Evaluation of (Anti)androgenic Activities of Environmental Xenobiotics in Milk Using a Human Liver Cell Line and Androgen Receptor-Based Promoter-Reporter Assay

Abstract

The recent reports on milk consumption and its associated risk with hormone related disorders necessitates the evaluation of dairy products for the presence of endocrine disrupting chemicals (EDCs) and ensure the safety of consumers. In view of this, we investigated the possible presence of (anti)androgenic contaminants in raw and commercialized milk samples. For this purpose, a novel HepARE-Luc cell line that stably expresses human androgen receptor (AR) and the androgen responsive luciferase reporter gene was generated and used in the present study. Treatment of this cell line with androgens and corresponding antiandrogen (flutamide) stimulated or inhibited expression of reporter luciferase, respectively. Real time polymerase chain reaction and immunostaining results exhibited transcription response and translocation of AR from the cytoplasm to the nucleus in response to androgen. Observations implied that a cell-based xenobiotic screening assay via AR response can be conducted for assessing the (anti)androgenic ligands present in food chain including milk. Therefore, the cell line was further used to screen the (anti)androgenic activity of a total of 40 milk fat samples procured as raw or commercial milk. Some of the raw and commercial milk fat samples distinctly showed antiandrogenic activities. Subsequently, some commonly used environmental chemicals were also evaluated for their (anti)androgenic activities. Initial observations with molecular docking studies of experimental compounds were performed to assess their interaction with AR ligand binding domain. Furthermore, (anti)androgenic activities of these compounds were confirmed by performing luciferase assay using the HepARE-Luc cell line. None of the test compounds showed androgenic activities rather some of them like Bisphenol A (BPA) and rifamycin showed antiandrogenic activities. In conclusion, our results provide a valuable information about the assessment of (anti)androgenic activities present in milk samples. Overall, it is proposed that a robust cell-based CALUX assay can be used to assess the (anti)androgenic activities present in milk which can be attributed to different environmental chemicals present therein.