Expansion microscopy revealed specific impacts of nano zinc oxide on early organ development in fish

Abstract

Nanomaterials exhibit significant advantages in biomedical applications. However, their potential risks to organisms cannot be overlooked, particularly during early development. Nano zinc oxide (nZnO), one of the most widely used metallic nanomaterials, can have toxic effects on the early development of aquatic organisms once released into the aquatic environment. Traditional organ-specific toxicity assessment methods have limitations in exploring the nanoscale differences between materials. The novel expansion microscopy (ExM) technique allows biological samples to expand approximately 4.5-fold in three dimensions, enabling nanoscale imaging of protein and nucleic acid targets in cells and tissues using conventional optical microscopy. This study systematically assesses the toxicological changes and sources of nZnO and Zn²⁺ in the visual, skeletal muscle, and digestive systems in medaka (Oryzias melastigma). Our results indicated that appropriate concentrations of nZnO supported the normal early development in the visual and skeletal muscle systems, while potentially leading to excessive toxicity in the medaka digestive system. Conversely, the concentrations of nZnO suitable for the development of the digestive system may be inadequate for the needs of the visual and skeletal muscle systems. This discrepancy may arise from differences in the solubility and bioaccessibility of nZnO in gastrointestinal fluids. Further RNA sequencing revealed differences in the sensitivity of various organs in medaka to nanomaterial exposure, highlighting the necessity of implementing comprehensive risk assessment strategies in toxicology. Overall, we visualized and quantified the subtle developmental toxicities of nZnO and Zn²⁺ across different fish organs for the first time. The application of the expansion microscopy technique offered a novel perspective for evaluating the toxicity of nanomaterials.