Amity Institute of Biotechnology, Amity University, Gurgaon 122413, India.
Amity Institute of Biotechnology, Amity University, Gurgaon 122413, India.
Sci Total Environ. 2021 Jun 1;771:144514. doi: 10.1016/j.scitotenv.2020.144514. Epub 2021 Jan 23.
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.
每年都有成千上万种化学物质排放到世界的水体中。这些化学物质会导致内分泌紊乱，并对人类和水生生物的健康产生不良影响。全球环境保护机构强调需要开发快速和特定的测试方法，以识别水中的这些内分泌干扰化学物质（EDCs）。检测干扰雄激素信号的化学物质尤为重要，因为在生命的特定阶段雄激素的输入对于男性的正常发育至关重要。基于效应的方法，如报告基因检测，是识别混合物中未知成分的 EDCs 的合适工具。本研究描述了一种稳定的、分泌型碱性蛋白酶（SEAP）报告基因检测系统，用于直观地检测水样中的雄激素/抗雄激素活性。该系统的一个新特点是，除了雄激素反应元件（3×HRE）、雄激素受体（AR）和 SEAP 报告基因的最佳组合外，还包含了共激活因子 GRIP1、CARM1、p300 和 mZac1b。共激活因子的加入使转录倍数变化达到 162 倍，使得在加入测试样品 1 小时内，可以在低得多的雄激素浓度（1 皮摩尔）下进行视觉检测。此外，非雄激素类类固醇，如雌激素、孕酮和地塞米松，除了在非常高的浓度下，不会引起明显的报告基因活性。该报告基因检测系统可以很容易地转化为高通量格式，同时用于多个样本的研究，反映了在哺乳动物细胞内可能发生的变化。该检测方法用于评估来自六个不同水源的雄激素活性。结果表明，靠近工业区的河流和湖泊中的水具有显著的雄激素活性，而含有造纸和纸浆厂废水的水中则观察到最高的雄激素活性。因此，该生物检测为水中雄激素/抗雄激素化合物的效应定向分析提供了一种快速、直观的检测工具。
