Eun-Jung Park

Silver nanoparticles induce cytotoxicity by a Trojan-horse type mechanism

Silver nanoparticles (AgNPs) are widely applied in many household products and medical uses. However, studies on the effects of AgNPs on human health and environmental implications are in the beginning stage. Furthermore, most data on the toxicity of AgNPs have been generated using nanoparticles modified with detergents to prevent agglomeration, which may alter their toxicities. In this study, we studied toxicity using AgNPs prepared by dispersing them in fetal bovine serum (FBS), biocompatible materials. AgNPs (average size; 68.9 nm, concentrations; 0.2, 0.4, 0.8, and 1.6 ppm, exposure time; 24, 48, 72, and 96h) showed cytotoxicity to cultured RAW264.7 cells by increasing sub G1 fraction, which indicates cellular apoptosis. AgNPs decreased intracellular glutathione level, increased NO secretion, increased TNF-alpha in protein and gene levels, and increased gene expression of matrix metalloproteinases (MMP-3, MMP-11, and MMP-19). When cells were treated with AgNPs, they were observed in the cytosol of the activated cells, but were not observed in the dead cells. It seemed that AgNPs were ionized in the cells to cause cytotoxicity by a Trojan-horse type mechanism suggested by previously reported studies.

Oxidative stress and apoptosis induced by titanium dioxide nanoparticles in cultured BEAS-2B cells

As the applications of industrial nanoparticles are being developed, the concerns on the environmental health are increasing. Cytotoxicities of titanium dioxide nanoparticles of different concentrations (5, 10, 20 and 40 microg/ml) were evaluated in this study using a cultured human bronchial epithelial cell line, BEAS-2B. Exposure of the cultured cells to nanoparticles led to cell death, reactive oxygen species (ROS) increase, reduced glutathione (GSH) decrease, and the induction of oxidative stress-related genes such as heme oxygenase-1, thioredoxin reductase, glutathione-S-transferase, catalase, and a hypoxia inducible gene. The ROS increase by titanium dioxide nanoparticles triggered the activation of cytosolic caspase-3 and chromatin condensation, which means that titanium dioxide nanoparticles exert cytotoxicity by an apoptotic process. Furthermore, the expressions of inflammation-related genes such as interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), TNF-a, and C-X-C motif ligand 2 (CXCL2) were also elevated. The induction of IL-8 by titanium dioxide nanoparticles was inhibited by the pre-treatment with SB203580 and PD98059, which means that the IL-8 was induced through p38 mitogen-activated protein kinase (MAPK) pathway and/or extracellular signal (ERK) pathway. Uptake of the nanoparticles into the cultured cells was observed and titanium dioxide nanoparticles seemed to penetrate into the cytoplasm and locate in the peri-region of the nucleus as aggregated particles, which may induce direct interactions between the particles and cellular molecules, to cause adverse biological responses.

Oxidative stress induced by cerium oxide nanoparticles in cultured BEAS-2B cells

Cerium oxide nanoparticles of different sizes (15, 25, 30, 45 nm) were prepared by the supercritical synthesis method, and cytotoxicity was evaluated using cultured human lung epithelial cells (BEAS-2B). Exposure of the cultured cells to nanoparticles (5, 10, 20, 40 microg/ml) led to cell death, ROS increase, GSH decrease, and the inductions of oxidative stress-related genes such as heme oxygenase-1, catalase, glutathione S-transferase, and thioredoxin reductase. The increased ROS by cerium oxide nanoparticles triggered the activation of cytosolic caspase-3 and chromatin condensation, which means that cerium oxide nanoparticles exert cytotoxicity by an apoptotic process. Uptake of the nanoparticles to the cultured cells was also tested. It was observed that cerium oxide nanoparticles penetrated into the cytoplasm and located in the peri-region of the nucleus as aggregated particles, which may induce the direct interaction between nanoparticles and cellular molecules to cause adverse cellular responses.

Oxidative stress and pro-inflammatory responses induced by silica nanoparticles in vivo and in vitro

Oxidative stress and inflammatory responses induced by silica nanoparticles were evaluated both in mice and in RAW264.7 cell line. Single treatment of silica nanoparticles (50mg/kg, i.p.) led to the activation of peritoneal macrophages, the increased blood level of IL-1beta and TNF-alpha, and the increased level of nitric oxide released from the peritoneal macrophages. mRNA expressions of inflammation-related genes such as IL-1, IL-6, TNF-alpha, iNOS, and COX-2 were also elevated in the cultured peritoneal macrophages harvested from the treated mice. When the viability of splenocytes from the mice treated with silica nanoparticles (50mg/kg, 100mg/kg, and 250mg/kg, i.p.) was measured, the viability of splenocytes was significantly decreased in the higher dose-treated groups (100mg/kg, 200mg/kg i.p.). However, cell proliferation without cytotoxicity was shown in group treated with relatively low dose of 50mg/kg i.p. When leukocyte subtypes of mouse spleen were evaluated using flow cytometry analysis, it was found that the distributions of NK cells and T cells were increased to 184.8% and 115.1% of control, respectively, while that of B cells was decreased to 87.7%. To elucidate the pro-inflammatory mechanism of silica nanoparticles in vivo, in vitro study using RAW 264.7 cell line which is derived from mouse peritoneal macrophage was done. Treatment of silica nanoparticles to the cultured RAW264.7 cells led to the reactive oxygen species (ROS) generation with a decreased intracellular GSH. In accordance with ROS generation, silica nanoparticles increased the level of nitric oxide released from the cultured macrophage cell line. These results suggested that silica nanoparticles generate ROS and the generated ROS may trigger the pro-inflammatory responses both in vivo and in vitro.

