Silver nanoparticles: a brief review of cytotoxicity ... - Wiley Online Library

Review Article Received: 18 January 2012,

Revised: 31 March 2012,

Accepted: 19 April 2012

Published online in Wiley Online Library: 13 June 2012

(wileyonlinelibrary.com) DOI 10.1002/jat.2780

Silver nanoparticles: a brief review of cytotoxicity and genotoxicity of chemically and biogenically synthesized nanoparticles Renata de Lima,a,b* Amedea B. Seabrac and Nelson Duránd,e ABSTRACT: In recent years interest in silver nanoparticles and their applications has increased mainly because of the important antimicrobial activities of these nanomaterials, allowing their use in several industrial sectors. However, together with these applications, there is increasing concerning related to the biological impacts of the use of silver nanoparticles on a large scale, and the possible risks to the environment and health. In this scenario, some recent studies have been published based on the investigation of potential inflammatory effects and diverse cellular impacts of silver nanoparticles. Another important issue related to nanoparticle toxicity in biological media is the capacity for increased damage to the genetic material, since nanoparticles are able to cross cell membranes and reach the cellular nucleus. In this regard, there is increasing interest in the analysis of potential nanoparticle genotoxicity, including the effects of different nanoparticle sizes and methods of synthesis. However, little is known about the genotoxicity of different silver nanoparticles and their effects on the DNA of organisms; thus further studies in this field are required. This mini-review aims to present and to discuss recent publications related to genotoxicity and the cytotoxicity of silver nanoparticles in order to better understand the possible applications of these nanomaterials in a safe manner. This present work concludes that biogenic silver nanoparticles are generally less cyto/genotoxic in vivo compared with chemically synthesized nanoparticles. Furthermore, human cells were found to have a greater resistance to the toxic effects of silver nanoparticles in comparison with other organisms. Copyright © 2012 John Wiley & Sons, Ltd. Keywords: nanotoxicology; silver nanoparticle; genotoxicity; nanotechnology; nanobiotechnology; biogenic nanoparticle; cytotoxicity

Introduction

J. Appl. Toxicol. 2012; 32: 867–879

*Correspondence to: Renata de Lima, Department of Biotechnology, University of Sorocaba, Rodovia Raposo Tavares S/N - km 92,5, CEP 18023-000, Sorocaba, S.P., Brazil. E-mail: [email protected] a Department of Biotechnology, University of Sorocaba, Rodovia Raposo Tavares S/N-km 92,5, CEP 18023-000, Sorocaba, S.P., Brazil b

Universidade Federal de São Carlos, UFSCar, Sorocaba, S.P., Brazil

c

Exact and Earth Sciences Department, Universidade Federal de São Paulo, UNIFESP, Diadema, S.P., Brazil d Center of Natural and Human Sciences, Universidade Federal do ABC, Santo André, S.P., Brazil e Institute of Chemistry, Biological Chemistry Laboratory, Universidade Estadual de Campinas, Campinas S.P., Brazil

Copyright © 2012 John Wiley & Sons, Ltd.

867

Nanotechnology has been expanding rapidly in recent years, impacting on diverse areas such as the economy and the environment. In this context, the number of commercial products comprising nanomaterials is increasing. Among the commercially available nano-sized materials, silver nanoparticles are by far the most used nanocompounds (Ahmed et al., 2008) owing to its potent antimicrobial activity (Durán et al., 2010). Indeed, silver nanoparticles have been used in commercial products such as personal care, household and medical products, as well in textiles and food production (Hood, 2004; Wijnhoven et al., 2009). An interesting characteristic of nanostructured materials is the possibility of different physicochemical properties, such as mechanical, chemical, magnetic, optical or electric properties compared with bulk materials (Wise et al., 2010). For example, gold nanoparticles are considered important scaffolds for drug delivery and act as strong catalysts in opposition to almost inert bulk gold. Similarly, the novel properties of nano-size materials may also reflect their potent toxic effects, since the same properties that make these nanoparticles very interesting for a wide range of applications might affect their toxicity (Wise et al., 2010). Therefore, there is an increasing concern about the evaluation of the toxicity of nanoparticles. The development of nanotechnology has resulted in a growing public debate on the toxicity and environmental impact of direct and indirect exposures to nanoparticles (Brayner, 2008; Panda et al., 2011). In fact, nanoparticles may have higher toxicity than bulk materials (Donaldson et al., 1999). Nanoparticle toxicity, including human health and environmental implications, is still

