluated. Kaolin clay was spiked with CdCl2 (1.28 mg Cd/g clay) and adult zebrafish were exposed to 0, 500, 1,000, and 2,000 mg/L Cd-contaminated clay in a continuous recirculation system. Cumulative mortality was evaluated as a function of exposure time, and LT50 values were calculated. Results of acute toxicity tests showed that an exposure to 1,000 mg/L Cd-contaminated clay resulted in LT50 values of 12 and 92 h (n = 2 experiments) and in a LT50 value of 22 h in both experiments with 2,000 mg/L Cd-contaminated clay. Positive control experiments with corresponding measured dissolved Cd concentrations were performed to evaluate by comparison the toxicity of the clay-bound Cd. These control experiments gave LT50 values higher than 144 h for both conditions. Moreover, no toxicity (LT50 〉 144 h) of 2,000 mg/L uncontaminated clay was observed. This study showed that cadmium present on clay particles can be bioavailable and exert a toxic effect to the zebrafish D. rerio.
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1432-136X
    Keywords: Haematology ; Osmotic changes ; Rainbow trout ; Low pH ; Aluminium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Elevated values of haematocrit were observed in rainbow trout (Oncorhynchus mykiss) during combined acid and aluminium exposure. Possible causes of this, i.e., decreased plasma volume, swelling of erythrocytes, and/or mobilization of erythrocytes into the blood were investigated. Slight haematocrit increases (10–20%) during mild acid stress (pH 5.0, 25 μmol Ca2+·1-1) were mostly caused by osmotic shifts. Both swelling of erythrocytes rocytes and a significant decrease of the plasma volume were demonstrated in fish at pH 5.0. These osmotic disturbined were greater during acid exposure (pH 5.0) combined with Al (60 μg·1-1; 200 μg·1-1). In addition, numbers of erythrocytes increased by 40% compared to acid exposure, which contributed to the severe haematocrit rise (35%) during Al exposure. A contraction of the spleen releasing erythrocytes into the blood is suggested to occur as an adrenergic response to hypoxia, which is observed in fish acutely exposed to Al.
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 1095-8649
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Some physiological parameters were measured in adult rainbow trout during a 10-day exposure to 180 μg Altotal l−1 in acid water (pH 4.7) with or without humic substances (10 mg l−). The fish were acclimatized to pH 5.0 for 7 days prior to the experimental treatments.Chemical analyses revealed that, in the presence of human substances, 74–80% of the A1 was organic bound, while in the absence of humic substances most of the Al(987percnt;) occurred in the inorganic form.Al bound to humic substances (13–150 μg l−1) did not alter the plasma NaCl-concentration, nor the haematocrit value, of rainbow trout during an exposure period of 10 days. This contrasts with the high death rate obtained within 2–3 days when most of the A1 (175 μg l−1) was in the inorganic form. The lethality was accompanied by a 25% decrease in the plasmaconcentration of NaCl and a doubling of the haematocrit value. Bulk analysis revealed that when the metal was present in inorganic forms the total Al content of the gills (75 μg A1 g−1 wet weight) was 15 times higher than when it was present as bound to the humic substances. These experiments showed that the accumulation of A1 at the gills was accompanied by physiological disturbances, both being a function of the chemical speciation of Al.
    Type of Medium: Electronic Resource
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