Abstract

The development and production of engineered 2D nanomaterials are expanding exponentially, increasing the risk of their release into the aquatic environment. A recent study showed 2D MnO₂ nanosheets, under development for energy and biomedical applications, dissolve upon interaction with biological reducing agents, resulting in depletion of intracellular glutathione levels within fish gill cells. However, little is known concerning their toxicity and interactions with subcellular organelles. To address this gap, we examined cellular uptake, cytotoxicity and mitochondrial effects of 2D MnO₂ nanosheets using an in vitro fish gill cell line to represent a target tissue of rainbow trout, a freshwater indicator species. The data demonstrate cellular uptake of MnO₂ nanosheets into lysosomes and potential mechanisms of dissolution within the lysosomal compartment. MnO₂ nanosheets induced severe mitochondrial dysfunction at sub-cytotoxic doses. Quantitative, single cell fluorescent imaging revealed mitochondrial fission and impaired mitochondrial membrane potential following MnO₂ nanosheet exposure. Seahorse analyses for cellular respiration revealed that MnO₂ nanosheets inhibited basal respiration, maximal respiration and the spare respiratory capacity of gill cells, indicating mitochondrial dysfunction and reduced cellular respiratory activity. MnO₂ nanosheet exposure also inhibited ATP production, further supporting the suppression of mitochondrial function and cellular respiration. Together, these observations indicate that 2D MnO₂ nanosheets impair the ability of gill cells to respond to energy demands or prolonged stress. Finally, our data demonstrate significant differences in the toxicity of the 2D MnO₂ nanosheets and their microparticle counterparts. This exemplifies the importance of considering the unique physical characteristics of 2D nanomaterials when conducting safety assessments.

 抽象的

工程二维纳米材料的开发和生产呈指数级增长，增加了其释放到水生环境中的风险。最近的一项研究表明，正在为能源和生物医学应用开发的2D MnO ₂纳米片在与生物还原剂相互作用时溶解，导致鱼鳃细胞内谷胱甘肽水平的消耗。然而，人们对它们的毒性以及与亚细胞细胞器的相互作用知之甚少。为了解决这一差距，我们使用体外鱼鳃细胞系来代表淡水指示物种虹鳟鱼的靶组织，检查了 2D MnO ₂纳米片的细胞摄取、细胞毒性和线粒体效应。数据表明细胞将MnO ₂纳米片摄取到溶酶体中以及在溶酶体室内溶解的潜在机制。 MnO ₂纳米片在亚细胞毒性剂量下会诱导严重的线粒体功能障碍。定量单细胞荧光成像显示，MnO ₂纳米片暴露后线粒体裂变和线粒体膜电位受损。海马细胞呼吸分析表明，MnO ₂纳米片抑制了鳃细胞的基础呼吸、最大呼吸和备用呼吸能力，表明线粒体功能障碍并降低了细胞呼吸活动。 MnO ₂纳米片暴露还抑制了 ATP 的产生，进一步支持了对线粒体功能和细胞呼吸的抑制。总之，这些观察结果表明，2D MnO ₂纳米片损害了鳃细胞响应能量需求或长期应激的能力。 最后，我们的数据证明了 2D MnO ₂纳米片及其微粒对应物的毒性存在显着差异。这说明了在进行安全评估时考虑二维纳米材料独特物理特性的重要性。