Genotoxic effects of silver nanoparticles stimulated by oxidative stress in human normal bronchial epithelial (BEAS-2B) cells

Abstract

Many classes of silver nanoparticles (Ag-NPs) have been synthesized and widely applied, but the genotoxicity of Ag-NPs and the factors leading to genotoxicity remain unknown. Therefore, the purpose of this study is to elucidate the genotoxic effects of Ag-NPs in lung and the role of oxidative stress on the genotoxic effects of Ag-NPs. For this, Ag-NPs were completely dispersed in medium by sonication and filtration. The Ag-NPs dispersed in medium were 43–260 nm in size. We observed distinct uptake of Ag-NPs into BEAS-2B cells. The Ag-NPs aggregates were wrapped with an endocytic vesicle within the cytoplasm and nucleus of BEAS-2B cells. In the comet assay and micronucleus (MN) assay for BEAS-2B cells, Ag-NPs stimulated DNA breakage and MN formation in a dose-dependent manner. The genotoxic effect of Ag-NPs was partially blocked by scavengers. In particular, of the scavengers tested, superoxide dismutase most significantly blocked the genotoxic effects in both the cytokinesis-block MN assay and the comet assay. In the modified comet assay, Ag-NPs induced a significant increase in oxidative DNA damage. Furthermore, in the oxidative stress assay, Ag-NPs significantly increased the reactive oxygen radicals. These results suggest that Ag-NPs have genotoxic effects in BEAS-2B cells and that oxidative stress stimulated by Ag-NPs may be an important factor in their genotoxic effects.

Introduction

Nanomaterials are defined by the U.S. National Nanotechnology Initiative as materials that are 1–100 nm in size in at least one dimension. In recent years, nanotechnology has been involved in the creation and manipulation of materials at nanoscale levels to generate products that exhibit novel properties [1]. In particular, nanoparticles have been proposed for the treatment of many diseases that require a constant drug concentration in the blood or specific drug targeting of cells or organs [2], [3]. With the development of nanotechnology, the size effects of particles have gradually been considered to be important. However, although the novel properties of nanomaterials are impressive from a physicochemical viewpoint, there are concerns about their possible adverse effects on biological systems. Nanoparticles may be more toxic than larger particles of the same substance because of their larger surface area and enhanced chemical reactivity [4]. Moreover, some nanoparticles readily travel throughout the body, deposit in target organs, penetrate cell membranes, lodge in mitochondria, and may trigger injurious responses [5], [6], [7].

Silver (Ag) is a naturally occurring element that has been widely used for thousands of years in applications such as jewellery, utensils, monetary currency, dental alloys, photography, and even explosives [8]. In addition, Ag is widely known as a catalyst, oxidizing methanol to formaldehyde and ethylene to ethylene oxide [9]. Strong growth is projected in the silver nanoparticles (Ag-NPs) market in the coming years [10]. This increasing use of Ag-NPs necessitated a health and environmental risk assessment [10], [11]. In particular, because pulmonary exposure to Ag-NPs occurs during handling of Ag-NPs, it was necessary to evaluate the toxic response in the respiratory tract. The results of 90-day inhalation studies with Ag-NPs indicated that lungs and liver are the major target tissues for Ag-NPs accumulation [12], [13]. It was recently reported that exposure of human lung epithelial cells to metal-containing nanoparticles generated reactive oxygen species (ROS), which can lead to oxidative stress and cellular damage [14]. Barillet et al. [15] reported that silicon carbide nanoparticles induced genotoxicity and oxidative stress in A549 lung epithelial cells. In addition, Kim et al. [16] reported that Ag-NPs induced oxidative stress-dependent toxicity in human liver cells. However, there is no report on the genotoxic effects of Ag-NPs or the role of oxidative stress in these effects in normal lung epithelial cells. Therefore, we examined the genotoxic potential of Ag-NPs and the role of oxidative stress in their effects in a human normal bronchial epithelial (BEAS-2B) cell line.

We purchased Ag nanopowder (<100 nm) and completely dispersed them in experimental medium by sonication. After this process, the Ag-NPs solution was filtrated using a membrane filter; the Ag-NPs were then physically characterized. First, prior to their genotoxic evaluation, the uptake of Ag-NPs was characterized along with their effects on cellular adhesion and their surface distribution. Second, in order to evaluate the genotoxic potential of Ag-NPs in human pulmonary exposure, cultured human lung cells (BEAS-2B cells) were used in the comet assay and the in vitro micronucleus (MN) assay. Third, we identified the genetic toxicity related to ROS generation by Ag-NPs. To measure ROS generation, an oxidative stress assay of intracellular oxidation of 2′,7′-dichlorofluorescein-diacetate (DCFH-DA) was performed [1], [17]. In addition, specific bacterial enzymes, formamidopyrimidine DNA glycosylase (FPG) and endonuclease III (Endo III), were used to measure oxidized purine and oxidized pyrimidine in the modified comet assay (Comet assay interest group website; http://cometassay.com/). To determine whether the genotoxic effect of Ag-NPs was stimulated by ROS, BEAS-2B cells were co-treated with both Ag-NPs (10 μg/ml) and scavengers [mannitol (Man), OH radical scavenger; catalase (CAT) and sodium selenite (SS), H₂O₂ radical scavengers; superoxide dismutase (SOD), superoxide radical scavenger] in the comet assay and the cytokinesis-block MN (CBMN) assay.