Zinc oxide nanoparticles (ZnO NPs) potentially undergo physicochemical transformation in the environment, which may lead to unexpected environmental and health risks. The "aging" process is essential for better understanding the toxicity and fate of NPs in the environment. However, the mutagenic effects of aged ZnO NPs are still unexplored. The present study focused on investigating the physicochemical transformation during aging process and clarifying the mutagenicity of naturally aged ZnO NPs in human-hamster hybrid (AL) cells. It was found that ZnO NPs underwent sophisticated physicochemical transformations with aging regardless of original morphology or size, such as the microstructural changes, the formation of hydrozincite (Zn5(CO3)2(OH)6) and the release of free zinc ions. Interestingly, the aged ZnO NPs were investigated to be able to result in much lower cytotoxicity while relatively high degree mutation than fresh ZnO NPs. With characterization of the soluble and insoluble fractions of aged ZnO NPs suspension, together with the control measurements using metal chelator (TPEN) and endocytosis inhibitor (Nystatin), it was revealed that the release of zinc ions and nanoparticle uptake made significantly different contributions to the mutagenicity of fresh and aged ZnO NPs. This study clearly demonstrated that the physicochemical transformation of ZnO NPs with aging plays important and comprehensive roles in the ZnO NPs-induced mutagenicity in mammalian cells.

With rapid development of the nanoindustry, studies focusing on the transformation of nanoparticles (NPs) are required to understand their stability and toxicity after being released into the environment. Here, we characterized the physicochemical properties of ZnO NPs and found that they are naturally alkalized in the presence of air (without the addition of exogenous alkaline substances). Energy dispersive X-ray/X-ray powder diffraction/Fourier transform infrared (EDX/XRD/FTIR)/Raman spectroscopy gave evidence for the formation of hydrozincite (Zn 5 (CO 3 ) 2 (OH) 6 ) and zinc hydroxide (Zn(OH) 2 ). Further, we comparatively evaluated the cellular toxicity of pristine and alkalized ZnO NPs. Cell viability testing (colony formation) showed that alkalization time-dependently decreased cytotoxicity. Alkalized NPs exhibited mutagenicity at multiple concentrations, as shown by a CD59 gene loci mutation assay. Variations in toxicity were associated with the chemical transformation of ZnO NPs, and Zn 2+ played a key role in the mutagenicity of alkalized NPs. These results indicate that NPs are chemically transformed in the environment. These transformations result in obvious variations in toxicity, suggesting that the NP transformation process should be considered more thoroughly when evaluating toxicity.

Compared to fresh zinc oxide NPs, aged zinc oxide NPs induce higher levels of ROS and DNA double strand breaks, as well as more pronounced cell malignant progression in the tyrosine phosphatase SHP2 gain-of-function mutant mouse embryonic fibroblast cells.