These bioactive mediators may have harmful effects on hepatocytes. To examine the hypothesis that activated KCs mediate hepatic injury, we established a noncontact coculture model using the supernatants of SiO2 NP-stimulated KCs to culture BRL cells. In addition to the morphological changes (Figure 4), we also observed that the viability of BRL cells was reduced, and leakage of LDH no and AST increased (Figure 3). LDH and AST are relatively stable enzymes in BRL cells, thus their leakage from the intracellular compartment of hepatocytes into the extracellular space indicates that plasma membrane disruption and hepatocyte damage have occurred.32,33 In this study, the elevated levels of these enzymes in the supernatants of BRL cells may be correlated with the increased release of ROS, TNF-��, and NO by activated KCs, as previously mentioned.

Studies have demonstrated that ROS are capable of causing oxidative damage to major cellular structures, in particular the mitochondria and the plasma membrane.34,35 NO has been reported to downregulate cytochrome P450 and to suppress liver protein and DNA synthesis, and these activities may contribute to hepatotoxicity.36 Additionally, TNF-��, a potent inflammatory cytokine produced by activated KCs, can induce multiple mechanisms to initiate apoptosis in hepatocytes that leads to hepatic injury.37 These data suggest that KCs activated by SiO2 NPs mediate hepatotoxicity and that the preliminary mechanism might occur through the release of ROS, TNF-��, and NO. An in vivo analysis was carried out to determine whether SiO2 NPs activate KCs and induce liver injury.

The results showed that the number of sinusoidal KCs increased after administration of SiO2 NPs (Figure 5E and F), which indicated that SiO2 NPs activated the phagocytic activity of sinusoidal cells by increasing the number of KCs to help remove accumulating nanoparticles.38 The observed hyperplasia and activation of KCs could also lead to the increased formation of bioactive mediators contributing to hepatic injury, such as ROS, TNF-��, and NO, which we demonstrated in the in vitro study. Additionally, we observed infiltration of inflammatory cells into the liver and increases in the serum levels of WBC, LYM, MONO, NEU, and TNF-��, which suggests that SiO2 NPs may induce inflammation.

Aderem39 reported that inflammation may result from the internalization of nanoparticles into macrophages and the subsequent activation of these macrophages. Because KCs can phagocytose SiO2 NPs and become activated, we deduced that KCs played a critical role in the inflammation that we observed. There is a link between inflammation and oxidative AV-951 stress where recruited inflammatory cells can generate oxidative stress by activating oxidative stress responsive transcription factors.40 In this study, we found that SiO2 NPs caused oxidative stress in the liver, with a decrease in GSH activity and an elevation in MDA levels.