Repeated-dose toxicity and inflammatory responses in mice by oral administration of silver nanoparticles

Toxicity of nanoparticles depends on many factors including size, shape, chemical composition, surface area, surface charge, and others. In this study, we compared the toxicity of different sized-silver nanoparticles (AgNPs) which are being widely used in consumer products due to its unique antimicrobial activity. When mice were treated with AgNPs 1mg/kg for 14 days by oral administration, small-sized AgNPs (22nm, 42nm, and 71nm) were distributed to the organs including brain, lung, liver, kidney, and testis while large-sized AgNPs (323nm) were not detected in those tissues. The levels of TGF-β in serum were also significantly increased in the treated group of small-sized AgNPs but not in large-sized AgNPs. In addition, B cell distribution was increased in small-sized AgNPs but not in large-sized-AgNPs by the phenotype analysis. However, body weight or in the ratio of organ/body weight were not different between the control group and all the AgNPs-treated groups. The repeated-dose toxicity of AgNPs (42nm) was also investigated in mice by oral administration for 28 days. By the administration of AgNPs (0.25mg/kg, 0.50mg/kg, 1.00mg/kg), adverse impacts on liver and kidney were observed in a high dose-treated group (1.00mg/kg), when determined by blood chemistry and histipathological analysis. Cytokines including IL-1, IL-6, IL-4, IL-10, IL-12, and TGF-β were also increased in a dose-dependent manner by repeated oral administration. In addition, B cell distribution in lymphocyte and IgE production were increased. Based on these results, it is suggested that repeated oral administration of nano-sized AgNPs may cause organ toxicity and inflammatory responses in mice.

Induction of chronic inflammation in mice treated with titanium dioxide nanoparticles by intratracheal instillation

Titanium dioxide nanoparticles (TNP) are nanomaterials which have various applications including photocatalysts, cosmetics, and pharmaceuticals because of their high stability, anticorrosiveness, and photocatalytic properties. Induction of cytokines and potential chronic inflammation were investigated in mice treated with TNP (5 mg/kg, 20 mg/kg, and 50 mg/kg) by a single intratracheal instillation. Pro-inflammatory cytokines such as IL-1, TNF-a, and IL-6 were significantly induced in a dose-dependent manner at day 1 after instillation. The levels of Th1-type cytokines (IL-12 and IFN-gamma) and Th2-type cytokines (IL4, IL-5 and IL-10) were also elevated dose-dependently at day 1 and the inflammatory responses were sustained until the remainder of experimental period for 14 days. By the induction of Th2-type cytokines, the increased B cell distributions both in spleen and in blood, and increased IgE production in BAL fluid and serum were observed. In lung tissue, increase of inflammatory proteins (MIP and MCP) and granuloma formation were observed. Furthermore, the expressions of genes related with antigen presentation (H2-T23, H2-T17, H2-K1, and H2-Eb1) and genes related with the induction of chemotaxis of immune cells (Ccl7, Ccl3, Cxcl1, Ccl4, Ccl2) were markedly increased using microarray analysis. From these data, it could be suggested that TNP possibly cause chronic inflammatory diseases through Th2-mediated pathway in mice.

Pro-inflammatory and potential allergic responses resulting from B cell activation in mice treated with multi-walled carbon nanotubes by intratracheal instillation.

The increased application of engineered carbon nanotubes (CNTs) has also raised the level of public concern regarding possible toxicities caused by exposure to these nanostructures. In this study, pulmonary and systemic immune responses induced by intratracheal instillation of multi-walled carbon nanotubes (MWCNTs) were investigated in mice. Total numbers of immune cells in bronchoalveolar lavage (BAL) fluid were significantly increased in treated groups (5, 20, and 50mg/kg doses of MWCNTs) and the distribution of neutrophils was elevated at day 1 after instillation. Pro-inflammatory cytokines (IL-1, TNF-alpha, IL-6, IL-4, IL-5, IL-10, IL-12, and IFN-gamma) were also increased in a dose-dependent manner, both in BAL fluid and in blood. Most of the cytokines showed the highest levels at day 1 after instillation and then decreased. Th2-type cytokines (IL-4, IL-5, and IL-10) were elevated in the treated group to levels higher than those of the Th1-type cytokines (IL-12 and IFN-gamma). Furthermore, distributions of B cells in spleen and blood were significantly increased at day 1 after instillation, indicating that Th2-type cytokines had activated B cells, causing them to proliferate. Along with the additional numbers of B cells, granuloma formation in the lung tissue and IgE production were also observed, with an intensity dependent on the dose of MWCNTs instilled. Based on these observations, it is suggested that MWCNTs may induce allergic responses in mice through B cell activation and production of IgE.