considered not completely elucidated and relatively unexplored (Nel et al., 2006; Lewinski et al., 2008; Ju-Nam and Lead, 2008). Concerning human health, studies have demonstrated that nanoparticles have toxic effects at the cellular, subcellular and biomolecular levels, such as genes and proteins (Gurr et al., 2005; Chi et al., 2009). The evaluation of toxicity of silver nanoparticles has been carried out in different cellular models, such as human lung fibroblasts (AshaRani et al., 2009). Indeed, oxidative stress and severe lipid peroxidation have been observed in fish brain tissue upon exposure to nanomaterials (Oberdörster, 2004). The proposed mechanism by which metallic nanoparticles lead to cytotoxicity was considered to be through the induction of reactive oxygen species (ROS; Foldbjerg et al., 2011). In this context, a reduction in glutathione levels, an increase of ROS levels

R. de Lima et al. and an observation of lipid peroxidation upon in vivo exposure to silver nanoparticles have already been reported (Arora et al., 2008; Kim et al., 2009a, b). This mini-review concludes that there are few publications focusing on nanoparticle genotoxicity. It has been observed that silver nanoparticles lead to an increase in ROS associated with DNA damage, apoptosis and necrosis (Arora et al., 2008; Kim et al., 2009a, b; Foldbjerg et al., 2011). Therefore, more studies of the toxicity of nanoparticles, in particular genotoxicity, are imperative. Interesting reviews that discuss the methodologies currently available for genotoxic studies were recently published. These reviews presented a survey of the in vitro and in vivo genotoxicological studies of nanomaterials conducted in recent years, including silver nanoparticles (Ng et al., 2010; Johnston et al., 2010; Rico et al., 2011). In this context, the aim of this review is to present and discuss the new discoveries of silver nanoparticles toxicity and highlight the importance of more detailed studies in the field. Uncapped and Capped Silver Nanoparticles In general, thermodynamic stabilization of nanoparticles is achieved by adding capping agents, which bind to the nanoparticle surface via covalent bonds or by chemical interaction. These capping agents are essential to prevent nanoparticle aggregation and increase the solubility of the nanosystem, and also can be used as a site for bioconjugation of the nanoparticle with important molecules (Sing et al., 2009; Seabra and Durán, 2010). Examples of capping agents include water-soluble polymers, oligosaccharides and polysaccharides. On the other hand, uncapped nanoparticles are bare nanoparticles.

Uncapped Silver Nanoparticles From the recent literature, considering uncapped silver nanoparticles, it is possible to conclude that, for nanoparticles in the size range of 20–50 nm, cytotoxicity and genotoxicity effects are only observed for nanoparticle concentrations less than 10 mg ml 1 (Griffitt et al., 2008, 2009; Wise et al., 2010; Park and Choi, 2010; Park et al., 2010; Nair et al., 2011), even in different culture cells. In animal models, the genotoxicity effects of silver nanoparticles are less effective compared with the genotoxicity effects in cell cultures. The tendency of Lethal Dose, 50% (LD50) is in the range of 1–7 mg ml 1 for both in vitro and in vivo studies, as evaluated for cell culture (Wise et al., 2010), crustaceans (Park and Choi, 2010) or larvae (Nair et al., 2011). For exposure of animals to silver nanoparticles at 5 mg ml 1, size 20–30 nm, through water ingestion (Griffitt et al., 2008, 2009), exposure of animals to silver nanoparticles (~1.0 mg ml 1, size 18 nm) through inhalation (Kim et al., 2011), and exposure of plants to silver nanoparticles (less than 25 mg ml 1, size 70 nm; Kumari et al., 2009) no genotoxicities were observed. Table 1 summarizes the cytotoxicity and genotoxicity effects of silver nanoparticles in different organisms. Chemical/uncapped Synthesis

868

Medaka fish (Oryzias latipes) cell line 0LHN12 was used to investigate the cytotoxicity and genotoxicity of silver nanospheres (size 20–30 nm synthesized by citrate method). Cytotoxicity (24 h treatment) was found to be dose-dependent (1% survival at 3 mg cm 2) in a colony-forming assay (Lethal Concentration, 50% (LC50) reported as 1.3 mg ml 1). The same profile with silver nanoparticles also induced chromosomal aberrations and aneuploidy (24 h

