Optimization and prevalidation of the in vitro ERalpha CALUX method to test estrogenic and antiestrogenic activity of compounds. Reprod Toxicol

BioDetection Systems BV, Science Park 406, Amsterdam, The Netherlands.
Reproductive Toxicology (Impact Factor: 3.23). 05/2010; 30(1):73-80. DOI: 10.1016/j.reprotox.2010.04.007
Source: PubMed


Estrogenicity of chemicals has received significant attention and is linked to endocrine-disrupting activities. However, there is a paucity of validated methods to assess estrogenicity in vitro. We have established a robust method to test estrogenic and antiestrogenic activity of compounds in vitro, as an alternative to using animal models such as the uterotrophic assay. To this end we optimized protocols to be used in combination with CALUX reporter gene assays and carried out an in house prevalidation, followed by two rounds of tests to establish transferability. Problems in the initial test with transferability were solved by isolation of a novel cell clone of the ERalpha CALUX line with greatly improved stability and luciferase levels. This cell line proved to be a very suitable and reliable predictor of estrogenicity of chemicals and was able to readily rank a range of chemicals on the basis of their EC50 values.

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    • "Testosterone TT EC 50 9.1 Â 10 À10 3 1 N.D. 11 AR-GeneBLAzer (Huang et al., 2011) ( Huang et al., 2011) Testosterone TT EC 50 1.6 Â 10 À9 6 4 TTEQ 14 ng/L 12 ERa-GeneBLAzer (Huang et al., 2011) ( Huang et al., 2011), this study 17b-Estradiol E2 EC 50 6.5 Â 10 À11 22 6 EEQ 1.8 ng/L 13 ER-CALUX (Sonneveld et al., 2005) ( Escher et al., 2014; Houtman et al., 2009, Houtman et al. 2006; Legler et al., 2002; Leusch et al., 2010, Leusch et al. 2014b; Schenk et al., 2010; Schreurs et al., 2005; Sonneveld et al., 2005, Sonneveld et al. 2006; van der Burg et al., 2010) 17b-Estradiol E2 EC 50 6.4 Â 10 À12 28 7 EEQ 0.2 ng/L 14 E-SCREEN (Soto et al., 1995) ( Behnisch et al., 2001; Escher et al., 2014; K€ orner et al., 2001; Leusch et al., 2010; Soto et al., 1995) 17b-Estradiol E2 EC 50 7.1 Â 10 À12 16 6 EEQ 0.9 ng/L 15 YES (Routledge and Sumpter, 1996) (Escher et al., 2014; Leusch et al., 2010; Rutishauser et al., 2004; Sanseverino et al., 2005; Vinggaard et al., 2000) 17b-Estradiol E2 EC 50 3.2 Â 10 À10 14 5 EEQ 12 ng/L 16 hERa-HeLa-9903 (OECD, 2009) ( Takeyoshi, 2006) 1 7 b-Estradiol E2 EC 50 8.2 Â 10 À12 8 7 EEQ 0.6 ng/L 17 PR-CALUX (Sonneveld et al., 2005) ( Houtman et al., 2009; Leusch et al., 2014b "
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