Kämpfer Angela, La Spina Rita, Gilliland Douglas, Valzacchi Sandro, Asturiol David, Stone Vicki, Kinsner-Ovaskainen Agnieszka
European Commission Joint Research Centre , Directorate for Health, Consumers and Reference Materials, Via E. Fermi 2749, TP 125, 21027 Ispra, Varese, Italy.
Nanosafety Research Group, School of Life Sciences, Heriot-Watt University , Edinburgh EH14 4AS, United Kingdom.
Chem Res Toxicol. 2017 Apr 17;30(4):1030-1037. doi: 10.1021/acs.chemrestox.6b00280. Epub 2017 Mar 24.
Silver (Ag) is the most common nanomaterial (NM) in consumer products. Much research has been focused on elucidating the potential impact of Ag-containing NMs on human health, e.g., cytotoxicity, genotoxicity, or proinflammatory responses. In the case of proinflammatory responses, a frequently used end point is the induction of nitric oxide (NO), which is indirectly quantified as nitrite (NO) with the Griess reaction. After preliminary studies in a macrophage-like cell culture system showed anomalous false negative results in the presence of silver nanoparticles (Ag NPs), we studied the influence of Ag on the detection of NO in a cell-free environment. Solutions containing a known concentration of NaNO were prepared in HO, PBS, or complete cell culture medium (CCM) and analyzed using the Griess reaction in the presence of Ag in its metallic or ionic state. In Milli-Q HO, the impact of salts on the detection was investigated using NaCl and KBr. After completion of the Griess reaction, the samples were analyzed spectrophotometrically or chromatographically. It was found that the presence of metallic but not ionic Ag interfered with the quantification of NO. The effect was more pronounced in PBS and HO containing NaCl or KBr. The chromatographical analysis provided evidence of a competing reaction consuming the intermediate diazonium salt, which is critical to the Griess reaction. These findings demonstrate yet another substantial interference of NMs with a frequently used in vitro assay. If gone unnoticed, this interference might cause false negative results and an impaired hazard assessment of Ag NMs.
银（Ag）是消费品中最常见的纳米材料（NM）。许多研究都集中在阐明含银纳米材料对人类健康的潜在影响，例如细胞毒性、遗传毒性或促炎反应。在促炎反应方面，一个常用的终点是一氧化氮（NO）的诱导，它通过格里斯反应间接定量为亚硝酸盐（NO₂⁻）。在巨噬细胞样细胞培养系统中进行的初步研究表明，在存在银纳米颗粒（Ag NPs）的情况下会出现异常的假阴性结果后，我们研究了银在无细胞环境中对NO检测的影响。在水、磷酸盐缓冲液（PBS）或完全细胞培养基（CCM）中制备含有已知浓度NaNO₂的溶液，并在金属态或离子态银存在的情况下使用格里斯反应进行分析。在超纯水（Milli-Q H₂O）中，使用氯化钠（NaCl）和溴化钾（KBr）研究了盐对检测的影响。格里斯反应完成后，对样品进行分光光度法或色谱分析。发现金属态而非离子态的银会干扰NO的定量。在含有NaCl或KBr的PBS和水中，这种影响更为明显。色谱分析提供了一个竞争性反应消耗对格里斯反应至关重要的中间重氮盐的证据。这些发现表明纳米材料对一种常用的体外检测方法又产生了重大干扰。如果不被注意到，这种干扰可能会导致假阴性结果，并损害对银纳米颗粒的危害评估。