wileyonlinelibrary.com/journal/jat

treatment) at 0.3 mg cm 2 (1.2 mg ml 1) in 15.8% of metaphases and 24 total aberrations in 100 metaphases, respectively. The authors suggested that, in those conditions, silver nanoparticles are cytotoxic and genotoxic to fish cells (Wise et al., 2010). The response of zebrafish (Danio rerio), daphnia (Daphnia pulex) and Pseudokirchneriella subcapitata to 20–30 nm commercial silver nanopowder (prepared by gas phase condensation and comprising a metal core with a thin metal oxide coating) was studied. Silver nanoparticles induced toxicity after 48 h with an LD50 of 7.0–7.2 mg ml 1 in zebrafish, depending on whether the exposure was to an adult or juvenile fish, and 0.040–0.067 mg ml 1 in daphnia. In P. subcapitata the LD50 was 0.19 mg ml 1. No effects on survival up to 5 mg ml 1 were observed. For soluble metal treatment, zebrafish presented an LD50 of 0.022 mg ml 1 and for daphnia the LD50 was found to be 0.008–0.16 mg ml 1. This result shows a greater toxicity of silver soluble metal compared with silver nanoparticles (Griffitt et al., 2008). In this treatment, after 24 and 48 h of exposition, nanosilver (at 1 mg ml 1) also induced changes in gene expression, as revealed by the microarray test. These results showed 66 upregulation genes and 82 downregulation genes during 24 h, and 126 upregulation genes and 336 downregulation genes during 48 h. In addition, among the observed genes some of them presented homology with human genes, most of them being involved with apoptosis, mitogenesis and proliferation signaling; however, they did not affect gill filament length (Griffitt et al., 2009). Silver nanoparticle (35 nm) effects on daphnia magna were studied and the results showed that 100% mortality occurred for 1 mg ml 1 during 96 h, and 43.33% for 0.1 mg ml 1 treatment. Chronic studies, in which Daphnia magna was exposed to lower concentrations of silver nanoparticles, showed significant toxicity at 0.001 mg ml 1 (Gaiser et al., 2011). In other work, (Geiser et al., 2012) studied micro and nano (600–1.600 and 35 nm) sized particles and compared the potential effects in a range of cells (human hepatocyte, intestinal and fish hepatocyte) and animals (Daphnia magna, Cyprinus carpio). This study showed similar biological response from different models. The author observed that Ag nanoparticles were found to be more toxic in comparison with larger micro-sized material. The commonalities in toxicity of these particle types across diverse biological systems suggest that cross-species extrapolations may be possible for metal nanoparticle test development in the future. Genotoxicity and ecotoxicity assessments of silver nanoparticles (size 50 nm, Sigma, pre-filtered) were studied on the freshwater crustacean Daphnia magna. Acute toxicity up to 2 mg ml 1 concentration showed 100% mortality (24 h; LD50 ~1.2 mg ml 1). DNA strand breaks increased after exposure to silver nanoparticles to approximately 4 and 8 times higher levels than the negative control at the concentrations of 1 and 1.5 mg ml 1, respectively. Increased mortality was concomitantly observed with DNA damage in the silver nanoparticles, which suggests that silver nanoparticle-induced DNA damage might provoke higher-level consequences. The results of the comparative toxicities of silver nanoparticles and silver ions suggested that the former were slightly more toxic (Park and Choi, 2010). In this context, silver nanoparticles (Sigma–Aldrich Chemicals, size 40–70 nm) were homogenously dispersed in deionized water by sonication, and stirred for several days, followed by filtration through a cellulose membrane (pore size 100 nm) to remove nanoparticle aggregations. Incubation of these silver nanoparticles with the fourth instar larvae of the aquatic midge, Chironomus riparius, did not result in acute toxicity (up to 1 mg ml 1). However,


J. Appl. Toxicol. 2012; 32: 867–879

869

J. Appl. Toxicol. 2012; 32: 867–879


Gas phase condensation and a metal core with a thin metal oxide coating Gas phase condensation and a metal core with a thin metal oxide coating

between 25 and Silver nanoparticles capped 50 mg ml 1 with soluble potato starch/ silver nanoparticles capped with BSA Sigma — Synthesized by borohydride red. polyvinyl alcohol (capping agent)

20–30

5–20

Zebrafish Danio rerio 6 animals Zebrafish Danio rerio 4 ind./replicate 3 replicates per concentration Zebrafish Danio rerio embryos

5–35

18

Zebrafish Danio rerio embryos

Rats Sprague–Dawley

Vertebrate/ Chordata/ Mammalian

Evaporation/condensation using a small ceramic heater

—

1 mg ml 1 24 h, 66 up- and 82 downregulated 48 h, 126 up- and 336 downregulated 7.0–7.2 mg ml 1

Phenotypic changes starting from 25 mg ml and increasing concentrations 0.8 mg ml 1

Nanoparticles inside the nucleus cells able to take alterations and breakings to DNA

Dose-dependent increase CrGbRH1, CrBR2.2 for upregulated and CrL15 downregulated gene.

