纳米氧化铜对酿酒酵母的细胞毒性机制研究

以模式生物酿酒酵母BY4741为研究对象,测定了不同粒径的纳米CuO(CuO-NPs)及常规CuO在SD-Ura培养基中的水合粒径和溶出的可溶性铜含量,研究了CuO-NPs、常规CuO和Cu2+对酿酒酵母的细胞毒性效应及胞内活性氧(Reactive Oxide Species,ROS)的产生.结果发现,CuO-NPs对酵母的细胞毒性远大于常规CuO,而Cu2的细胞毒性最强,不同粒径的CuO-NPs对酵母的细胞毒性无显著性差异.酵母受试不同形态的铜4h后,常规CuO有30.75％～51.25％的可溶性铜溶出,而CuO-NPs溶出的可溶性铜高达77.32％～ 100％,且溶出的可溶性铜受pH的影响.Cu2产生的ROS最多,CuO-NPs产生的ROS的平均荧光强度(Mean fluorescence intensity,MFI)在1265～2329之间,但两者之间无显著性差异,常规CuO产生的ROS的平均荧光强度最低,约1000.上述试验表明,CuO-NPs对酵母的细胞毒性主要是由可溶性铜和ROS引起的,团聚作用和尺寸效应对CuO-NPs的毒性影响不明显.该结果可为纳米金属氧化物的安全性评价提供科学依据.

The cytotoxicity mechanism of CuO nanoparticles to yeast Saccharomyces cerevisiae BY4741

In this work, yeast Saccharomyces cerevisiae BY4741 was used as a model organism. The diameter of hydrated products and soluble Cu concentration of CuO-NPs and bulk CuO of different diameters in SD-Ura medium were measured. The effects of CuO-NPs, bulk CuO and Cu²⁺ on yeast cytotoxicity and the production of reactive oxide species (ROS) were studied. The results indicated that the cytotoxicity of CuO-NPs was much higher than bulk CuO, while Cu²⁺ had the highest cytotoxicity. The cytotoxicity of CuO-NPs of different diameters showed no significant difference. After 4 h exposure, about 30.75%~51.25% of bulk CuO and 77.32%~100% of CuO-NPs were existed in dissolved form in the medium with yeast BY4741, and the amount of soluble Cu is greatly affected by pH. Exposed to bulk CuO, yeast BY4741 intracellular ROS generation showed no dose-dependent effects and the florescence intensity of ROS was about 1000 at all concentrations. The florescence intensity ranged from 1265 to 2329 for CuO-NPs. The highest florescence intensity was founded for Cu²⁺. However, there was no significant difference on ROS generation between Cu²⁺ and CuO-NPs. This work demonstrated that the cytotoxicity of CuO-NPs was mainly caused by soluble Cu and intracellular ROS generation, while the agglomeration and size effects were less important. In general, this research provided scientific support for nanomaterials safety evaluation.