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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