Development of Androgen- and Estrogen-Responsive Bioassays, Members of a Panel of Human Cell Line-Based Highly Selective Steroid-Responsive Bioassays

BioDetection Systems B.V., Badhuisweg 3, 1031 CM Amsterdam, The Netherlands.
Toxicological Sciences (Impact Factor: 3.85). 02/2005; 83(1):136-48. DOI: 10.1093/toxsci/kfi005
Source: PubMed


We have established highly sensitive and specific androgen and estrogen reporter cell lines which we have named AR (androgen receptor) and ERalpha (estrogen receptor alpha) CALUX (Chemically Activated LUciferase eXpression), respectively. Both bioassays are member of a panel of CALUX reporter cell lines derived from the human U2-OS osteosarcoma cell line, all using highly selective reporter constructs based with a basal promoter element linked to multimerized response elements, allowing efficient and specific measurement of compounds interfering with androgen, estrogen, progesterone, and glucocorticoid receptors. The AR CALUX bioassay contains the human androgen receptor and a luciferase reporter construct containing three androgen-responsive elements coupled to a minimal TATA promoter. This cell line was characterized by its stable expression of AR protein, its highly selective response to low levels of different natural and synthetic androgens, and its insignificant response to other nuclear hormone receptor ligands such as estrogens, progestins, and glucocorticoids. The EC50 of dihydrotestosterone (DHT) was found to be 0.13 nM, consistent with the high affinity of this ligand to the human AR. Flutamide, cyproterone acetate, and the environmental contaminants vinclozolin, DDT, methoxychlor, its metabolite HPTE, and penta-BFR showed clear antagonistic activity in the AR CALUX bioassay, competitively inhibiting DHT-mediated transactivation. The established AR CALUX bioassay proved to excel in terms of easy cell line maintenance, high fold induction range (typical 30 times over solvent control), low minimal detection limit (3.6 pM), and high androgen selectivity. Potential applications such as testing the androgenic or estrogenic activity of pure chemicals and pharmaceuticals and complex mixtures (environmental, food, feed, and clinical) are discussed.

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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.
    Water Research 09/2015; 81. DOI:10.1016/j.watres.2015.05.049 · 5.53 Impact Factor
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    • "For the reporter cell lines for the quantification of (anti-)endocrine activities mediated by the estrogen receptor Fig. 1 Graphical representation of the Kanibadam POP and obsolete pesticide dumpsite and its surroundings. Stars indicate locations from which samples were taken that were analyzed both by chemical and by CALUX analyses alpha (ERα CALUX), the progestin receptor (PR CALUX), and the androgen receptor (AR CALUX), the respective receptor , under control of a constitutive promoter, was also introduced into the cell line (Sonneveld et al. 2005). "
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    Environmental Science and Pollution Research 05/2015; 22(19). DOI:10.1007/s11356-015-4739-5 · 2.83 Impact Factor
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    • "Stable receptor-driven reporter cell lines such as those implemented in the present study could help to determine possible modes of action of other classes of progestins and thus support the in vivo studies necessary to establish possible risks to fish heath posed by the presence of synthetic progestins in the aquatic environment. The cell lines described here could also be useful for the routine screening of environmental samples for androgenic and progestogenic activity with greater relevance to possible effects in fish than other assays currently used for such purposes, which rely on human-derived receptors (Sonneveld et al., 2005, 2011; van der Linden et al., 2008 "
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    Aquatic toxicology (Amsterdam, Netherlands) 03/2015; 163:97-101. DOI:10.1016/j.aquatox.2015.03.021 · 3.45 Impact Factor
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