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Bioactivity screening studies often face sample amount limitation with respect to the need for reliable, reproducible and quantitative results. Therefore approaches that minimize sample use are needed. Low-volume exposure and chemical dilution procedures were applied in an androgen receptor reporter gene human cell line assay to evaluate environmental contaminants and androgen receptor modulators, which were the agonist 5α-dihydrotestosterone (DHT); and the antagonists flutamide, bisphenol A, 1-hydroxypyrene and triclosan. Cells were exposed in around 1/3 of the medium volume recommended by the protocol (70 μL/well). Further, chemical losses during pipetting steps were minimized by applying a low-volume method for compound dilution in medium (250 μL for triplicate wells) inside microvolume glass inserts. Simultaneously, compounds were evaluated following conventional procedures (200 μL/well, dilution in 24-well plates) for comparison of results. Low-volume exposure tests produced DHT EC 50 (3.4–3.7 × 10 − 10 M) and flutamide IC 50 (2.2–3.3 × 10 − 7 M) values very similar to those from regular assays (3.1–4.2 × 10 − 10 and 2.1–3.3 × 10 − 7 M respectively) and previous studies. Also, results were within assay acceptance criteria, supporting the relevance of the downscaling setup for agonistic and antagonistic tests. The low-volume exposure was also successful in determining IC 50 values for 1-hydroxypyrene (2.1–2.8 × 10 − 6 M), bisphenol A (2.6–3.3 × 10 − 6 M), and triclosan (1.2–1.9 × 10 − 6 M) in agreement with values obtained through high-volume exposure (2.3–2.8, 2.5–3.4 and 1.0–1.3 × 10 − 6 M respectively). Finally, experiments following both low-volume dosing and exposure produced flutamide and triclosan IC 50 values similar to those from regular tests. The low-volume experimental procedures provide a simple and effective solution for studies that need to minimize bioassay sample use while maintaining method reliability. The downscaling methods can be applied for the evaluation of samples, fractions or chemicals which require minimal losses during the steps of pipetting, transference to medium and exposure in bioassays. [ABSTRACT FROM AUTHOR]

Among all the nuclear-receptor mediated endocrine disruptive effects, antiandrogenicity is frequently observed in aquatic environments and may pose a risk to aquatic organisms. Linking these effects to responsible chemicals is challenging and a great share of antiandrogenic activity detected in the environment has not been explained yet. To identify drivers of this effect at a hot spot of antiandrogenicity in the German river Holtemme, we applied effect-directed analysis (EDA) including a parallel fractionation approach, a downscaled luciferase reporter gene cell-based anti-AR-CALUX assay and LC-HRMS/MS nontarget screening. We identified and confirmed the highly potent antiandrogen 4-methyl-7-diethylaminocoumarin (C47) and two derivatives in the active fractions. The relative potency of C47 to the reference compound flutamide was over 5.2, whereas the derivatives were less potent. C47 was detected at a concentration of 13.7 μg/L, equal to 71.4 ±g flutamide equivalents per liter (FEq/L) in the nonconcentrated water extract that was posing an antiandrogenic activity equal to 45.5 (±13.7 SD) FEq/L. Thus, C47 was quantitatively confirmed as the major cause of the measured effect in vitro. Finally, the antiandrogenic activity of C47 and one derivate was confirmed in vivo in spiggin-gfp Medaka. An endocrine disrupting effect of C47 was observed already at the concentration equal to the concentration in the nonconcentrated water extract, underlining the high risk posed by this compound to the aquatic ecosystem. This is of some concern since C47 is used in a number of consumer products indicating environmental as well as human exposure. [ABSTRACT FROM AUTHOR]