http://informahealthcare.com/nan ISSN: 1743-5390 (print), 1743-5404 (electronic) Nanotoxicology, 2014; 8(S1): 36–45 ! 2014 Informa UK Ltd. DOI: 10.3109/17435390.2013.855827

ORIGINAL ARTICLE

Cytotoxicity and genotoxicity assessment of silver nanoparticles in mouse Yan Li1, Javed A. Bhalli1y, Wei Ding1, Jian Yan1, Mason G. Pearce1, Rakhshinda Sadiq2, Candice K. Cunningham3z, M. Yvonne Jones3, William A. Monroe3, Paul C. Howard3, Tong Zhou4, and Tao Chen1 1

Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR, USA, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan, 3Nanotechnology Core Facility, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR, USA, and 4Center for Veterinary Medicine, U.S. Food and Drug Administration, Rockville, MD, USA

Nanotoxicology Downloaded from informahealthcare.com by Thammasat University on 10/08/14 For personal use only.

2

Abstract

Keywords

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 polyvinylpyrrolidone (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.

Clastogenicity, in vivo comet assay, in vivo micronucleus assay, in vivo pig-a gene mutation assay, mutagenicity, silver nanoparticles

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 nanomaterials, 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 broadspectrum antibacterial, antifungal and antiviral agent. At nanoscale, AgNPs recently have been recognized as more potent antimicrobial than bulk silver (Agarwal et al., 2009; Madhumathi

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. [email protected]

History Received 26 April 2013 Revised 13 August 2013 Accepted 22 September 2013 Published online 22 November 2013

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 specificity (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 distribution in the body (Gonzalez et al., 2008; Landsiedel et al., 2009;

Nanotoxicology Downloaded from informahealthcare.com by Thammasat University on 10/08/14 For personal use only.

DOI: 10.3109/17435390.2013.855827

Oesch & Landsiedel, 2012). The in vitro data alone may not be sufficient for hazard identification of nanomaterials. Hence, it is recommended to perform genotoxicity evaluations of nanomaterials in vivo and apply in vivo results with in vitro results (Gonzalez et al., 2008; Landsiedel et al., 2009). However, in vivo genotoxicity studies on AgNPs are currently limited. Studies for evaluation of in vivo genotoxicity of AgNPs are required to fill this regulatory gap. Currently, several in vivo genotoxicity tests are being used for evaluation of mutagenicity, clastogenicity and aneugenicity of a test agent. Rodent micronucleus (MN) assay and Pig-a assay measure genotoxicity in the surrogate tissue bone marrow while the in vivo Comet assay can test genotoxicity in any tissue. The MN test in rodents has been widely used and acts as a standard in vivo assay for the detection of chromosome damage caused by clastogenicity or aneugenicity of agents (OECD, 1997). Pig-a assay has emerged in the last few years as a new in vivo gene mutation assay, detecting mutations in the phosphatidylinositol glycan, Class A gene (Dertinger et al., 2011a). International evaluation of the Pig-a gene mutation assay has showed its promising potential for measuring the in vivo gene mutation and reproducibility and transferability of the assay (Dertinger et al., 2011b). The in vivo Comet assay (single cell gel electrophoresis) has been widely used in genotoxicity testing especially because of its applicability to various tissues and/or special cell types (Burlinson, 2012). To obtain a broad scope of information on the in vivo genotoxicity of AgNPs, the in vivo MN assay, in vivo Pig-a assay and in vivo Comet assay were conducted in mice treated with different sizes and coatings of AgNPs in this study.

Materials and methods Characterization of AgNPs AgNPs were purchased from NanoComposix, Inc. (San Diego, CA, USA). The materials had characterized by the manufacturer. Size of AgNPs was determined using JEOL 1010 transmission electron microscopy (TEM) from JEOL Ltd. (Tokyo, Japan). The spectral property of AgNPs was analyzed using an Agilent 8453 UV–visible spectrometer (Agilent Technologies, Inc., Santa Clara CA, USA). Hydrodynamic diameter and zeta potential of AgNPs were measured in water using a Zetasizer Nano ZS (Malvern, Worcestershire, UK). All of the above characterization were also confirmed in the Nanotechnology Core Facility at NCTR as previously described (Li et al., 2012a) with JEM-2100 (JEOL Ltd., Tokyo, Japan), Lambda 45 UV/Vis spectroscopy (PerkinElmer, Waltham, MA, USA), and Zetasizer Nano ZS (Malvern, Worcestershire, UK) before the animal treatment. Suspension of the NPs was obtained with vigorous mixing and sonication for 1 min. Animal treatment All experimental procedures involving animals were approved by the NCTR Animal Care and Use Committee. Male B6C3F1 mice from the NCTR breeding colony were used in this study. The mice were housed 2–3 per cage in conventional animal rooms and were identified by ear clipping. Water and food were available ad libitum throughout the acclimation and experimental period. Each experimental group consisted of five 7-week-old male mice (weighing 25–30 g). Two sets of experiments were performed according to size, coating of the AgNPs and the dose selection was based on the highest available stock solution and i.v. injection volume that a mouse could accept, as well as the results from our previous in vitro studies. For set one, the animals were treated intravenously (i.v.) once with 5 nm PVP-coated AgNPs at doses of 0.5, 1.0, 2.5, 5.0, 10.0 or 20.0 mg/kg to evaluate the

