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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    ABSTRACT: Cell-based bioassays are becoming increasingly popular in water quality assessment. The new generations of reporter-gene assays are very sensitive and effects are often detected in very clean water types such as drinking water and recycled water. For monitoring applications it is therefore imperative to derive trigger values that differentiate between acceptable and unacceptable effect levels. In this proof-of-concept paper, we propose a statistical method to read directly across from chemical guideline values to trigger values without the need to perform in vitro to in vivo extrapolations. The derivation is based on matching effect concentrations with existing chemical guideline values and filtering out appropriate chemicals that are responsive in the given bioassays at concentrations in the range of the guideline values. To account for the mixture effects of many chemicals acting together in a complex water sample, we propose bioanalytical equivalents that integrate the effects of groups of chemicals with the same mode of action that act in a concentration-additive manner. Statistical distribution methods are proposed to derive a specific effect-based trigger bioanalytical equivalent concentration (EBT-BEQ) for each bioassay of environmental interest that targets receptor-mediated toxicity. Even bioassays that are indicative of the same mode of action have slightly different numeric trigger values due to differences in their inherent sensitivity. The algorithm was applied to 18 cell-based bioassays and 11 provisional effect-based trigger bioanalytical equivalents were derived as an illustrative example using the 349 chemical guideline values protective for human health of the Australian Guidelines for Water Recycling. We illustrate the applicability using the example of a diverse set of water samples including recycled water. Most recycled water samples were compliant with the proposed triggers while wastewater effluent would not have been compliant with a few. The approach is readily adaptable to any water type and guideline or regulatory framework and can be expanded from the protection goal of human health to environmental protection targets. While this work constitutes a proof of principle, the applicability remains limited at present due to insufficient experimental bioassay data on individual regulated chemicals and the derived effect-based trigger values are of course only provisional. Once the experimental database is expanded and made more robust, the proposed effect-based trigger values may provide guidance in a regulatory context. Copyright © 2015 Elsevier Ltd. All rights reserved.
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    • "The experiments were performed with the human bone cell line U2-OS and are based on the determination of emission of light by use of a firefly luciferase coupled to responsive elements (REs) as a reporter gene. The measurements were conducted according to the instructions of the manufacturers with a 24-h incubation period (van der Burg et al. 2010 "
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