Delaval Mathilde, Wohlleben Wendel, Landsiedel Robert, Baeza-Squiban Armelle, Boland Sonja
Unit of Functional and Adaptive Biology (BFA), Lab of Molecular and Cellular Responses to Xenobiotics, UMR CNRS 8251, Univ-Paris-Diderot (Sorbonne Paris Cité), 5 rue Thomas Mann, 75205, Paris Cedex 13, France.
Material Physics GMC/R, BASF SE, 67056, Ludwigshafen am Rhein, Germany.
Arch Toxicol. 2017 Jan;91(1):163-177. doi: 10.1007/s00204-016-1701-3. Epub 2016 Apr 8.
Oxidative stress has increasingly been demonstrated as playing a key role in the biological response induced by nanoparticles (NPs). The acellular cytochrome c oxidation assay has been proposed to determine the intrinsic oxidant-generating capacity of NPs. Yet, there is a need to improve this method to allow a rapid screening to classify NPs in terms of toxicity. We adapted the cytochrome c assay to take into account NP interference, to improve its sensitivity and to develop a high-throughput method. The intrinsic oxidative ability of a panel of NPs (carbon black, MnO, Cu, Ag, BaSO, CeO, TiO and ZnO) was measured with this enhanced test and compared to other acellular redox assays. To assess whether their oxidative potential correlates with cellular responses, we studied the effect of insoluble NPs on the human bronchial epithelial cell line NCI-H292 by measuring the cytotoxicity (WST-1 assay), pro-inflammatory response (IL-8 cytokine production and expression) and antioxidant defense induction (SOD2 and HO-1 expression). The adapted cytochrome c assay had a greatly increased sensitivity allowing the ranking of NPs in terms of their oxidative potential by using the developed high-throughput technique. Besides, a high oxidative potential revealed to be predictive for toxic effects as MnO NPs induced a strong oxidation of cytochrome c and a dose-dependent cytotoxicity, pro-inflammatory response and antioxidant enzyme expression. BaSO, which presented no intrinsic oxidative potential, had no cellular effects. Nevertheless, CeO and TiO NPs demonstrated no acellular oxidant-generating capacity but induced moderate cellular responses. In conclusion, the novel cytochrome c oxidation assay could be used for high-throughput screening of the intrinsic oxidative potential of NPs. However, acellular redox assays may not be sufficient to discriminate among low-toxicity NPs, and additional tests are thus needed.
氧化应激在纳米颗粒（NPs）诱导的生物学反应中所起的关键作用已得到越来越多的证实。已有人提出采用无细胞细胞色素c氧化试验来测定NPs的内在产生活性氧能力。然而，有必要改进该方法，以便能够快速筛选并根据毒性对NPs进行分类。我们对细胞色素c试验进行了改进，以考虑NPs的干扰，提高其灵敏度，并开发出一种高通量方法。用这种改进后的试验测定了一组NPs（炭黑、MnO、Cu、Ag、BaSO、CeO、TiO和ZnO）的内在氧化能力，并与其他无细胞氧化还原试验进行了比较。为了评估它们的氧化潜力是否与细胞反应相关，我们通过测量细胞毒性（WST-1试验）炎症反应（IL-8细胞因子产生和表达）和抗氧化防御诱导（SOD2和HO-1表达），研究了不溶性NPs对人支气管上皮细胞系NCI-H292的影响。改进后的细胞色素c试验灵敏度大大提高，通过使用开发的高通量技术，可以根据氧化潜力对NPs进行排名。此外，高氧化潜力被证明可预测毒性作用，因为MnO NPs诱导细胞色素c强烈氧化以及剂量依赖性细胞毒性、炎症反应和抗氧化酶表达。没有内在氧化潜力的BaSO对细胞没有影响。然而，CeO和TiO NPs虽然没有无细胞产生活性氧的能力，但却诱导了适度的细胞反应。总之，新型细胞色素c氧化试验可用于高通量筛选NPs的内在氧化潜力。然而，无细胞氧化还原试验可能不足以区分低毒性NPs，因此需要进行额外的试验。