In vivo genotoxicity of of AgNPs

37

possible dose-response effect. For set two, 15–100 nm PVP- or 10–80 nm silicon-coated AgNPs were i.v. delivered to mice with a single 25 mg/kg dose or 25 mg/kg/day for 3 consecutive days to evaluate the possible effect of size and coating. Additional groups of mice, serving as the negative and positive controls, were i.v. administered once with water or i.p. administered with 140 mg/kg N-ethyl-N-nitrosourea (ENU), respectively. Blood and bone marrow sampling for in vivo micronucleus assay and Pig-a assay Approximately 30 ml blood samples were collected through submandibular bleeding on Day 2, and on Weeks 2, 4 and 6 for the Pig-a assay in experiment set one. Immediately upon collection, the blood was mixed with 100 ml of heparin solution. Blood sampled at 48 h after the last treatment was used to measure in vivo micronucleus induction. When the Pig-a assay was performed, an additional 30 ml of blood was collected from one of the vehicle control animals and used to prepare the instrument calibration standard (ICS). In experiment set two, the animals were sacrificed at 48 h after the last treatment and the blood samples were collected by submandibular bleeding and cardiac puncture for the MN assay and inductively coupled plasma-mass spectrometry (ICP-MS) and transmission electron microscopy (TEM) analyses, respectively. The bone marrow was also collected for ICP-MS and TEM analyses by flushing the femur with 1 ml of PBS. Determination of silver concentration in the bone marrow, blood and liver ICP-MS was used to determine the amount of Ag element in the bone marrow, blood and liver Bone marrow was elutriated as described above, and the amount of the biological samples were quantified based on the protein content. An aliquot of the bone marrow suspension was treated with RIPA lysis buffer system (ChemCruz, Santa Cruz, CA, USA), and the protein was quantified using a Coomassie Plus Bradford Assay kit (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. The total Ag level in the bone marrow and blood samples was quantified using the following procedure: 300 ml of bone marrow or total 3 ml of blood sample were added to a vessel containing 3 ml HNO3 and 1 ml HCl for microwave assisted acid digestion using a CEM MARSXpress system (CEM, Matthews, NC). Following digestion, samples were diluted to 10 ml with 18 M water. Samples and standards were then prepared for analysis as follows: 1 ml of sample or standard was added to 3.9 ml 18 M water then spiked with 100 ml of internal standard (100 mg/l indium). Appropriate reagent and matrix blanks were treated similarly. Liver samples were cut into three sections weighing 5300 mg, individually added to a vessel containing 3 ml HNO3 and 1 ml HCl and processed by microwave digestion. Vessels were capped and vortexed prior to digestion in a CEM MARSXpress system. Following digestion, samples were diluted to 5 ml with 18 M

water. The second and third sets were diluted to 10 ml with 18 M water. Samples and standards were then prepared for analysis as follows: 1 ml of sample or standard was added to 3.8 ml 18 M water then spiked with 200 ml of internal standard. All the bone marrow, blood and liver samples and standards were analyzed for silver isotope, Ag-107, on an Agilent Technologies 7700x ICP-MS. The method limit of detection (LOD) was determined to be 0.0191 mg/l. Sample concentrations below this LOD are reported as 0 mg/l for blood, 0 mg/mg protein for bone marrow and 0 mg/mg mass weight for liver.

38

Li et al.

Nanotoxicology Downloaded from informahealthcare.com by Thammasat University on 10/08/14 For personal use only.

TEM analysis was used to determine the presence of AgNPs in the bone marrow and liver Bone marrow was collected and spun down to a 1 mm pellet. Bone marrow pellets and five small pieces of liver were subsequently treated in 4% EM grade glutaraldehyde in Sorenson’s phosphate buffer, pH 7.4 at 4  C overnight. Once fixed and washed one time for 10 min in buffer, the bone marrow pellets and liver pieces were then post-fixed and stained in 1% Osmium tetroxide for 1 h on a rotor. After washing the samples once more in buffer, they were dehydrated in a graded series of ethanol washes consisting of 10 min each of 50, 70, 85 and 95% ethanol. This was followed by two 15-min washes in a 100% ethanol and two 15-min washes in propylene oxide. Plastic infiltration of the bone marrow pellets and liver pieces were then performed by exposing the dehydrated bone marrow pellets and liver pieces for 1 h each: first to a 1:1 dilution of propylene oxide to Epon/Araldite resin and then to a 1:3 dilution of propylene oxide to Epon/Araldite resin. This was followed by a 2-h to overnight exposure to 100% Epon/Araldite at room temperature under vacuum. Polymerization of the plastic was then performed by transferring the bone marrow pellets and liver pieces into a polymerization oven heated to 65  C and polymerizing overnight. Once embedded and polymerized, the bone marrow pellets and liver pieces were mounted and trimmed in a Leica UC7 ultramicrotome and sectioned for electron microscopy. The sections were then placed on 300 mesh copper/carbon coated EM grids. The prepared grids were placed into a JEOL JEM-2100 electron microscope operating at 80 keV and imaged with a Gatan US4000 4 k  4 k CCD camera. Silver Nanoparticles were identified by using EDAX X-Ray Analyzer. In vivo micronucleus assay Flow cytometry scoring of micronuclei in erythrocytes was used in our study since it has been well developed and proven to be a more efficient and reproducible method compared to the traditional microscopy-based assay (Dertinger et al., 2011c). The frequency of micronuclei in reticulocytes (% MN-RETs) in blood was determined with a FACS Canto II flow cytometer (BD Biosciences, San Jose, CA, USA), following the protocol from In Vivo Mouse MicroFlowÕ kit (Litron Laboratories, Rochester, NY, USA). Kit-supplied malaria-infected erythrocytes served as a biological standard to guide instrument settings on each day of analysis (Dertinger et al., 2000; Tometsko et al., 1993). The % MN-RETs frequency was determined by the acquisition of approximately 2  104 CD71þ RETs for each animal. In vivo Pig-a assay The Pig-a assay was conducted on the same day when the blood was collected, as previously described (Bhalli et al., 2011). Briefly, the entire 130 ml of the blood/anticoagulant mixture were layered on top of 3 ml of LympholyteÕ -M (Cedarlane Laboratories, Burlington, NC, USA) in a 15 ml conical polypropylene tube. To enrich for erythrocytes, the tubes were centrifuged at 800 g for 20 min. The resulting supernatant was carefully aspirated, and the surface of the pellet was washed twice by carefully adding 300 ml of PBS and aspirating without disturbing the pellet. After the second wash, 155 ml of PBS were added to each pellet and the cells were resuspended. Approximately 150 ml of gradient-processed blood were then transferred to a tube containing 5 ml of stock anti-mouse CD24-PE antibody (1 mg) from BD Biosciences (San Jose, CA, USA) and 95 ml of PBS supplemented with 2% v/v fetal bovine serum. After 30 min of incubation on ice, the cell suspension was transferred to

