Intra- and Intercellular Silver Nanoparticle Translocation and Transformation in Oyster Gill Filaments: Coupling Nanoscale Secondary Ion Mass Spectrometry and Dual Stable Isotope Tracing Study

The extensive application of silver nanoparticles (AgNPs) requires a full examination of their biological impacts, especially in aquatic systems where AgNPs are likely to end up. Despite numerous toxicity studies from molecular to individual levels, it is still a daunting challenge to achieve in situ subcellular imaging of Ag and to determine the sites of AgNP interaction with organelles or macromolecules simultaneously. Here, by coupling high-resolution nanoscale secondary ion mass spectrometry elemental mapping with scanning electron microscopy ultrastructural characterization, we successfully visualized the subcellular localization and the potential toxicity effects of AgNPs in the oyster gill filaments. The stable isotope tracing method was also adopted to investigate the respective uptake and transport mechanisms of differently labeled (109)AgNPs and Ag-107(+) ions. Ag-109 hotspots were colocalized with endosomes or lysosomes, proving an endocytosis-based entry of AgNPs which passed through the barrier of oyster gill epithelium. These Ag-109 hotspots showed a strong colocalization with S-32(-). For the first time, we provided visualized evidence of AgNP-induced autophagy in the oyster gill cells. We further identified two categories of hemocytes (blood cells) and illustrated their roles in AgNP transport and sequestration. The integration of morphological and functional aspects of Ag subcellular distribution in different target cells suggested that oysters were equipped with a specialized endolysosomal (epithelial cells) or phagolysosomal system (hemocytes) in regulating the cellular process of AgNPs, during which the lysosome was the most involved organelle and sulfur was the most relevant macronutrient element. This study highlighted not only the intracellular but also the intercellular AgNP translocation and transformation, providing important subcellular imaging of silver and reliable methodology regarding bio-nano interactions in natural environments.

Automated summary

This study utilized high-resolution nanoscale secondary ion mass spectrometry and scanning electron microscopy to visualize the subcellular localization and potential toxicity effects of AgNPs in oyster gill filaments. The findings demonstrated an endocytosis-based entry of AgNPs into cells and identified AgNP-induced autophagy in oyster gill cells. Additionally, the study revealed the transport and sequestration roles of different hemocytes in AgNP distribution.