Novel use of coactivators to enhance sensitivity of SEAP-based reporter assay system for visual monitoring and quantitation of androgens and antiandrogens in water

Abstract

Every year thousands of chemicals get discharged into the waterbodies of the world. These chemicals cause endocrine disruption and induce adverse health effects in human and aquatic life. Global environmental protection agencies emphasise the need to develop rapid and specific tests for identification of these endocrine disruptive chemicals (EDCs) in water. Detection of chemicals that disrupt androgen signaling is especially important because androgen input at specific phases of life is critical for proper male development. Effect-based methods such as reporter assays are suitable tools for identification of EDCs in mixtures of unknown composition. The current study describes a stable, secreted alkaline protease (SEAP)-based reporter assay system, for visual detection of androgenic/antiandrogenic activity present in water samples. A novel feature of this system is the inclusion of coactivators, GRIP1, CARM1, p300 and mZac1b, in addition to an optimal combination of androgen response element (3× HRE), androgen receptor (AR) and the SEAP reporter gene. Incorporation of the coactivators resulted in a transcriptional fold change of 162 folds, enabling visual detection at much lower concentrations of androgen (1 picomolar) within 1 h of addition of test sample. Also, non-androgenic steroids such as estrogen, progesterone and Dexamethasone did not induce significant reporter activity, except at very high concentrations. This reporter assay can be readily converted into a high throughput format for investigation in multiple samples simultaneously, and reflects the changes that can be expected to occur inside a mammalian cell. The androgenic activity in six different water sources was evaluated using this assay. The results reveal significant androgenic activity in rivers and lakes close to Industrial areas, whereas the highest androgenic activity was observed in water containing paper and pulp mill effluents. This bioassay therefore provides a rapid, visual detection tool for effect-directed analysis of androgenic/antiandrogenic compounds in water. IMPACT STATEMENT: The current SEAP-based assay allows visual detection of androgens/antiandrogens in water, at concentrations as low as 1 picomolar, within a 1 h time period, in a high throughput format, providing a very useful technique for field users and regulatory bodies.

Keywords: AR coactivators; And endocrine disruptive chemicals (EDCs); Androgens; Secreted alkaline protease (SEAP) gene.