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 109AgNPs and 107Ag+ ions. 109Ag hotspots were colocalized with endosomes or lysosomes, proving an endocytosis-based entry of AgNPs which passed through the barrier of oyster gill epithelium. These 109Ag hotspots showed a strong colocalization with 32S-. 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.