Elsevier

Toxicology Letters

Volume 184, Issue 1, 10 January 2009, Pages 18-25
Toxicology Letters

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

https://doi.org/10.1016/j.toxlet.2008.10.012Get rights and content

Abstract

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 (50 mg/kg, i.p.) led to the activation of peritoneal macrophages, the increased blood level of IL-1β and TNF-α, 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-α, 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 (50 mg/kg, 100 mg/kg, and 250 mg/kg, i.p.) was measured, the viability of splenocytes was significantly decreased in the higher dose-treated groups (100 mg/kg, 200 mg/kg i.p.). However, cell proliferation without cytotoxicity was shown in group treated with relatively low dose of 50 mg/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.

Introduction

Silicas (SiO2) are the most abundant compounds in the earth's crust except carbon and they can be divided into crystalline or non-crystalline (amorphous) silica. Amorphous silicas are divided into naturally occurring amorphous silicas and synthetic forms. Synthetic amorphous silicas (SAS) are intentionally manufactured and it has been known that SAS do not contain measurable levels of crystalline silica which causes adverse health effects such as silicosis (Arts et al., 2007). Based on this knowledge, SAS are used in various industrial fields and are being used as the materials for nanoparticles. Various nanoparticles made from SAS are also widely used in chemical and biomedical products such as printer toners, varnishes, cancer therapy, DNA delivery, and enzyme immobilization (Barik et al., 2008). With the rapid increase of nanoparticle applications, the concerns on the health impacts caused by amorphous silica nanoparticles are also increasing.

Regarding the toxicity of crystalline silica particles, inhalation of the crystalline form of silica has been a well-known exposure route and historically associated with the development of a severe respiratory disease, silicosis which is lung-pneumoconiosis characterized by alveolar proteinosis and diffused fibrosis (Hamilton et al., 2008, Iyer et al., 1996). Based on the evidence obtained from both animal models and epidemiological studies, the IARC (International Agency for Research on Cancer) has concluded that there are sufficient evidences that inhaled crystalline silica from occupational sources, in the form of quartz, cristobalite or tridymite is carcinogenic to humans (IARC, 1997, Cocco et al., 2007). There are many reports on the pathogenesis of silicosis induced by crystalline silica. Investigators have studied the effects of crystalline silica particles on the induction of cytokines such as IL-1β, IL-6, IL-10, TNF-α and transforming growth factor (TGF); chemokines such as monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2(MIP-2); the reactive oxygen species (ROS), reactive nitrogen species (RNS) and nitric oxide (NO)-generated mainly through iNOS (Rimal et al., 2005, Øvrevik et al., 2006).

However, the toxicities of the amorphous synthetic silica particles, micro-sized particles, and nano-sized particles, have not been widely studied (Cho et al., 2007). Recently, data on the growth inhibition of silica nanoparticles on the green alga Pseudokirchneriella subcapitata were published (Van Hoecke et al., 2008). Silica nanoparticles also showed cytotoxicity in different types of cultured mammalian cell lines (Chang et al., 2007, Jin et al., 2007, Lin et al., 2006). As in vivo studies, the acute and subacute lung toxicities of ultrafine colloidal silica particles were assessed using mice. When cellular and biochemical parameters in bronchoalveolar lavage fluid (BALF) were assessed in the mice intratracheally instilled with ultrafine silica particles, moderate to severe pulmonary inflammation was observed (Kaewamatawong et al., 2006).

It seems that pro-inflammatory responses induced by nanoparticles have been focused as one of the toxic mechanisms. Recently, a few types of nanoparticles such as titanium dioxide and carbon black showed pro-inflammatory effects on epithelial cells in vitro (Monteiller et al., 2007). But, information on the pro-inflammatory responses induced by amorphous silica nanoparticles has not been fully released yet.

In this study, we investigated the oxidative stress and pro-inflammatory responses both in mice and in RAW 264.7 cell line to evaluate the toxicity and possible mechanisms of amorphous silica nanoparticles.

Section snippets

Maintenance of animals, cell culture and nanoparticle treatment

ICR mice were purchased from Orient-Bio Animal Company (Seongnam, Gyeonggi, Korea) and were maintained for adaptation in animal room before the study. The environmental conditions of animal room are maintained as follows; temperature, 23 ± 1 °C; relative humidity, 55 ± 5%; 12 h light/dark cycle. Silica nanoparticles was intraperitoneally treated to the mice with the dosages of 50 mg/kg, 100 mg/kg, and 250 mg/kg for the splenocytes proliferation test. For the test of macrophage activation, NO synthesis,

Activation of peritoneal macrophages in mice

Mice were treated with silica nanoparticles 50 mg/kg through intraperitoneal injection, and were sacrificed at 12 h, 24 h, 48 h, 72 h after treatments, respectively. Macrophages were harvested from the peritoneal cavity of mouse and were incubated in CO2 incubator for 3 h to observe the morphological changes. Activated macrophages, which showed the cytoplasmic spreading, were observed in the mice sacrificed at 12 h after silica nanoparticle treatment. However, cytotoxic effect was also shown in the

Discussion

Toxicological studies are rapidly increasing both in engineered nanomaterials and in naturally occurring particles (Kipen and Laskin, 2005, Kagan et al., 2005, Curtis et al., 2006, Hardman, 2006). As one of the toxic mechanisms of nanoparticles, ROS generation may be the most widely studied. Recently, ROS generation in cultured cells treated with C60 fullerenes, single-walled nanotubes (SWNTs), cerium oxide nanoparticles, and other metal particles have been reported (Hussain et al., 2005, Park

Conflict of interest statement

None.

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