# Cellular Effects of Silver Nanoparticle Suspensions on Lung Epithelial Cells and Macrophages
## 4 Discussion  
In many cases, cellular uptake and intracellular metal ion release are important factors for the cytotoxicity of metal-based nanoparticles [18; 21]. In the present study, basic cellular responses caused by two silver nanoparticle suspensions were similar in both A549 and dTHP-1 cells. The exposure of silver nanoparticle suspensions caused an enhancement of IL-8 expression, intracellular oxidative stress, and MT2A gene expression. In contrast, cell viability was hardly affected by silver nanoparticle suspensions. Cellular responses tended to be stronger by AgNP-A than AgNP-B. When the AgNP suspension was diluted to 10 \(\mathrm{\SIUnitSymbolMicro g}\)/mL in the culture medium, the extracellular Ag\({}^{2+}\) concentration was approximately 5-6 \(\mathrm{\SIUnitSymbolMicro m}\)ol/L. However, the efficacies of 10 \(\mathrm{\SIUnitSymbolMicro m}\)ol/L AgNO\({}_{3}\) solution in inducing IL-8 expression and intracellular oxidative stress were weaker than those of AgNP suspensions. AgNO\({}_{3}\) solution at 10 \(\mathrm{\SIUnitSymbolMicro m}\)ol/L did not affect the gene expression of IL-8 or cellular oxidative stress. AgNO\({}_{3}\) hardly showed any cellular effect at a concentration of 10 \(\mathrm{\SIUnitSymbolMicro m}\)ol/L. These results suggest that extracellular dissolved Ag\({}^{2+}\) is not a major factor in the cellular responses caused by AgNPs. In particular, AgNP-A were more soluble under acidic conditions. Therefore, it is possible that intracellular AgNPs release silver ions at the phagolysosome, which is acidic. The cellular uptake of AgNP-A was observed in both A549 and dTHP-1 cells. In contrast, the cellular uptake of AgNP-B was hardly observed in A549 cells. However, interestingly, the intracellular Ag\({}^{2+}\) concentration increased in both AgNP-A- and AgNP-B-treated A549 cells. Moreover, intracellular Ag\({}^{2+}\) levels were higher in A549 cells than in dTHP-1 cells. The intracellular silver concentration in A549 and dTHP-1 cells was approximately 3.4 and 1.9 \(\mathrm{\SIUnitSymbolMicro m}\)ol/L, respectively. Furthermore, the gene expression of MT2A was remarkably enhanced by AgNP suspension. MT2A expression was also enhanced in AgNP-B-treated A549 cells. Therefore, there is a possibility that intracellular Ag\({}^{2+}\) levels were increased in  
Figure 5: Effects of AgNPs and AgNO\({}_{3}\) on intracellular oxidative stress. (**a**) Intracellular ROS levels. A549 and dTHP-1 cells were treated with AgNP suspensions at 10 \(\mathrm{\SIUnitSymbolMicro g}\)/mL or AgNO\({}_{3}\) at 10 \(\mathrm{\SIUnitSymbolMicro m}\)ol/L. After incubation for 24 h, intracellular ROS levels were measured by the DCFH assay. The value of DCF fluorescence in standardized untreated cells was 1. (**b**) HO-1 gene expression. A549 and dTHP-1 cells were treated with AgNP suspensions at 10 \(\mathrm{\SIUnitSymbolMicro g}\)/mL or AgNO\({}_{3}\) at 10 \(\mathrm{\SIUnitSymbolMicro m}\)ol/L. After incubation for 24 h, HO-1 gene expression was determined by real-time PCR. ** \(p<0.01\), * \(p<0.05\) (vs. untreated cells, ANOVA and Dunnett’s test). AgNP-B-treated A549 cells. However, intracellular AgNP-B were not detected. Although intracellular Ag\({}^{2+}\) may be important for cellular responses caused by silver nanoparticles, the mechanism of intracellular Ag\({}^{2+}\) induction is unclear. The reason for the increase in intracellular Ag\({}^{2+}\) levels in AgNP-B-treated A549 cells is unknown. In any of these cases, both AgNP suspensions elevated intracellular ROS levels and IL-8 expression. These cellular responses have previously been observed in cells exposed to cytotoxic metal-based nanoparticles, such as ZnO and CuO nanoparticles [24]. However, the effect of AgNP exposure on mitochondrial activity was weak. The cytotoxic ZnO and CuO nanoparticles can decrease mitochondrial activity at a concentration of 10 \(\mu\)g/mL, and one of the two types of AgNPs used in this study also reduced mitochondrial activity. AgNP-A decreased mitochondrial activity in both A549 and dTHP-1 cells.