Study On The Toxicity Mechanism Of Silver Nanoparticles Involving Oxidative Stress

With the fast development of nanotechnology, products of nanomaterials have been constantly developed and widely applied. At nanoscale, silver exhibits remarkably unusual physical, chemical and biological properties. Silver nanoparticles (AgNPs) have became one of engineered nanomaterials with the highest degree of commercialization. It has been widely used in the fields of optics, hygiene, health care, medicine and so on. With the increasing development and application of AgNPs products, the body may be in direct contact with AgNPs-related products or expose to the circumstance containing AgNPs, which may induce a potential threat to humans. In vivo studies have shown that AgNPs can enter to the organism by different routes, and distribute to different tissues and organs, which can induce adverse effects on the body. Meanwhile, in vivo studies suggest that the accumulation and target organs of AgNPs may be liver and lung. In vitro studies have shown that AgNPs can induce cytotoxicity, genotoxicity, neurotoxicity and immunotoxicity in different cells. Although there are many studies on the toxicity of AgNPs, the mechanisms of AgNP toxicity remain unclear, and there are many problems need to be solved. For example, what is the relationship between cytotoxicity and oxidative stress induced by AgNPs; whether the toxicity is owing to nanoparticle itself or is related to Ag+ stemmed from the AgNP dissolution.Therefore, human lung cancer cells (A549) and human hepatoma cells (HepG2), which derive from the target organ-lung and liver, were selected as the research objects in this study. The aim of this research was to explore the cytotoxicity of AgNPs on the two cells, and explore the relationship between oxidative stress and cytotoxicity, distinguish the role of cytotoxicity on Ag+ release from AgNPs, and further study the molecular mechanism of apoptosis.1. Characterization of silver nanoparticles and stability study in vitro. The morphology of the PVP-coated AgNPs used in this study was observed through transmission electron microscope (TEM). The results indicated that the silver particles was in spherical shape in good dispersion, with an average size ot 23.44±4.92 nm. The hydrodynamic diameter (HDD) of AgNPs in different dispersion media was detected using dynamic light scattering (DLS) method. The mean hydrodynamic diameter of AgNPs in serum-free culture medium was 61.89 nm, while the mean hydrodynamic diameter of AgNPs in 10% FBS culture medium was 51.58 nm. It showed that AgNPs was aggregated in culture medium, serum was helpful to the dispersion of silver particles. Furthermore, the maximum UV absorption peak and the HDD were unchanged when AgNPs was dispersed in ultrapure water, PBS and saline at 4?, 25? and 37? within 48 h. However, silver particles in DMEM culture medium had a good dispersity in 4?, the obvious agglomeration was detected in 25? and 37?. It showed that dispersity of AgNPs in vitro was related to dispersion medium, time and temperature.2. Study on the cytotoxicity of silver nanoparticles in A549 and HepG2 cells. After 24 or 48 h of treatment, the survival rate of A549 and HepG2 showed time-and dose-dependent decreased. Under the same exposure conditions, A549 cells was more sensitive to AgNPs than HepG2 cells. The morphology of A549 and HepG2 cells exposed to different dosage AgNPs (20,40,80,160 ug/mL) for 24 h or 48 h was observed via inverted microscopy. It was found that the morphology of two cells was obviously changed and the cells became shrinking and round, increasing in floating cells and intracellular vacuoles after exposed to 80,160 ?g/mL AgNPs. Nevertheless, there was no apparent influence of 20,40 ?g/mL AgNPs on the morphology of two cells. Apoptosis was assessed by flow cytometry. It was found that after 24 h of treatment, the apoptosis rate of HepG2 cells were significantly higher than the control group, while the apoptosis rate of A549 cells had not significantly changed. After 48 h of treatment, the apoptosis rate of A549 cells (except for 20 ?g/mL group) and HepG2 cells (160 ?g/mL group) were significantly higher than the control group. Furthermore, compared with the 160 ?g/mL AgNPs under the same exposure time, the apoptosis rate of two cells was