Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA, 52242, USA.
Free Radical and Radiation Biology Program, Department of Radiation Oncology, The University of Iowa, Iowa City, IA, 52242, USA.
Redox Biol. 2020 May;32:101518. doi: 10.1016/j.redox.2020.101518. Epub 2020 Mar 29.
Selenium is a metalloid trace element essential for maintaining the optimal redox environment in cells and tissues. It is structurally incorporated into over 25 selenoproteins and enzymes. The glutathione peroxidase (GPx) family of enzymes has a critical role in human health because of its antioxidant function. The recommended daily allowance (RDA) for selenium intake in humans was established to maximize the activity of GPx in plasma. Suboptimal availability of selenium can limit the expression and activities of GPxs leading to a compromised redox environment. This can cause detrimental oxidative distress that could be prevented by increasing the availability of selenium. In cell culture studies, the medium is typically deficient in selenium; supplementation with selenium can increase selenoenzyme activities. However, the optimal level of supplementation in cell culture media has not been well characterized. We performed dose-response experiments for the activities of GPx1 and GPx4 vs. the level of selenium supplementation in cell culture medium. For this, we advanced an assay to determine the activities of both GPx1 and GPx4 efficiently in a single run. During the optimization process, we found that the observed activities of GPx1 and GPx4 depend greatly on the pH of the assay buffer; the observed activities increase with increasing pH, with pH 8 being optimal. Using the combination assay, we also found that the expression and activities for both GPx1 and GPx4 can be maximized in exponentially growing cells by supplementing cell culture media with ≈ 200 nM seleno-l-methionine, without concerns for toxicity. Optimizing the availability of selenium in cell culture to maximize the expression and activities GPx1 and GPx4 may allow for better translation of information from cell culture work to in vivo settings.
硒是一种必需的类金属微量元素，对于维持细胞和组织中的最佳氧化还原环境至关重要。它结构上被纳入超过 25 种硒蛋白和酶中。谷胱甘肽过氧化物酶 (GPx) 酶家族因其抗氧化功能而对人类健康具有重要作用。人类硒摄入量的推荐日允许量 (RDA) 是为了最大限度地提高血浆中 GPx 的活性而建立的。硒的供应不足会限制 GPxs 的表达和活性，导致氧化还原环境受损。这可能导致有害的氧化应激，通过增加硒的供应可以预防这种情况。在细胞培养研究中，培养基通常缺乏硒；补充硒可以增加硒酶的活性。然而，细胞培养基中补充硒的最佳水平尚未得到很好的描述。我们进行了剂量反应实验，以研究 GPx1 和 GPx4 的活性与细胞培养基中硒补充水平的关系。为此，我们开发了一种测定方法，可以在单次运行中有效地测定 GPx1 和 GPx4 的活性。在优化过程中，我们发现 GPx1 和 GPx4 的观察活性在很大程度上取决于测定缓冲液的 pH 值；观察到的活性随 pH 值的增加而增加，pH 值为 8 时最佳。使用组合测定法，我们还发现通过向细胞培养基中补充约 200 nM 硒代蛋氨酸，可以最大程度地提高指数生长期细胞中 GPx1 和 GPx4 的表达和活性，而不会担心毒性。优化细胞培养中硒的可用性，以最大程度地提高 GPx1 和 GPx4 的表达和活性，可能有助于更好地将细胞培养工作中的信息转化为体内环境。