1

Kim et al., 2011

Continues

AshaRani et al., 2011

AshaRani et al., 2008

Griffitt et al., 2008


Nair et al., 2011

1

2 mg ml

40–70

20–30

Gaiser et al., 2011


Gaiser et al., 2012

Cvaehironomus riparius, lar

Sigma–Aldrich Chemical

—

—

Reference

Significant uptake was detected in liver, intestine, and gallbladder

Commercial Sigma

35

1

(96 h)

Genotoxicity

1 and 1,5 mg ml-1 Park and Choi, 2010 4 and 8 times bigger than the negative control 96 h, 100% mortality 10–1 mg ml 1 Gaiser et al., 2012 21 days, 30% mortality 0.1 mg ml 1

1.2 mg ml

1

Cyprius carpio 8 fish

50

Daphnia magna Daphnia Daphnia magna

0.1 mg ml

0.040 – 0.067 mg ml 1

Cytotoxicity (LD50)

Commercial Sigma

35

Daphnia

Gas phase condensation and a metal core with a thin metal oxide coating Without coating or surface modifications Sigma Commercial Sigma, pre-filtered

Synthesis

Daphnia 35 10 each concentration

20–30

Size (nm)

Daphnia

Organism

Vertebrate/ Chordata/ Fish

Vertebrate/ Chordata/ Fish

Vertebrate/ Chordata/ Fish Vertebrate/ Chordata/ Fish

Invertebrate/ Arthropoda/ Crustacea Invertebrate/ Arthropoda/ Crustacea Invertebrate/ Arthropoda/ Crustacea Invertebrate/ Arthropoda/ Crustacea Vertebrate/ Chordata/ Fish Invertebrate/ Arthropoda/ Insect

Classification

Table 1. In vivo cytotoxicity and genotoxicity effects of silver nanoparticles in different organismsa

Genotoxicity of silver nanoparticles


Tail DNA%, Quantification of damage. It must be noted that many of the systems referred to in Table 1 are not validated.

mice

870 wileyonlinelibrary.com/journal/jat

a

Reference Genotoxicity

Tail DNA% (170 mg ml 1), 12 h, 32.8% 24 h, 26.3% Tail DNA% (negative control) 16.2% 1

5 and 3.3 mg ml (24 h)

Cytotoxicity (LD50) Synthesis

Reverse micelles, AOT [bis-(2-ethylhexyl) sodium sulphosuccinate, sodium salt,quercetin] with anionic surfactant (AOT) 3–15 BALB

Vertebrate/ Chordata/ Mammalian

Classification

Table 1. (Continued)

Organism

Size (nm)

Ordzhonikidze et al., 2009

R. de Lima et al. chronic toxicity on larvae development and reproduction was significantly decreased upon exposure to 1 mg ml 1. The genotoxicity (comet assay) was found to be dose-dependent. The gene expression, which was assessed by differential display PCR, based on the annealing control primer technique and confirmed by real-time PCR, showed CrGbRH1, CrBR2.2 upregulated and CrL15 downregulated genes. The last was observed to affect the ribosomal assembly and consequently protein synthesis. The observed impairment in the larvae reproduction was attributed to upregulation of the gonadotrophin release hormone gene (CrGnRH1). Similarly, upregulation of the Balbiani ring protein gene (CrBR2.2) was considered an indication of the protection of the larvae against the effects of silver nanoparticles (Nair et al., 2011). In another study, an in vivo genotoxicity test of silver nanoparticles (size 18 nm, generated by evaporation/condensation using a small ceramic heater) was carried out. Male and female Sprague– Dawley rats were exposed to silver nanoparticles (0.8 mg ml 1) by inhalation for 90 days with a 6 h per day dose (OECD 474, in vivo micronuclei test). Twenty-four hours after the last administration, bone marrows were collected to evaluate micronucleus induction. All the results showed no genetic toxicity in male or female rat bone marrow at 0.8 mg ml 1 concentration (Kim et al., 2011). However, cytotoxicity and genotoxicity of silver nanoparticles in the root tip cells of Allium cepa as an indicator organism were observed. Tip cells were treated with different concentrations of engineered silver nanoparticles (Sigma, below 100 nm size). The results showed that silver nanoparticles could penetrate the plant system and may impair stages of cell division causing chromatin bridge, stickiness, disturbed metaphase, multiple chromosomal breaks and cell disintegration at 100 mg ml 1 (mitotic index: control, 55–65%; at 25 mg ml 1, 33–40%; at 50 mg ml 1, 36–40%; at 100 mg ml 1, 25–30%; Kumari et al., 2009). Taken together, these different papers indicate that daphnia is a more susceptible organism to genotoxicity, compared with others organisms. Moreover, it can be concluded that the potential toxicity of silver nanoparticles, either in animals or in plants apparently depends on nanoparticle size. In the case of evaluation of genotoxicity of cellular cultures, the results were found to be similar for control and treated groups, and mammalian cells were found to be more resistant to silver nanoparticles, compared with other culture cells.

