Agrawal Himanshu, Thakur Keshav, Mitra Shreyasi, Mitra Debarghya, Keswani Chetan, Sircar Debabrata, Onteru Suneel, Singh Dheer, Singh Surya P, Tyagi Rakesh K, Roy Partha
Department of Bioscience and Bioengineering, Indian Institute of Technology Roorkee, Roorkee - 247667, Uttarakhand, India.
Special Center for Molecular Medicine, Jawaharlal Nehru University, New Delhi - 110067, Delhi, India.
ACS Omega. 2022 Nov 4;7(45):41531-41547. doi: 10.1021/acsomega.2c05344. eCollection 2022 Nov 15.
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 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.
近期有关牛奶消费及其与激素相关疾病的关联风险的报告，使得有必要对乳制品中是否存在内分泌干扰化学物质（EDCs）进行评估，并确保消费者的安全。鉴于此，我们调查了生牛奶和商业化牛奶样品中可能存在的（抗）雄激素污染物。为此，我们构建并在本研究中使用了一种稳定表达人雄激素受体（AR）和雄激素反应性荧光素酶报告基因的新型HepARE-Luc细胞系。用雄激素和相应的抗雄激素（氟他胺）处理该细胞系，分别刺激或抑制报告荧光素酶的表达。实时聚合酶链反应和免疫染色结果显示，雄激素刺激后，AR出现转录反应并从细胞质转移至细胞核。这些观察结果表明，可以通过基于细胞的异生物质筛选试验——AR反应，来评估包括牛奶在内的食物链中存在的（抗）雄激素配体。因此，该细胞系进一步用于筛选总共40个作为生牛奶或商业牛奶采购的乳脂肪样品的（抗）雄激素活性。一些生牛奶和商业牛奶的乳脂肪样品明显显示出抗雄激素活性。随后，还评估了一些常用环境化学物质的（抗）雄激素活性。通过对实验化合物进行分子对接研究进行初步观察，以评估它们与AR配体结合域的相互作用。此外，使用HepARE-Luc细胞系进行荧光素酶测定，证实了这些化合物的（抗）雄激素活性。所测试的化合物均未显示出雄激素活性，相反，其中一些化合物如双酚A（BPA）和利福霉素显示出抗雄激素活性。总之，我们的结果为评估牛奶样品中存在的（抗）雄激素活性提供了有价值的信息。总体而言，建议可以使用强大的基于细胞的化学激活荧光素酶报告基因检测法（CALUX）来评估牛奶中存在的（抗）雄激素活性，这可能归因于其中存在的不同环境化学物质。