Nanotoxicology, 2014; 8(S1): 36–45

a 15 ml polypropylene tube prefilled with 10 ml PBS, and centrifuged at 300 g for 5 min. The supernatant was aspirated and the pellet was resuspended in 1 ml of PBS containing 150 nM SYTO 13 (Invitrogen, Carlsbad, CA) and incubated at 37  C for 30 min. SYTO-labeled samples were kept on ice until flow cytometry analysis. On each experimental day, an ICS was prepared as described elsewhere (Phonethepswath et al., 2010). The ICS was used for optimizing photomultiplier tube voltages and fluorescence compensation settings, and to define the vertical demarcation line that distinguished mutant erythrocytes and/or reticulocytes (RETs) (CD24) from wild type (CD24þ) events. The frequency of RBCCD24 mutants was determined by acquiring data for 1  106 RBCs and 3  105 RETs. In vivo alkaline Comet assay A portion of the left lateral lobe of the liver was removed 3 h after the last treatment from the treated group with 15–100 nm PVP- or 10–80 nm silicon-coated AgNPs with 25 mg/kg/day for 3 consecutive days. Liver sample was washed in the cold mincing buffer until as much blood as possible has been removed. The portion was minced with a pair of fine scissors to release the cells. The cell suspension was strained through a 40 mm cell strainer (Fisher Scientific, Pittsburg, PA, USA) to remove lumps and the remaining suspension will be placed on ice, and the supernatant will be used to prepare Comet slides. The standard alkaline Comet assay was performed according to the protocol used for International Interlaboratory Validation based on the recommendation of Japanese Center for the Validation of Alternative methods (JaCVAM, 2009). One-hundred microliters of the single cell suspensions derived from the mouse livers were mixed with 1 ml 0.5% (w/v) low-melting agarose gel (Lonza, NuSieve GTG Agarose) in PBS at 37  C, and 100 ml of this suspension were applied to microscope slides (VWR, Radnor, PA, USA) previously coated with 1% agarose (Fisher Scientific, St. Louis, MO, USA). Cover slips were placed on the slides, and the slides were stored at 4  C for 30 min to solidify the agarose. After the cover slips were gently removed, the slides were placed in freshly prepared lysis buffer (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris, with 10% dimethylsulfoxide and 1% Triton X-100 added just before use) and stored at 4  C overnight. The slides then were transferred into a chilled alkaline solution (300 mM NaOH, 1 mM EDTA, pH 13), and allowed to remain in the solution for 20 min in the dark to unwind the DNA. After unwinding, electrophoresis was performed in the same solution at 4  C in the dark for 20 min at 0.7 V/cm and 300 mA. The slides then were washed with neutralizing buffer (0.4 M Tris, adjusted to pH 7.5 with HCl) three times for 5 min each to neutralize the remaining alkali and remove detergent; the slides next were fixed with ice-cold ethanol (100%) and dried overnight. Prior to scoring, the slides were stained with SYBR Gold (Invitrogen, Carlsbad, CA) (1:10 000 dilutions in TBE buffer). Two slides were scored from each sample; at least 150 cells were selected randomly for each sample and scored using a system consisting of a Nikon 501 fluorescent microscope and Comet IV digital imaging software (Perceptive Instruments, Wiltshire, UK). Percent (%) DNA in tail, defined as the fraction of DNA in the tail divided by the total amount of DNA associated with a cell multiplied by 100, was used as the parameter for DNA damage analysis. Enzyme-modified Comet assay with addition of Endonuclease III (ENDOIII) and human 8-oxoguanine DNA N-glycosylase 1 (hOGG1) was performed as described previously (Ding et al., 2011). Oxidative DNA damage was calculated using the protocol of Dusinska (Dusinska, 2000).

Nanotoxicology Downloaded from informahealthcare.com by Thammasat University on 10/08/14 For personal use only.

DOI: 10.3109/17435390.2013.855827

In vivo genotoxicity of of AgNPs

39

Figure 1. Transmission electron microscopy (TEM) characterization of the silver nanoparticles (AgNPs). (A) Representative image of the 5 nm PVP-coated AgNPs taken at 60 000  from manufacture. (B) Representative image of the 5 nm PVP-coated AgNPs characterized immediately before the treatment, taken at 60 000. (C) Representative image of the 15–100 nm PVP-coated AgNPs taken at 25 000. (D) Representative image of the 10–80 nm silicon-coated AgNPs taken at 25 000.

Statistical analyses Statistical analysis of all data was performed using SigmaPlotÕ 11 (SPSS, Chicago, IL, USA). These data were analyzed by one-way analysis of variance (ANOVA), followed by comparisons between the vehicle control and the individual treatment groups using Dunnett’s test.

