http://informahealthcare.com/nan ISSN: 1743-5390 (print), 1743-5404 (electronic) Nanotoxicology, Early Online: 1–10 ! 2013 Informa UK Ltd. DOI: 10.3109/17435390.2013.855827 ORIGINAL ARTICLE Cytotoxicity and genotoxicity assessment of silver nanoparticles in mouse Yan Li 1 , Javed A. Bhalli 1 y, Wei Ding 1 , Jian Yan 1 , Mason G. Pearce 1 , Rakhshinda Sadiq 2 , Candice K. Cunningham 3 z, M. Yvonne Jones 3 , William A. Monroe 3 , Paul C. Howard 3 , Tong Zhou 4 , and Tao Chen 1 1 Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR, USA, 2 National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan, 3 Nanotechnology Core Facility, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR, USA, and 4 Center for Veterinary Medicine, U.S. Food and Drug Administration, Rockville, MD, USA Abstract Silver nanoparticles (AgNPs) are among the most commercially used nanomaterials and their toxicity and genotoxicity are controversial. Although many in vitro studies have been conducted to evaluate the genotoxicity of AgNPs, in vivo genotoxicity studies on the nanomaterials are limited. Given the unique physicochemical properties and complex pharmacokinetics behavior of nanoparticles (NPs), in vivo genotoxicity assessment of AgNPs is badly needed. In this study, the clastogenicity and mutagenicity of AgNPs with different sizes and coatings were evaluated using mouse micronucleus (MN) assay, Pig-a assay and Comet assay. Five 7-week-old male B6C3F1 mice per group were treated with 5 nm polyvinylpyrroli- done (PVP)-coated AgNPs at a single dose of 0.5, 1.0, 2.5, 5.0, 10.0 or 20.0 mg/kg body weight (bw) via intravenous injection for both the MN and Pig-a assays; or with 15–100 nm PVP- or 10–80 nm silicon-coated AgNPs at a single or 3-day repeated dose of 25.0 mg/kg bw for the MN assay and Comet assay in mouse liver. Inductively coupled plasma mass spectrometry (ICP-MS) and transmission electron microscopy (TEM) analyses indicated that AgNPs reached the testing tissues (bone marrow for the MN and Pig-a assays and liver for the Comet assay). Although there was a reduction of reticulocytes in the PVP-coated AgNPs-treated animals, indicating cytotoxicity of the AgNPs, none of the treatments resulted in a significant increase of either mutant frequencies in the Pig-a gene or the percent of micronucleated reticulocyte over the concurrent controls. However, both the PVP- and silicon-coated AgNPs induced oxidative DNA damage in mouse liver. These results demonstrate that the AgNPs can reach mouse bone marrow and liver, and generate cytotoxicity to the reticulocytes and oxidative DNA damage to the liver. Keywords Clastogenicity, in vivo comet assay, in vivo micronucleus assay, in vivo pig-a gene mutation assay, mutagenicity, silver nanoparticles History Received 26 April 2013 Revised 13 August 2013 Accepted 22 September 2013 Published online 22 November 2013 Introduction Nanotechnology has developed rapidly and nanomaterials are being produced at an increasing rate. These nanomaterials have been applied in a wide variety of commercial products including aerospace engineering, electronics, environmental remediation and healthcare (Singh et al., 2009b). Among different nanomater- ials, silver nanoparticles (AgNPs) have been most commonly used in both daily life and the medical arena (Woodrow Wilson International Center, 2011). Silver has been used as a broad- spectrum antibacterial, antifungal and antiviral agent. At nano- scale, AgNPs recently have been recognized as more potent antimicrobial than bulk silver (Agarwal et al., 2009; Madhumathi et al., 2009). With the increasing use of AgNPs, issues on their safety and potential risk to human health have been raised (ICTA 2006, 2008). It shows that more scientific research is required to evaluate the potential toxicity and genotoxicity of AgNPs (Kux, 2012). As one of the important components for hazard identification, genotoxicity of AgNPs has been widely assessed in the in vitro level. In our previous studies, 5 nm AgNPs induced mutations in the Tk gene of mouse lymphoma L5178Y cells (Mei et al., 2012) and micronuclei in TK6 cells (Li et al., 2012a). The genotoxicity of AgNPs has also been reported in other cell types, including A549 human lung cancer cell line (Foldbjerg et al., 2011), normal human lung fibroblast (IMR-90) and human glioblastoma (U251) cells (Asharani et al., 2009), testicular cells (Asare et al., 2012) and human normal bronchial epithelial (BEAS-2B) cells (Kim et al., 2011a). Generally, mammalian cell assays are sensitive for detecting the genotoxicity of testing substances with low speci- ficity (Singh et al., 2009a). For the evaluation of nanomaterials, the in vitro assays may not appropriately reflect in vivo toxicity of the nanomaterials due to the complex nature of nanomaterials and the complicated processes of uptake, deposition and distri- bution in the body (Gonzalez et al., 2008; Landsiedel et al., 2009; yPresent Address: Covance Laboratories Inc. 671 S. Meridian Rd. Greenfield, IN 46140, USA zPresent Address: RJ Reynolds, 950 Reynolds Blvd, Winston Salem, NC 27105, USA Correspondence: Tao Chen, Division of Genetic and Molecular Toxicology, US FDA/NCTR, 3900 NCTR Rd, Jefferson, AR 72079, USA. Tel: +1-870-543-7954. Fax: +1-870-543-7494. E-mail: tao. chen@fda.hhs.gov Nanotoxicology Downloaded from informahealthcare.com by FDA on 12/27/13 For personal use only.