Unraveling pathways of cyto and genotoxicity of AgNP in lung and bone cells: in vitro and in vivo approach

Silver nanoparticles (AgNPs) have been extensively used due to their antimicrobial and anti-inflammatory properties. The unique properties have been exploited in a wide range of fields, such as, medical, scientific, industrial, and consumer products allowing the exposure through various routes including inhalation, ingestion and dermal. Although, is still not clear whether their toxicity is attributed to AgNPs intrinsic toxicity and/or from the released ions and their fate and behavior in the organism. Therefore, this study was sub-divided in two main hypothesis a) Small differences on AgNPs size enough to induce different toxicity profiles in in vitro lung and bone cells. b) The toxicity, distribution and excretion kinetics AgNPs after inhalation are size. Therefore, in the in vitro work, A549 and MG-63 cell lines were exposed to AgNPs with a diameter of 10 nm (AgNP10) and 20 nm (AgNP20) (up to 100μg/mL), as well as ionic silver (AgNO3). The effects on cell viability, proliferation, induced apoptosis, DNA damage and cell cycle dynamics were assessed. Also, the contribution of ionic silver (due to AgNP dissociation) on the toxicity of AgNPs was determined. Results for A549 cell line showed that for concentrations 50μg/mL, AgNP10 induced severe DNA damage (comet class 3-4), cell cycle arrest at G2 and induction of late-apoptosis. Contrarily, AgNP20 induced arrest at S phase and increase in the % sub-G1, that was not recovered after 48h. AgNP20 also induced late-apoptosis/necrosis. MG-63 results, showed that AgNPs10 are more reactive and prone to form large aggregates in α-MEM medium. AgNP20 were found to induce a cell cycle arrest at G0/G1 and apoptosis, while AgNP10 did not induce a cytostatic effect, but rather induced necrosis. Finally, combining both A549 and MG-63 results, we concluded that A549 are more sensitive to AgNPs than MG-63, and that small difference in AgNP size is enough to induce different responses in both cell lines. AgNO3 in short exposure (48h) to is more toxic compared to AgNPs, however, when exposure lasts longer we observed that, the AgNPs turn out being more toxic. A completed characterization of the particles, before the toxicity assessment is crucial to understand AgNPs effects in different cell lines. AgNPs toxicity cannot be attributed to the dissociated Ag+ alone. AgNPs toxicity for A549 it is highly dependent on size and concentration of the AgNPs. For MG-63 cell line the size dependent AgNPs toxicity may be associated in part with the NPs interference with the cell membranes and consequent uptake/adsorption processes. Silver toxicity, distribution and excretion of two different sizes of AgNPs (5 and 50nm) and ionic silver (AgNO3) were assessed in mice. Two experiments were performed: A) acute exposure - endpoint evaluation after 1 or 2 intratracheal instillation (IT) and B) chronic exposure - endpoint evaluation after repeated ITs, once a week for 5 weeks. Mice were allowed to recover for 1, 2, 7, 14, 21 or 28 days after the last instillation (dpi). At the end of both studies, blood samples were collected for hematology, and the organs (brain, lung, liver, heart, spleen and kidney), urine and feces were collected to evaluate the silver concentration by ICP-MS. Lung and liver tissues were collected to GSH analysis. For the acute study only, lung tissues were collected to study the effects on the metabolic profile by NMR and for the chronic study only, data was gathered to build a PBPK model. Overall, for the acute study the effects of the instilled AgNPs were dependent on size and number of instillations. The AgNP5 shared a similar inflammatory effect with AgNO3, while AgNP50, seems to have higher influence on the innate immune system. Regarding the biodistribution results, the highest concentration of silver obtained was in the lungs, followed by blood, spleen, kidneys and liver. AgNP5 showed a faster and higher distribution to all organs, whereas larger particles remained mostly in the blood and only a small fraction was obtained in the organs. AgNP50 showed higher excretion, as evaluated by higher silver levels in the urine and faeces of AgNP50-exposed animals. After 2 IT of AgNPs or AgNO3, the lungs of exposed mice showed increased GSSG levels and a, suggesting an oxidative stress response. GSH was increased in the liver after 1 IT with AgNP50 or AgNO3, which could be related to biliary excretion of silver as Ag-GSH complex. NMR profiling of lung tissues revealed several Ag-induced alterations in metabolites involved in different pathways, such as glycolysis and TCA cycle amino acid metabolism, phospholipid metabolism and antioxidant defense. Notably, most of the metabolic changes observed after 1 IT were either absent or reversed in animals subjected to 2 IT, suggesting adaptation mechanisms to cope with the initial insult and recover homeostasis. Additionally, AgNO3 showed no significant alterations after 2 IT. For the chronic study, haematology results showed that AgNP5 induced higher toxicity to RBC, neutrophils and basophils which indicate an inflammatory reaction, while AgNP50 showed eosinophilia and possible blockage in the monocyte-macrophage differentiation. Overall, after chronic exposures the GSSG:Total GSH ratio was increased in the lung for 14 and 21 dpi, while in the liver there was an increase in the GSSG:Total GSH ratio shortly after 1 and 2 dpi for AgNPs, followed by a decrease at 14 and 21 dpi, for all treatments. The major route for excretion seems to be through the urine, despite a high concentration of AgNP5 was also found in faeces. A Physiologically based pharmacokinetic (PBPK) model from 7 specific tissues proved to be successful for the heart, liver and kidney compartments with the modelled silver concentrations in line with the in vivo data. Overall, this study suggests that even small differences on AgNPs size are enough to induce different outcomes and effects in human cell lines. Additionally, when comparing bigger sizes differences, along with the size, the number of exposures seems to play a role in the AgNPs effects, distribution and elimination from the body.status: publishe