Developing High Throughput Reporter Assay for Detection of Endocrine Disruptive Chemicals

Abstract

Every year thousands of chemicals get discharged into the water bodies 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. newlineThe 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 (3X 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 hour 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. newline