Results Characterization of AgNPs Five nm PVP-coated AgNPs were well distributed and dispersed in water. Agglomeration seldom occurred in the water-diluted samples according to TEM analysis (Figure 1A). Also, analysis with UV–visible spectroscopy indicated that the 5 nm PVP-coated AgNPs had an absorbance maximum at about 417 nm (Table 1), suggesting that the NPs had a relatively narrow size distribution. The characterization of 5 nm PVP-coated AgNPs was also confirmed and a similar average size of 4.13  1.53 nm was obtained by TEM before treatment (Figure 1B). AgNPs coated with PVP or silicon (Figure 1C and D) were also characterized with TEM examination of size distribution, UV–visible spectroscopy analysis of absorbance spectrum, DLS (dynamic light scattering) analysis of hydrodynamic diameter and Zetasizer Nano ZS detection of Zeta potential (Table 1). Average primary size for PVP-coated AgNPs and silicon-coated AgNPs is 51.4  21.8 nm and 45.5  20.9 nm by TEM analysis, respectively.

Tissue distribution of AgNPs in bone marrow, peripheral blood and liver Distribution analysis of the Ag level in bone marrow, peripheral blood and liver was conducted with the mice treated with 15–100 nm PVP- or 10–80 nm silicon-coated AgNPs. As shown in Table 2, Ag levels in the treated groups increased over the control in both the bone marrow and blood in a dose-dependent manner. The amount of Ag for PVP-coated AgNPs was detected in a higher concentration in bone marrow (449.40  85.98 ng/mg protein for the single-dose treatment and 470.60  177.5 ng/mg protein for the three-dose treatment) than silicon-coated AgNPs (3.40  0.16 ng/mg protein for the single-dose treatment and 158.20  37.16 ng/mg protein for the three-dose treatment). Ag level in circulating blood was also elevated with the increase of the dose for the two types of coated AgNPs (Table 2). The data suggested that the AgNPs circulated in blood and reached the bone marrow, the target tissue for the genotoxicity assays in this study. ICP-MS analysis data also showed that a great amount of Ag was accumulated in liver after 3-day continuous treatment (Table 2). According to ICP-MS analysis, the group that repeatedly treated with PVP-coated AgNPs has the highest concentration of Ag in bone marrow. Meanwhile, PVP-coated and silicon-coated AgNPs repeatedly treated groups showed a high level of Ag element in liver (Table 2). To confirm the ICP-MS results, we conducted TEM images together with element analysis for

40

Li et al.

Nanotoxicology, 2014; 8(S1): 36–45

Table 1. Characterization of the three types of silver nanoparticles. Coating materials PVP PVP Silica

Size distribution (nm)

Primary size (nm)

Hydrodynamic Diameter (nm)

Maximum Wavelength (nm)

Zeta Potential (mV)

3–8 15–100 10–80

5.4  1.2 51.4  21.8 45.5  20.9

N/A* 101.6  2.5 1343  61.8

417 426 472

N/A* 12.4  0.4 4.04  1.3

*The particle size is too small to be suitable for DLS or Zeta Potential analysis due to the detection limits of the current instrumentation for metal nanoparticles. Table 2. Silver levels in the bone marrow, peripheral blood and liver of mice (n ¼ 5) treated with a dose of 25 mg/kg of PVP- or silicon-coated silver nanoparticles (AgNPs) once or in triplicate for 3 consecutive days.

Nanotoxicology Downloaded from informahealthcare.com by Thammasat University on 10/08/14 For personal use only.

Treatment group Vehicle control Single dose Silicon coated AgNPs PVP coated AgNPs Repeated Dose Silicon coated AgNPs PVP coated AgNPs

Sampling time after final treatment (h) 48

Concentration of silver in bone marrow (ng/mg protein)

Concentration of silver in blood (ng/ml blood)

3.67  0.01

0.07  0.07

Concentration of silver in liver (ng/mg liver) 0.29  0.31

48 48

3.40  0.16### 449.40  85.98***,###

20.67  7.21* 35.92  18.87**

– –

48 48

158.20  37.16** 470.60  177.55***

72.60  18.04*** 65.11  21.54***

696.64  75.93*** 774.33  104.54***

The data are expressed as mean  SD. *, ** and *** indicate p50.05, p50.01 and p50.001, respectively for the treatment group vs. vehicle control group. ### represent p50.001 for the comparison between silicon-coated and PVP-coated AgNPs at the same treatment schedule.

these samples. No AgNPs were observed by TEM analysis in the bone marrow (Figure 2A) and liver (Figure 2B) of the control group, whereas AgNPs were clearly accumulated in bone marrow (Figure 2C) and liver (Figure 2E and G) in the TEM images. These particles in treated samples were confirmed to be AgNPs by the EDS analysis (Figure 2D, F and H). Evaluation of in vivo genotoxicity of AgNPs In vivo micronucleus assay According to the OECD guideline 474 (OECD, 1997), a reduction in the proportion of immature erythrocytes among total erythrocytes in the bone marrow can be used to indicate toxicity to the bone marrow. Hence, in our study, reticulocytes percentage was used as an index of bone marrow toxicity. PVP-coated AgNPs (5 nm) caused a statistically significant decrease (maximum 30%) in % reticulocytes at all the concentrations tested from 0.5 to 20 mg/kg, indicating toxicity to the bone marrow (Figure 3A). Similarly, 15–100 nm PVP-coated AgNPs also caused 33 and 22% reductions in % reticulocytes over the control in the single and repeated treatments, respectively (Figure 3C), while no toxicity to the bone marrow was found following both the single and repeated treatment of silicon-coated AgNPs (Figure 3C). Although 5 nm and 15–100 nm PVP-coated AgNPs caused cytotoxicity in the bone marrow, no increase was observed in % MN-RET when the treatments were compared with the vehicle control (Figure 3B and D), indicating these AgNPs did not exert genotoxicity in mice by in vivo micronucleus assay. In contrast, the positive control (140 mg/kg ENU) caused a significant reduction in % reticulocytes and a significant increase in % MN-RETs when compared to the control (Figure 3A and C).

