Evaluacija Hersbergova biotesta na glodavcima s pomoću tri referentna (anti) androgena

Under the umbrella of the Organization for Economic Cooperation and Development (OECD) the rodent Hershberger bioassay is being validated as an in vivo screen for the detection of (anti)androgens. As part of the validation work we studied trenbolone (TREN), 1,1-bis-(4-chlorophenyl)-2,2-dichloroethylene (p,p\u27-DDE) and vinclozolin (VIN). Oral intubation of castrated rats with TREN [0.3-1.5-8-40 mg kg-1 body weight (b.w.)] for ten days increased androgen-sensitive tissue weights (ASTW) at the high dose. p,p\u27-DDE (5-16-50-160 mg kg-1 b.w.) and VIN (0-3-10-30-100 mg kg-1 b.w.) orally administered for ten days produced a dose-dependent decrease in ASTW in castrated rats subcutaneously supplemented with testosterone propionate (0.4 mg kg-1 b.w.). p,p\u27-DDE also strongly increased liver weights and induced hepatocellular hypertrophy and thyroid follicular cell hypertrophy that was most likely mediated by liver enzyme induction. Our data strongly suggest that the OECD protocol of the rodent Hershberger bioassay describes a sensitive in vivo screen, capable of detecting weakly active (anti)androgens. Furthermore, our data may also indicate that thyroid effects could be assessed, if the protocol is amended accordingly.Hershbergerov biotest na glodavcima potvrđen je u sklopu OECD-ove međ unarodne studije kao in vivo test probira za otkrivanje tvari s afinitetom za androgene receptore. Našem laboratoriju povjereno je provođenje istraživanja s anaboličkim agensom trenbolonom (TREN) te s 1,1-bis-(4-klorfenil)-2,2-dikloretilenom (p,p\u27-DDE) i vinklozolinom (VIN). Samo visoka doza TREN primijenjena na kastriranim štakorima oralnom intubacijom [0,3 - 1,5 - 8 - 40 mg kg-1 tjelesne mase (t. m.)] tijekom deset dana, jako povećava masu tkiva osjetljivih na androgen. Oralno primijenjeni p,p\u27-DDE (5 - 16 - 50 - 160 mg kg-1 t. m.) i VIN (0 - 3 - 10 - 30 - 100 mg kg-1 t. m.) tijekom deset dana u štakora koji su dodatno primali testosteron propionat (0,4 mg kg-1 t. m., sc.), izazvali su ovisno o dozi smanjenje težine svih tkiva osjetljivih na androgen. Uz to je p,p\u27-DDE, takođ er ovisno o dozi, izazvao izrazito povećanje težine jetre i hipertrofiju stanica jetre i folikularnih stanica štitnjače, što je najvjerojatnije posljedica indukcije enzima jetre. Podaci u radu pokazuju da je opisani protokol osjetljiv za probir in vivo te sposoban za otkrivanje slabih (anti)androgena. Nadalje, podaci također upućuju na to da bi se mogli procjenjivati i učinci na štitnjaču ako se ovaj protokol prikladno unaprijedi

Report from the BfR expert hearing on practicability of hormonal measurements: recommendations for experimental design of toxicological studies with integrated hormonal end points

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Assessment of a robust model protocol with accelerated throughput for a human recombinant full length estrogen receptor-α binding assay: Protocol optimization and intralaboratory assay performance as initial steps towards validation

Despite about two decades of research in the field of endocrine active compounds, still no validated human recombinant (hr) estrogen receptor- (ERα) binding assay is available, although hr-ERα is available from several sources. In a joint effort, US EPA and Bayer Schering Pharma with funding from the EU-sponsored 6th framework project, ReProTect, developed a model protocol for such a binding assay. Important features of this assay are the use of a full length hr-ERα and performance in a 96-well plate format. A full length hr-ERα was chosen, as it was considered to provide the most accurate and human-relevant results, whereas truncated receptors could perform differently. Besides three reference compounds [17- estradiol, norethynodrel, dibutylphthalate] nine test compounds with different affinities for the ERα [diethylstilbestrol (DES), ethynylestradiol, meso-hexestrol, equol, genistein, o,p-DDT, nonylphenol, nbutylparaben, and corticosterone] were used to explore the performance of the assay. Three independent experiments per compound were performed on different days, and dilutions of test compounds from deep-frozen stocks, solutions of radiolabeled ligand and receptor preparation were freshly prepared for each experiment. The ERα binding properties of reference and test compounds were well detected. As expected dibutylphthalate and corticosterone were non-binders in this assay. In terms of the relative ranking of binding affinities, there was good agreement with published data obtained from experiments using a human recombinant ERα ligand binding domain. Irrespective of the chemical nature of the compound, individual IC50-values for a given compound varied by not more than a factor of 2.5. Our data demonstrate that the assay was robust and reliably ranked compounds with strong, weak, and no affinity for the ER[1] with high accuracy. It avoids the manipulation and use of animals, i.e., the preparation of uterine cytosol as receptor source from ovariectomized rats, as a recombinant protein is used and thus contributes to the 3R concept (reduce, replace, and refine). Furthermore, in contrast to other assays, this assay could be adjusted to an intermediate/high throughput format. On the whole, this assay is a promising candidate for further validation

The assessment of estrogenic or anti-estrogenic activity of chemicals by the human stably transfected estrogen sensitive MELN cell line: Results of test performance and transferability

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 which were stably transfected with the estrogen responsive gene ERE-ßGlob-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-¿ 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.JRC.I.2-Validation of Alternative Method

Long-term simulation of lead concentrations in agricultural soils in relation to human adverse health effects

Lead (Pb) exposure of consumers and the environment has been reduced over the past decades. Despite all measures taken, immission of Pb onto agricultural soils still occurs, with fertilizer application, lead shot from hunting activities, and Pb from air deposition representing major sources. Little is known about the intermediate and long-term consequences of these emissions. To gain more insight, we established a mathematical model that considers input from fertilizer, ammunition, deposition from air, uptake of Pb by crops, and wash-out to simulate the resulting Pb concentrations in soil over extended periods. In a further step, human oral exposure by crop-based food was simulated and blood concentrations were derived to estimate the margin of exposure to Pb-induced toxic effects. Simulating current farming scenarios, a new equilibrium concentration of Pb in soil would be established after several centuries. Developmental neurotoxicity represents the most critical toxicological effect of Pb for humans. According to our model, a Pb concentration of ~ 5 mg/kg in agricultural soil leads to an intake of approximately 10 µg Pb per person per day by the consumption of agricultural products, the dose corresponding to the tolerable daily intake (TDI). Therefore, 5 mg Pb/kg represents a critical concentration in soil that should not be exceeded. Starting with a soil concentration of 0.1 mg/kg, the current control level for crop fields, our simulation predicts periods of ~ 50 and ~ 175 years for two Pb immission scenarios for mass of Pb per area and year [scenario 1: ~ 400 g Pb/(ha × a); scenario 2: ~ 175 g Pb/(ha × a)], until the critical concentration of ~ 5 mg/kg Pb in soil would be reached. The two scenarios, which differ in their Pb input via fertilizer, represent relatively high but not unrealistic Pb immissions. From these scenarios, we calculated that the annual deposition of Pb onto soil should remain below ~ 100 g/(ha × a) in order not to exceed the critical soil level of 5 mg/kg. We propose as efficient measures to reduce Pb input into agricultural soil to lower the Pb content of compost and to use alternatives to Pb ammunition for hunting.publishe