Optimization and prevalidation of the in vitro ERα CALUX method to test estrogenic and antiestrogenic activity of compounds
Introduction
Because of existing scientific data showing interference with estrogen and androgen signalling by chemicals, pre-screening for estrogenicity and androgenicity has been assigned priority in the determination of endocrine activity of compounds [1], [2]. Several types of assays have been proposed, including in vivo screens using rodents, in particular the classical Hershberger assay for determining androgenicity and the uterotrophic assay to determine estrogenicity. These assays are relatively specific in vivo assays for androgens and estrogens only, and it seems possible that they can be replaced to a large extent by suitable in vitro assays.
Insight in the mechanism of action of steroid hormones has provided the tools to develop excellent alternatives to classical in vivo assays in rodents. Steroid hormones exert their action through activation of specific receptors in target cells [3]. Upon ligand binding these receptors become activated, they enter the nucleus and bind to recognition sequences in promoter regions of target genes, called hormone responsive element. The DNA-bound receptor will activate or repress transcription of target genes. This will lead to altered protein synthesis and cellular functioning. When hormone-responsive elements are linked to the gene of an easily measurable protein like luciferase (the so-called “reporter gene”) and introduced into a suitable cell line, a reporter cell line can be generated that allows the precise quantification of in vitro hormonal activity of chemicals. Robust reporter gene assays have been developed with the same cellular background and high sensitivity, selectivity and responsiveness for estrogens and androgens, expressing the human ERα receptor and human androgen receptor, respectively [4], [5], [6], [7].
The novel in vitro methods also have discrete advantages that go beyond increased speed, reduced costs, and animal use, such as the possibility to use human cells and receptors [5]. These may reduce the correlation with the animal model, but may enhance the predictive value of the in vitro model. Therefore, the expected purpose of the in vitro methods is to be at least at the level of inclusion of the first tier of a hierarchical approach in the context of regulatory guidelines, and even for full replacement of the in vivo methods.
In the ReProTect program and with the assistance of the ECVAM Endocrine Disruptor Task force, the PALM [7], MELN [6] and CALUX [5] methods were selected as tests that have the potential to reduce the in vivo testing for androgenic (AR CALUX and PALM) and estrogenic (ERα CALUX and MELN) compounds. To determine the suitability of the tests for this purpose a validation under the auspices of ECVAM is envisaged. However, before this can be undertaken, a successful prevalidation exercise was needed, along the lines proposed by Curren et al. [8], using a modular approach [9]. In this paper we describe this prevalidation phase, including a preliminary assessment on the test method with respect to protocol refinement, robustness, reproducibility and transferability.
Section snippets
Cells and cell culture procedures
The ERα CALUX® cell line (referred to as clone 1), as described by Sonneveld et al. [5] is a human U2-OS line stably co-transfected with an expression construct for the human ERα (pSG5-neo-hERα) and a pGL3 (Promega)-based reporter construct containing 3 EREs in front of a TATA box (pGL3-3xEREtataLuc). Cells were cultured as described by [5]. To select a subclone with higher and more stable luciferase expression stably transfected ERα CALUX cells were seeded in 96-well plates, 1 cell/500 μl in 100
Protocol optimization and test design
The test conditions selected were based on extensive previous experience with U2-OS-based reporter gene assays for estrogenic compounds [5], [13], [14], [15], [16], [17]. The optimal number of cells to be plated was tested, since in our experience this may influence test results (in particular levels of luciferase activity). An initial plating density of 0.9 × 104 cells/well was found to be optimal and was used in subsequent tests (data not shown). A pre-screening method was developed at the lead
Discussion
We have developed efficient and reliable methods to determine the estrogenic and antiestrogenic potency of chemicals, and transferred those to three other laboratories. Initial problems at one of the laboratories seemed related to low and relatively unstable luciferase expression in the original cell clone. This was solved by selecting a stable high luciferase-expressing clone, which performed excellent in three different laboratories.
Relatively low luciferase expression as was found in the
Conflict of interest
None declared.
Acknowledgements
This work was carried out in part with financial support from the Commission of the European Communities, integrated project “Development of a novel approach in hazard and risk assessment of reproductive toxicity by a combination and application of in vitro, tissue and sensor technologies” (ReProTect; LSHB-CT-2004-503257), ChemScreen, (GA 244236), and FND07007 (Linda Gijsbers). Statistical analysis done by Marc Weimer was done as contract work for ECVAM (CCR.IHCP.C432976.XO). We thank Snezana
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