Deciphering silver nanoparticle fate in liver up to biliary excretion using HepG2/C3A spheroids in scenarios mimicking different exposure pathways

Silver nanoparticles (AgNP) are heavily used in consumer products and medical devices, including face masks to fight against COVID-19. AgNP effects on mammals have been studied both using cellular and animal models. The former elucidate molecular mechanisms occurring at the subcellular level, while the latter bring insight into the toxicity and accumulation at the organ level. It has not yet been possible to reconcile these two kinds of studies in a translational approach. In the current study, we use 3D HepG2/C3A hepatocyte cultures that partially reproduce liver architecture, leading to the formation of active bile canaliculi and exposure scenarios mimicking parenteral or oral exposure to AgNP. By using a combination of cutting edge imaging techniques, atomic spectroscopy, and cell biology, we reveal the formation of Ag sulphide or Ag-organothiol complexes in the different conditions, the storage of excess Ag into vacuoles, the excretion of Ag(i) ions into bile canaliculi through the ATP7B Cu transporter, and modifications of the mitochondrial network. These findings, made possible by the use of a pseudo-organ as a model, reveal exposure-dependent mechanisms that can inspire future studies on nanomaterial metabolism in mammals and risk-assessment, and thereof regulations. Moreover, they can help bridge the gap between cellular and animal studies, bringing significant advances into the understanding of AgNP fate in mammals.