No increase was observed in RBCCD24 and RETCD24 frequencies in AgNPs-treated animals (Figure 4B and C). However, animals treated with ENU (140 mg/kg) as the positive control produced a highly significant response in RBCCD24 and RETCD24 frequencies at all the time points measured. Maximum RBCCD24 (190.13  106) and RETCD24 frequencies (550.00  106) were observed at Week 2; the mutant frequencies started dropping by Week 4 and Week 6 (Figure 4B and C). A reduction in %RETs as measured by CD71-PE during the MN assay was observed after 48 h, suggesting treatment-related cytotoxicity (Figure 3A), but a rebound in % RETs as measured by SYTO 13 staining in the Pig-a assay was recorded at Week 2 then returned normal in the later time points. A similar but more pronounced effect was observed in ENU-treated animals (Figure 4A). In vivo Comet assay Considering that the liver was the primary site of clearance for the AgNPs and most likely to be the main site of the particle accumulation following the i.v. treatment, the Comet assay was conducted to examine whether the genotoxicity damage resulted from the AgNP exposure. The liver samples were collected from the mice that were treated with 25 mg/kg bw PVP- or siliconcoated AgNPs, or the vehicle for 3 consecutive days and assayed for DNA damage 3 h after the last treatment (Figure 5). No DNA strand breaks were detected in liver for both PVP- and silicon-coated AgNPs in the standard Comet assay (Figure 5A) while significant induction of oxidative DNA damage by the AgNPs treatment were found in the enzyme-modified Comet assay (Figure 5B).

Discussion

Pig-a assay CD24

and Over the 6 weeks of the study period, average RBC average RETCD24 frequencies in the vehicle control animals ranged from 0 to 0.4  106 and 0 to 1.3  106, respectively.

AgNPs are known for their superior antimicrobial activity (Feng et al., 1999). Although a number of publications are available on the in vitro genotoxicity of AgNPs, the information is

In vivo genotoxicity of of AgNPs

41

Nanotoxicology Downloaded from informahealthcare.com by Thammasat University on 10/08/14 For personal use only.

DOI: 10.3109/17435390.2013.855827

Figure 2. TEM and EDS analyses of AgNPs in the bone marrow and liver of mice treated with different types of AgNPs. The transmission electron microscope (TEM) images of the bone marrow cells (A) and liver cells (B) were from the vehicle treated group. The representative TEM images were for the samples from the bone marrow cells treated with PVP-coated AgNPs (C), the liver cells treated with PVP-coated AgNPs (E) and liver cells treated with silicon-coated AgNPs (G). The scale shown in the graph is 200 nm. Chemical composition analysis of the particles inside the cells in (C), (E) and (G) using energy-dispersive X-ray spectroscopy (EDS) were shown in (D), (F) and (H), respectively. The areas that were analyzed by EDS on the TEM images are indicated by the red boxes in (C), (E) and (G). The red boxes in (D), (F) and (H) indicate the Ag element.

Nanotoxicology Downloaded from informahealthcare.com by Thammasat University on 10/08/14 For personal use only.

42

Li et al.

Nanotoxicology, 2014; 8(S1): 36–45

Figure 3. Cytoxicity and genotoxicity of AgNPs assessed by the in vivo micronucleus (MN) assay. (A) Cytotoxicity of 5 nm PVP-coated AgNPs in mice after a single treatment. Percent reticulocytes (RETs) measured using CD71-PE of control was used to determine the cytotoxicity in the micronucleus (MN) assay. (B) Reticulocyte micronucleus frequency (% MN-RET) induced by 5 nm PVP-coated AgNPs in mice after a single treatment. (C) Cytotoxicity of 15–100 nm PVP- or 10–80 nm silicon-coated AgNPs in mice after a single or a three-day repeated treatment; (D) % MN-RET induced by 15–100 nm PVP- or 10–80 nm silicon-coated AgNPs in mice after a single or a three-day repeated treatment. ENU was used as the positive control. ***Indicates that there was a significant difference between the treatment and their concurrent control (p50.001).

inadequate for drawing general conclusions partially due to lack of genotoxicity data from in vivo assays (Gonzalez et al., 2008; Landsiedel et al., 2009). Hence, in this study, we performed the in vivo genotoxicity assays to evaluate whether AgNPs could cause gene mutation, chromosomal damage and DNA damage in vivo and whether the genotoxicity was related to sizes and coating materials of AgNPs. Our data showed that all the different types of AgNPs could reach the bone marrow and liver; PVPcoated AgNPs were more cytotoxic than the silica-coated AgNPs in the bone marrow; and although the AgNPs did not induce genotoxicity when measured with in vivo micronucleus assay and Pig-a assay under our experimental conditions, they did cause oxidative DNA damage in liver. NPs can distribute through nearly all tissues and organs with various administration routes (Hagens et al., 2007). In the 28-day inhalation and 28-day oral-dose studies with both male and female Sprague-Dawley rats, a dose-dependent distribution and deposition of AgNPs was observed in the rat tissues including blood, stomach, brain, liver, kidneys, lungs and testis (Kim et al., 2008; Ji et al., 2007). In another 28-day repeat-dose oral study with PVP-coated AgNPs in Wistar Hannover GAlAs rats, AgNPs were found in the small intestine, stomach, kidneys and liver (Loeschner et al., 2011). Although it is assumed that AgNPs can reach the bone marrow (Kim et al., 2011b), prior to our current study, no data provided the direct evidence that AgNPs can reach the bone marrow. Because the target tissue for both the in vivo MN assay and Pig-a assay is bone marrow, it is very important to ensure that the NPs can reach the tissue so that a negative result can be claimed. Our preliminary studies suggested that i.v. treatment was the best route for delivering AgNPs to bone

