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.
Witters
H
H
Flemish Institute for Technological Research (VITO NV) & CARDAM, Mol, Belgium. hilda.witters@vito.be
Freyberger
A
A
Smits
K
K
Vangenechten
C
C
Lofink
W
W
Weimer
M
M
Bremer
S
S
Ahr
P H-J
PH
Berckmans
P
P
D000826:Animal Testing Alternatives; D001681:Biological Assay / Q000379:methods* / Q000592:standards; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004965:Estrogen Antagonists / Q000737:chemistry / Q000494:pharmacology*; D047628:Estrogen Receptor alpha / Q000819:agonists / Q000037:antagonists & inhibitors* / Q000235:genetics; D047629:Estrogen Receptor beta / Q000819:agonists / Q000037:antagonists & inhibitors* / Q000235:genetics; D006801:Humans; D008156:Luciferases / Q000235:genetics; D011485:Protein Binding; D015203:Reproducibility of Results; D014162:Transfection
D004965:Estrogen Antagonists; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D008156:Luciferases