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 (ER+, estrogen receptor positive) which were stably transfected with the estrogen responsive gene ERE-betaGlob-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.

Screening for (anti)androgenic properties using a standard operation protocol based on the human stably transfected androgen sensitive PALM cell line. First steps towards validation.

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.