marrow. Thus, the i.v. treatment of the NPs was used for this study. ICP-MS was used for determining Ag concentration in mouse bone marrow and blood 48 h after the treatment (Table 2). In conjunction with the results from TEM and EDS analyses (Figure 2), AgNPs were confirmed to accumulate in the bone marrow. A higher Ag level in the bone marrow was detected in mice treated with PVP-coated AgNPs than in the mice treated with the silicon-coated AgNPs (Table 2), which could be due to the effects of the different surface chemistry. First, polymers such as PVP prolonged the circulation time of NPs and increased plasma halflife compared to other coating materials (Monfardini & Veronese, 1998). Additionally, silicon-coated AgNPs are more easily flocculated than PVP coated AgNPs, which make their hydrodynamic diameter much bigger under normal conditions (1343  61.8 nm) than in the base solution (177.8  3.2 nm, unpublished data). The large size of silicon-coated AgNPs limits their access into the bone marrow since the bone marrow has a discontinuous endothelium with pores of 50–100 nm in size (Li & Reineke, 2012). With the relative high concentration in the bone marrow, the PVP-coated AgNPs caused cytotoxicity in the bone marrow while the silicon-coated AgNPs did not produce any cytotoxicity (Figure 3A and C). These results suggest that different coatings of NPs can have significant effects on their distribution and cytotoxicity in tissues. In our study, AgNPs did not increase MN or Pig-a frequency. The negative results for genotoxicity of AgNPs using these two assays are consistent with those from other in vivo studies. In either a 28 days oral toxicity study or a 90 days inhalation study, AgNPs did not cause chromosome damage in Sprague-Dawley

DOI: 10.3109/17435390.2013.855827

Nanotoxicology Downloaded from informahealthcare.com by Thammasat University on 10/08/14 For personal use only.

rats (Kim et al., 2008, 2011b). AgNPs capped with starch, bovine serum albumin (Asharani et al., 2008) and polyvinyl alcohol (Asharani et al., 2011) were not genotoxic to zebra fish embryos in the zebra fish model. Although no DNA strand breaks observed in liver in the standard Comet assay, which is

Figure 4. Cytoxicity and mutagenicity of AgNPs assessed by Pig-a gene mutation assay. (A) Cytotoxicity of 5 nm PVP-coated AgNPs in mice after a single dose of treatment. Percentage RETs of control measured by the SYTO 13 staining was used to measure the cytotoxicity in Pig-a assay. (B) Mutant frequency of CD24-negative total red blood cell (RBCCD24) frequencies in mice treated with 5 nm PVP-coated AgNPs. (C) Mutant frequency of CD24-negative reticulocyte (RETCD24) frequencies in mice treated with 5 nm PVP-coated AgNPs. A single dose of ENU (140 mg/kg) was used as the positive control, and purified water was used as the vehicle control. ***Indicates that there was a significant difference between the treatment and their concurrent control (p50.001).

In vivo genotoxicity of of AgNPs

43

consistent with previous findings in mouse spleen (Ordzhonikidze et al., 2009), PVP- and silicon-coated AgNPs were found to cause DNA breaks in liver in the enzyme-modified Comet assay (Figure 5). The nuclease ENDOIII recognizes oxidized pyrimidines, including thymine glycol and uracil glycol while nuclease hOGG1 targets oxidative guanine DNA adducts such as 8-oxoguanine. The DNA breaks resulted from the additional nuclease enzymes in the Comet assay suggest that the AgNPs can cause oxidative DNA damage. In our previous study, 5 nm uncoated AgNPs increased the micronucleus frequency in TK6 cells (Li et al., 2012a) and mutation frequency in the Tk gene of mouse lymphoma cells (Mei et al., 2012). Loss of heterozygosity analysis of the Tk mutants revealed that treatments with the 5 nm uncoated AgNPs induced mainly chromosomal alterations spanning less than 34 mega base pairs on chromosome 11 (Mei et al., 2012). The same PVP-coated AgNPs used in current study also induced micronuclei in TK6 cells (Li et al., 2012b). It is suggested that oxidative stress caused by NPs is responsible for the DNA damage in the in vitro systems (Mei et al., 2012; Petersen & Nelson, 2010). NPs may act considerably different under in vivo conditions and in vitro conditions (Klien & Godnic-Cvar, 2012). AgNPs have been reported to produce oxidative stress in the nose, blood and liver (Ferna´ndez-Urrusuno et al., 1997; Genter et al., 2012), but the stress was found to be transient and reversible (Genter et al., 2012) or resistible by organs along with a significant increase of catalase (Ferna´ndez-Urrusuno et al., 1997). Oxidative DNA damage in liver was also observed in our study (Figure 5B). These findings suggest that NPs can induce oxidative stress in vivo. However, many different antioxidant enzymes and systems that do not exist in in vitro systems may scavenge the oxidative species. These could be the reasons for different results from the in vivo and in vitro MN assays and mutation assays. Additional investigations of oxidative stress and antioxidative systems in vivo and in vitro are needed to understand the mode of action of AgNPs genotoxicity. The inconsistence between the positive genotoxicity results from the Comet assay in mouse liver and negative genotoxicity data from MN and Pig-a assays in mouse bone marrow could reflect a tissue-specific genotoxicity of AgNPs or different sensitivities of these assays to genotoxicity of AgNPs. In summary, although PVP-coated AgNPs cause cytotoxicity, the AgNPs tested under our experimental conditions showed no genotoxicity in mouse bone marrow. However, the AgNPs induced oxidative DNA damage in mouse liver measured with the in vivo Comet assay. Our results highlight the importance of evaluating the in vivo genotoxicity of nanomaterials and correlating the findings with the in vitro genotoxicity results. The general conclusion on genotoxicity of NPs is far from certain

Figure 5. Effect of AgNPs on DNA damage in the liver evaluated using the in vivo Comet assay. (A) Effect of AgNPs on DNA damage in the standard Comet assay, 100 mg/kg methyl methane sulphonate (MMS) was used as the positive control. (B) Effect of AgNPs on DNA damage in the EndoIII and hOOG1-modified Comet assay. In vivo Comet assay were conducted only in repeated dosing group of 15–100 nm PVP- or 10–80 nm silicon-coated AgNPs. The accumulated dose of AgNPs was 75 mg/kg. ***Indicates that there was a significant difference between the treatment and control groups (p50.001).

