Comparison of in vitro and in vivo screening models for androgenic and estrogenic activities.

Identification of nuclear receptor-mediated endocrine activities is important in a variety of fields, ranging from pharmacological and clinical screening, to food and feed safety, toxicological monitoring, and risk assessment. Traditionally animal studies such as the Hershberger and Allen-Doisy tests are used for the assessment of androgenic and estrogenic potencies, respectively. To allow fast analysis of the activities of new chemicals, food additives, and pharmaceutical compounds, high-throughput screening strategies have been developed. Here, a panel of mainly steroidal compounds, screened in different in vitro assays, was compared with two human U2-OS cell line-based CALUX (Chemically Activated LUciferase eXpression) reporter gene assays for androgens (AR CALUX) and estrogens (ERalpha CALUX). Correlations found between the data of these two CALUX reporter gene assays and data obtained with other in vitro screening assays measuring receptor binding or reporter gene activation (CHO cell line-based) were good (correlation coefficients (r2) between 0.54 and 0.76; p < 0.0001). Good correlations were also found between the in vitro and in vivo data (correlation coefficient r2 = 0.46 for the AR CALUX vs. Hershberger assay and r2 = 0.87 for the ERalpha CALUX vs. Allen-Doisy assay). The variations in the results obtained with the reporter gene assays (CALUX vs. CHO cell line based) were relatively small, showing the robustness of these types of assays. Using hierarchical clustering, bioactivity relationships between compounds but also relationships between various bioassays were determined. The in vitro assays were found to be good predictors of in vivo androgenic or estrogenic activity of a range of compounds, allowing prescreen and/or possible reduction of animal studies.

Development of androgen- and estrogen-responsive bioassays, members of a panel of human cell line-based highly selective steroid-responsive bioassays.

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.