Inflammatory responses may be induced by a single intratracheal instillation of iron nanoparticles in mice

Magnetite iron nanoparticles have been widely used as contrast agents and in thermal therapy for cancer. However, their adverse effects on human health have not been fully investigated. In this study, iron oxide nanoparticles were prepared using inorganic iron chloride (size: 5.3+/-3.6 nm in phosphate buffered saline, surface charge: 23.14 mV), and their inflammatory responses were investigated. When mice were treated with iron oxide nanoparticles (250 microg/kg, 500 microg/kg, and 1mg/kg) by a single intratracheal instillation, the level of intracellular reduced glutathione (GSH) was decreased in the cells of bronchoalveolar lavage (BAL) fluid. The arrest of cell cycles in G1 phase was observed, but S-phase was significantly decreased. The concentrations of pro-inflammatory cytokines (IL-1, TNF-alpha, and IL-6) were dose-dependently increased at day 1 after instillation in the BAL fluid and in the blood. During the experimental period of 28 days, pro-inflammatory cytokines (IL-1, TNF-alpha, and IL-6), Th0 cytokine (IL-2), Th1 type cytokine (IL-12), Th2 type cytokines (IL-4 and IL-5), TGF-beta, and IgE were also elevated. Expressions of many genes related with inflammation or tissue damage such as heat shock protein, matrix metalloproteinase, tissue inhibitors of metalloproteinases, and serum amyloid A were significantly induced. Formation of microgranuloma, which is one of the indicators for chronic inflammatory response, was observed in the alveolar space. In addition, distribution of B cell and CD8+ T cell in blood lymphocytes was increased at day 28. Based on the result, iron oxide nanoparticles may subchronic induce inflammatory responses via oxidative stress in mice by a single intratracheal instillation.

Bioavailability and toxicokinetics of citrate-coated silver nanoparticles in rats.

Bioavailability, tissue distribution, blood concentration, and excretion of citrate-coated silver nanoparticles (AgNPs; size, 7.9 ± 0.95 nm by TEM diameter) were investigated. Male SD rats were treated by a single oral or intravenous administration of either 1 or 10 mg/kg AgNPs. Silver concentration of blood was determined at 10 min, and at 1, 2, 4, 8, 24, 48, and 96 h after treatment. Silver in the liver, lungs, and kidneys was also measured at 24 and 96 h after treatment. Excretion of silver nanoparticles via feces and urine was determined at 24 h after treatment. After oral administration, most AgNPs were found in feces, and their blood concentration was very low. This suggests that absorption through the gastrointestinal tract was not good. However, a high level of silver in the blood was detected after tail vein injection. When rats were injected with 1 mg/kg AgNPs, the silver concentration of blood was significantly elevated at 10 min after injection; the level subsequently decreased. In the rats treated with 10 mg/kg AgNPs, the elevated level did not decrease, but was maintained during the experimental period. On the basis of the values of AUCoral/AUCiv, the bioavailability of orally administered AgNPs was 1.2% in the group treated with 1 mg/kg AgNPs and 4.2% in the group treated with 10 mg/kg AgNPs. AgNPs accumulated in the liver, lungs, and kidneys; the accumulated AgNPs were released into the blood stream. AgNP levels in the urine were extremely low compared to the levels in the feces. When rats were injected with AgNPs, these particles were also detected in feces at 24 h after treatment, which suggests bile secretion of AgNPs.

Asian Dust Storm and pulmonary function of school children in Seoul

Health effects of the Asian Dust Storm (ADS) have not been evaluated adequately, even though it may affect health of people in East Asia. This study was conducted to evaluate whether the ADS affects particulate air pollution and pulmonary function of children. We studied 110 school children in Seoul, Korea with daily measurement of peak expiratory flow rate (PEFR) from May 13 to June 15, 2007. PM(2.5), PM(10) and metals bound to the particles were also determined daily during the study period in Ala Shan and Beijing (China) as well as in Seoul (Korea). Three-day back trajectories showed that air parcels arrived at Seoul mostly from the desert areas in China and Mongolia through eastern China during ADS event affecting levels of particulate pollutants in the areas. Analysis of metals bound to the particulates showed that natural metal levels were much higher than the anthropogenic metal levels. We found that ambient concentrations of PM(2.5) and PM(10) were not significantly associated with PEFR in school children except asthmatics during the study period (P>0.05). However, most of the metal concentrations bound to the particulates were significantly associated with decrease of the childrens PEFR (P<0.05). The effect of anthropogenic metals was not different from natural components of metals for reduction of PEFR. This result indicates that exposure to the metals bound to particles during the ADS period reduces childrens pulmonary function, but there was no difference of potency for reduction of the pulmonary function between natural and anthropogenic metal components.