44

Li et al.

given the inconsistencies. More in vivo genotoxicity studies and researches in understanding of the mode of action are warranted for the risk assessment of nanomaterials.

Acknowledgements The authors would like to thank Christopher K. Dugard (Nanotechnology Core Facility, Jefferson, AR) for analyzing the silver content in liver by ICP-MS.

Declaration of interest

Nanotoxicology Downloaded from informahealthcare.com by Thammasat University on 10/08/14 For personal use only.

The authors declare that they have no competing interests. YL was supported by the appointment to the Postgraduate Research Program at the National Center for Toxicological Research administered by Oak Ridge Institute for Science Education through an interagency agreement between the U.S. Department of Energy and the U.S. FDA. The views presented in this article do not necessarily reflect those of the Food and Drug Administration.

References Agarwal A, Weis TL, Schurr MJ, Faith NG, Czuprynski CJ, McAnulty JF, et al. 2009. Surfaces modified with nanometer-thick silver-impregnated polymeric films that kill bacteria but support growth of mammalian cells. Biomaterials 31:680–90. Asare N, Instanes C, Sandberg WJ, Refsnes M, Schwarze P, Kruszewski M, Brunborg G. 2012. Cytotoxic and genotoxic effects of silver nanoparticles in testicular cells. Toxicology 291:65–72. Asharani PV, Low Kah Mun G, Hande MP, Valiyaveettil S. 2009. Cytotoxicity and genotoxicity of silver nanoparticles in human cells. ACS Nano 3:279–90. Asharani PV, Wu YL, Gong Z, Valiyaveettil S. 2008. Toxicity of silver nanoparticles in zebrafish models. Nanotechnology 19:255102. Asharani PV, Wu YL, Gong Z, Valiyaveettil S. 2011. Comparison of the toxicity of silver, gold and platinum nanoparticles in developing zebrafish embryos. Nanotoxicology 5:43–54. Bhalli JA, Pearce MG, Dobrovolsky VN, Heflich RH. 2011. Manifestation and persistence of Pig-a mutant red blood cells in C57BL/6 mice following single and split doses of N-ethyl-Nnitrosourea. Environ Mol Mutagen 52:766–73. Burlinson B. 2012. The in vitro and in vivo comet assays. Methods Mol Biol 817:143–63. Dertinger SD, Miura D, Hayashi M, MacGregor JT. 2011a. The in vivo cytogenetic damage. Mutagenesis 26:139–45. Dertinger SD, Phonethepswath S, Weller P, Nicolette J, Murray J, Sonders P, et al. 2011b. International Pig-a gene mutation assay trial: evaluation of transferability across 14 laboratories. Environ Mol Mutagen 52: 690–8. Dertinger SD, Torous DK, Hall NE, Tometsko CR, Gasiewicz TA. 2000. Malaria-infected erythrocytes serve as biological standards to ensure reliable and consistent scoring of micronucleated erythrocytes by flow cytometry. Mutat Res 464:195–200. Dertinger SD, Torous DK, Hayashi M, MacGregor JT. 2011c. Flow cytometric scoring of micronucleated erythrocytes: an efficient platform for assessing in vivo cytogenetic damage. Mutagenesis 26: 139–45. Ding W, Levy DD, Bishop ME, Lyn-Cook Lascelles E, Kulkarni R, Chang CW, et al. 2011. Methyleugenol genotoxicity in the Fischer 344 rat using the comet assay and pathway-focused gene expression profiling. Toxicol Sci 123:103–12. Dusinska M. 2000. The Comet assay, modified for detection of oxidised bases with the use of bacterial repair endonucleases. [Online] Available at: http://www.cometassayindia.org/Dusinska-protocol.PDF [Last accessed 9 August 2013]. Feng QL, Cui FZ, Kim TN, Kim JW. 1999. Ag-substituted hydroxyapatite coatings with both antimicrobial effects and biocompatibility. J Mater Sci Lett 18:559–61. Ferna´ndez-Urrusuno R, Fattal E, Fe´ger J, Couvreur P, The´rond P. 1997. Evaluation of hepatic antioxidant systems after intravenous administration of polymeric nanoparticles. Biomaterials 18:511–17. Foldbjerg R, Dang DA, Autrup H. 2011. Cytotoxicity and genotoxicity of silver nanoparticles in the human lung cancer cell line, A549. Arch Toxicol 85:743–50.

