Intracellular accumulation dynamics and fate of zinc ions in alveolar epithelial cells exposed to airborne ZnO nanoparticles at the air–liquid interface

Abstract

Airborne nanoparticles (NPs) that enter the respiratory tract are likely to reach the alveolar region. Accumulating observations support a role for zinc oxide (ZnO) NP dissolution in toxicity, but the majority of in-vitro studies were conducted in cells exposed to NPs in growth media, where large doses of dissolved ions are shed into the exposure solution. To determine the precise intracellular accumulation dynamics and fate of zinc ions (Zn²⁺) shed by airborne NPs in the cellular environment, we exposed alveolar epithelial cells to aerosolized NPs at the air–liquid interface (ALI). Using a fluorescent indicator for Zn²⁺, together with organelle-specific fluorescent proteins, we quantified Zn²⁺ in single cells and organelles over time. We found that at the ALI, intracellular Zn²⁺ values peaked 3 h post exposure and decayed to normal values by 12 h, while in submerged cultures, intracellular Zn²⁺ values continued to increase over time. The lowest toxic NP dose at the ALI generated peak intracellular Zn²⁺ values that were nearly three-folds lower than the peak values generated by the lowest toxic dose of NPs in submerged cultures, and eight-folds lower than the peak values generated by the lowest toxic dose of ZnSO₄ or Zn²⁺. At the ALI, the majority of intracellular Zn²⁺ was found in endosomes and lysosomes as early as 1 h post exposure. In contrast, the majority of intracellular Zn²⁺ following exposures to ZnSO₄ was found in other larger vesicles, with less than 10% in endosomes and lysosomes. Together, our observations indicate that low but critical levels of intracellular Zn²⁺ have to be reached, concentrated specifically in endosomes and lysosomes, for toxicity to occur, and point to the focal dissolution of the NPs in the cellular environment and the accumulation of the ions specifically in endosomes and lysosomes as the processes underlying the potent toxicity of airborne ZnO NPs.

• Airborne nanoparticles
• air–liquid interface
• endosomes
• FluoZin-3
• intracellular Zn²⁺
• lysosomes

Acknowledgements

We thank Dr. Zink at the University of California, Los Angeles for providing the ZnO nanoparticles as part of the National Institute of Environmental Health Science (NIEHS) Nanotechnology Environmental Health and Safety consortium effort.

Declaration of interest

This work was funded by NIEHS grant 1RC2ES018786-01 to GO, and by Pacific Northwest National Laboratory (PNNL) research and development funds. The research was performed using EMSL, a national scientific user facility sponsored by the Department of Energy’s Office of Biological and Environmental Research and located at PNNL.

Supplementary material available online

Supplementary Figures S1–S5