Planar chromatography-bioassays for the parallel and sensitive detection of androgenicity, anti-androgenicity and cytotoxicity

Abstract

Anti-androgens entering the aquatic environment, e.g., by effluents from wastewater treatment plants or agricultural settings are contributing to endocrine disruption in wildlife and humans. Due to the simultaneous presence of agonistic compounds, common in vitro bioassays can underestimate the risk posed by androgen antagonists. On the other hand, cytotoxic effects might lead to false positive assessments of anti-androgenic effects in conventional bioassays. In the present study, a combination of normal phase high-performance thin-layer chromatography (NP-HPTLC) with a yeast-based reporter gene assay is established for the detection of anti-androgenicity as a promising tool to reduce interferences of androgenic and anti-androgenic compounds present in the same sample. To avoid a misinterpretation of anti-androgenicity with cytotoxic effects, cell viability was assessed in parallel on the same plate using a resazurin viability assay adapted to HPTLC plates. The method was characterized by establishing dose-response curves for the model compounds flutamide and bisphenol A. Calculated effective doses at 10% (ED10) were 27.9 ± 1.3 ng zone^-1 for flutamide and 20.1 ± 5.1 ng zone^-1 for bisphenol A. Successful distinction between anti-androgenicity and cytotoxicity was exemplarily demonstrated with 4-nitroquinoline 1-oxide. As a proof of concept, the detection and quantification of anti-androgenicity in an extract of a landfill leachate is demonstrated. This study shows that the hyphenation of HPTLC with the yeast anti-androgen screen is a matrix-robust, cost-efficient and fast screening tool for the sensitive and simultaneous detection of anti-androgenic and cytotoxic effects in environmental samples. The method offers a wide range of possible applications in environmental monitoring and contributes to the identification of anti-androgenicity drivers in the course of an effect-directed analysis.

Keywords: Androgenicity; Anti-androgenicity; Cytotoxicity; Effect-directed analysis; High-performance thin-layer chromatography.