Nanotoxicology, 2014; 8(S1): 36–45

Genter MB, Newman NC, Shertzer HG, Ali SF, Bolon B. 2012. Distribution and systemic effects of intranasally administered 25 nm silver nanoparticles in adult mice. Toxicol Pathol 40: 1004–13. Gonzalez L, Lison D, Kirsch-Volders M. 2008. Genotoxicity of engineered nanomaterials: a critical review. Nanotoxicology 2:252–73. Hagens WI, Oomen AG, de Jong WH, Cassee FR, Sips AJAM. 2007. What do we (need to) know about the kinetic properties of nanoparticles in the body? Regul Toxicol Pharmacol 49:217–29. ICTA. 2006. Petition requesting FDA amend its regulations for products composed of engineered nanoparticles generally and sunscreen drug products composed of engineered nanoparticles specifically. [Online] Available at: http://www.icta.org/doc/Nano%20FDA%20petition% 20final.pdf [Last accessed 9 August 2013]. ICTA. 2008. Groups demand EPA stop sale of 200þ potentially dangerous nano-silver products. [Online] Available at: http://www. icta.org/files/2011/12/CTA_nano-silver_press_release_5_1_08.pdf [Last accessed 9 August 2013]. JaCVAM. 2009. International validation of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens. [Online] Available at: http://cometassay.com/JaCVAM.pdf [Last accessed 9 August 2013]. Ji JH, Jung JH, Kim SS, Yoon JU, Park JD, Choi BS, et al. 2007. Twenty-eight-day inhalation toxicity study of silver nanoparticles in Sprague-Dawley rats. Inhal Toxicol 19:857–71. Kim YS, Kim JS, Cho HS, Rha DS, Kim JM, Park JD, et al. 2008. Twenty-eight-day oral toxicity, genotoxicity, and gender-related tissue distribution of silver nanoparticles in Sprague-Dawley rats. Inhal Toxicol 20:575–83. Kim HR, Kim MJ, Lee SY, Oh SM, Chung KH. 2011a. Genotoxic effects of silver nanoparticles stimulated by oxidative stress in human normal bronchial epithelial (BEAS-2B) cells. Mutat Res 726:129–35. Kim JS, Sung JH, Ji JH, Song KS, Lee JH, Kang CS, Yu IJ. 2011b. In vivo genotoxicity of silver nanoparticles after 90-day silver nanoparticle inhalation exposure. Saf Health Work 2:34–8. Klien K, Godnic-Cvar J. 2012. Genotoxicity of metal nanoparticles: focus on in vivo studies. Arh Hig Rada Toksikol 63:133–45. Kux L. 2012. FDA response to 2006-P-0213-citizen petition. [Online] Available at: http://www.icta.org/doc/Andrew%20 Kimbrell-FDA-2006-P-0213-Citizen%20Petition.pdf [Last accessed 9 August 2013] Landsiedel R, Kapp MD, Schulz M, Wiench K, Oesch F. 2009. Genotoxicity investigations on nanomaterials: methods, preparation and characterization of test material, potential artifacts and limitations– many questions, some answers. Mutat Res 681:241–58. Li M, Reineke J. 2012. Physiologically based pharmacokinetic modeling for nanoparticle toxicity study. Methods Mol Biol 926:369–382. Li Y, Chen DH, Yan J, Chen Y, Mittelstaedt RA, Zhang Y, et al. 2012a. Genotoxicity of silver nanoparticles evaluated using the Ames test and in vitro micronucleus assay. Mutat Res 745:4–10. Li Y, Yan J, Bhalli J, Sadiq R, Ding W, Chen T. 2012b. Micronuclei in TK6 cells are increased by silver nanoparticles in a dose, size and surface coating-dependent manner. Toxicologist 100:140. Loeschner K, Hadrup N, Qvortrup K, Larsen A, Gao X, Vogel U, et al. 2011. Distribution of silver in rats following 28 days of repeated oral exposure to silver nanoparticles or silver acetate. Part Fibre Toxicol 8:18. Madhumathi K, Sudheesh Kumar PT, Abhilash S, Sreeja V, Tamura H, Manzoor K, et al. 2009. Development of novel chitin/nanosilver composite scaffolds for wound dressing applications. J Mater Sci Mater Med 21:807–13. Mei N, Zhang Y, Chen Y, Guo X, Ding W, Ali SF, et al. 2012. Silver nanoparticle-induced mutations and oxidative stress in mouse lymphoma cells. Environ Mol Mutagen 53:409–19. Monfardini C, Veronese FM. 1998. Stabilization of substances in circulation. Bioconjug Chem 9:418–50. OECD. 1997. Guideline for the testing of chemicals, guideline 474 mammalian erythrocyte micronucleus test. [Online] Available at: http:// www.oecd.org/chemicalsafety/assessmentofchemicals/1948442.pdf [Last accessed 9 August 2013]. Oesch F, Landsiedel R. 2012. Genotoxicity investigations on nanomaterials. Arch Toxicol 86:985–94. Ordzhonikidze CG, Ramaiyya LK, Egorova EM, Rubanovich AV. 2009. Genotoxic effects of silver nanoparticles on mice in vivo. Acta Naturae 1:99–101.

DOI: 10.3109/17435390.2013.855827

Nanotoxicology Downloaded from informahealthcare.com by Thammasat University on 10/08/14 For personal use only.

Petersen EJ, Nelson BC. 2010. Mechanisms and measurements of nanomaterial-induced oxidative damage to DNA. Anal Bioanal Chem 398:613–50. Phonethepswath S, Franklin D, Torous DK, Bryce SM, Bemis JC, Raja S, et al. 2010. Pig-a mutation: kinetics in rat erythrocytes following exposure to five prototypical mutagens. Toxicol Sci 114: 59–70. Singh N, Aardema M, Henderson L, Muller L. 2009a. Evaluation of the ability of a battery of engineered nanomaterials. Mutat Res 30: 1–256.

In vivo genotoxicity of of AgNPs

45

Singh N, Manshian B, Jenkins GJ, Griffiths SM, Williams PM, Maffeis TG, et al. 2009b. NanoGenotoxicology: the DNA damaging potential of engineered nanomaterials. Biomaterials 30:3891–914. Tometsko AM, Dertinger SD, Torous DK. 1993. Analysis of micronucleated cells by flow cytometry. 2. Evaluating the accuracy of high-speed scoring. Mutat Res 292:137–43. Woodrow Wilson International Center. 2011. A nanotechnology consumer products inventory. [Online] Available at: http://www. nanotechproject.org/inventories/consumer/analysis_draft/. Accessed on 9 August 2013.