Manganese dioxide nanosheets induce mitochondrial toxicity in fish gill epithelial cells

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.