efficiently prevented by use of N-Acetyl-L-cysteine (NAC). These studies indicated that AgNPs could induce the proliferation inhibition, morphological changes and apoptosis of two cells, which might be related to the increase of intracellular reactive oxygen species.3. Effect of silver nanoparticles on oxidative stress in A549 and HepG2 cells:The lipid peroxidation detection showed that a significant increase in 80?160 ?g/mL dose groups; the MDA content of HepG2 cells in all dose groups did not increase. The SOD and GSH detection found that SOD activity of A549 cells had not changed, and GSH levels of A549 cells in all dose groups increased after 24 h of treatment. While SOD activity and GSH levels decreased in 40?160 ?g/mL AgNPs groups after 48 h of treatment. After 24 or 48 h of treatment, SOD activity and GSH levels showed increased in HepG2 cells. Furthermore, the expression of stress protein Hsp70 and HO-1 significant increase in two cells after exposed to AgNPs for 48 h. The study showed that the AgNPs could induce more pronounced lipid peroxidation in A549 cells, which might be related to the different level of oxidative stress in the two cells.4. Quantitative analysis of cellular uptake of silver nanoparticles and distribution research:after 24 or 48 h of treatment, cellular uptake of AgNPs was measured by inductively coupled plasma mass spectrometry (ICP-MS). Quantitative analysis revealed that intracellular Ag contents showed a dose dependent increase. Cellular uptake of the Ag contents in 24 h is lower than that in 48 h when exposed to 20 g?/mL AgNPs, while cellular uptake of the Ag contents in 24 h is higher than that in 48 h when exposed to 80 and 160 ?g/mL AgNPs. The amount of Ag+ release from the AgNPs showed a dose dependent increase after 0 h incubation in DMEM culture medium containing 10% fetal bovine serum, and the amount of Ag+ released from 160 ?g/mL AgNPs about 37 ng/mL. However, only in 160 ?g/mL AgNPs could detect trace amounts of Ag+ after 24 h or 48 h of incubation at 37 ?. Separation of AgNPs and Ag+ in biological matrices was performed by the method of ultracentrifugation and ultrafiltration. Intracellular Ag+ contents of A549 cells showed a time and dose dependent increase, but Intracellular Ag+ contents of HepG2 cells showed a dose dependent increase. When using silver nitrate with the same silver content as control, the results showed that the cytotoxicity of silver nitrate was much higher than that of AgNPs. Transmission electron microscopy images indicated that A549 and HepG2 cells internalized AgNPs by endocytosis. The aggregates AgNPs were found in cytoplasm and lysosomes in A549 cells. The aggregates AgNPs were found to distribute throughout the cytoplasm, inside lysosomes and nucleus in HepG2 cells. The study showed that AgNPs could be taken up by endocytosis, and AgNPs would be oxidized and release Ag+ in cells, which could induce cytotoxicity.5. Study on the mechanism of cell apoptosis induced by silver nanoparticles: Apoptosis-related protein expression in two cells were examined via western blotting method. The results showed that the levels of Cytochrome C, AIF, Caspase3, Caspase9, Caspase8 protein expression were significantly increased in two cells, and the ratio of Bcl-2/Bax significantly decreased after 48 h of treatment. The study indicated that AgNPs-mediated apoptosis were related to mitochondrial pathway and tumor necrosis receptor pathway in A549 and HepG2 cells.In conclusion, AgNPs exhibited cytotoxicity to A549 and HepG2 cells. A549 cells was more sensitive to AgNPs than HepG2 cells after two cells were exposed to 40-160 ?g/mL AgNPs for 48 h. AgNPs could be taken up by endocytosis, and AgNPs were ionized in the cells to cause cytotoxicity. AgNPs-mediated apoptosis was related to mitochondrial pathway and tumor necrosis receptor pathway in two cells. The reason for different toxicity of the two cells were that Ag+ release from intracellular silver particles in A549 cells was higher than HepG2 cells, and A549 cells undergone more stronger oxidative stress than HepG2 cells. This study explored the possible molecular mechanisms of cytotoxicity and the potential reasons of different toxicity induced by AgNPs in A549 and HepG2 cells. The available toxicological data could be used for the safety evaluation and applications of AgNPs.