Capped Silver Nanoparticles Chemical/capped Synthesis A study of the genotoxicity of coated silver nanoparticles (synthesized by reduction of silver ions by a solution of polysaccharides from acacia gum of 25 nm) and uncoated silver nanoparticles (plasma synthesized silver nanoparticles of 25 nm treated with hydrocarbons to avoid sintering) on mammalian cells was reported. A previous study of the effects of silver nanoparticles on immortalized rat liver cells (BRL 3A) showed no toxic effect up to 10 mg ml 1 of silver nanoparticles, and only 50 mg ml 1 of silver nanoparticles showed toxic effects (Hussain et al., 2005). In a subsequent study, the authors decided to investigate possible toxic effects of silver nanoparticles on mouse embryonic stem (mES) cells and mouse embryonic fibroblasts (MEF) (Ahmed et al., 2008). It was reported that 50 mg ml 1 of silver nanoparticles led to upregulation of the cell cycle checkpoint protein p53, DNA damage repair proteins Rad51, and phosphorylated -H2AX expression. Both


J. Appl. Toxicol. 2012; 32: 867–879

Genotoxicity of silver nanoparticles

J. Appl. Toxicol. 2012; 32: 867–879

In a similar study, the genotoxicity of uncapped silver nanoparticles (size 20–50 nm), synthesized by the citrate method, was found to be weak on calf thymus DNA (ctDNA; at nanoparticle concentrations of 3.3 mg ml 1). However, capped silver nanoparticles (size 10–20 nm) with the detergent cetylpyridine bromide (CPB) showed genotoxicity (Table 1). The methodology followed in this case was simulated in vitro using resonance light scattering (RLS), absorption spectra and transmission electron microscopy (TEM), where the assembly of the contaminants on ctDNA was considered a sign of genotoxicity. The combined materials had a strong co-effect on ctDNA at a concentration of 3.3 mg ml 1 of silver nanoparticles and 6.0 mmol ml 1 of CPB. ctDNA addition to the silver nanoparticles–CPB system showed scattering and diameter decreases, which indirectly revealed that this complex system had genotoxicity (Chi et al., 2009). A toxicological evaluation of changes in cell morphology, cell viability, metabolic activity and oxidative stress of normal human lung fibroblast cells (IMR-90) and human glioblastoma cells (U251) was evaluated by incubation with starch-coated silver nanoparticles (in the size of 6–20 nm synthesized by sodium borohydride, followed by addition of potatoe starch). Mitochondrial damage through reactive oxygen species (15–20% decrease of ATP level on both cells at 50 mg ml 1 of nanoparticles) and DNA damage (tail moments at a 50 mg ml 1 of IMR-90 and U251 were ~5 and ~10 mm, respectively) were more prominent in the cancer cells compared with normal cells. The micronucleus study showed that the number of micronucleus responses in U251 cells was higher than in IMR-90 cells (100 mg ml 1). TEM demonstrated the presence of internal silver nanoparticles in the cells. The nanoparticle treatment caused cell cycle arrest in the G2/M phase, possibly owing to repair of damaged DNA, and the Annexin-V propidium iodide method showed no massive apoptosis or necrosis. The normal fibroblast cells showed 16% late apoptosis plus necrosis, 59% live cell and 25% early apoptosis at a 50 mg ml 1 nanoparticle concentration. The authors suggested that the toxicity mechanisms observed could be attributed to the disruption of the mitochondrial respiratory chain, disruption of ATP synthesis, and DNA damage (AshaRani et al., 2009). A comparison of toxicity and genotoxicity of silver nanoparticles (size 3–15 nm) reverse micelle, AOT [bis-(2-ethylhexyl) sodium sulphosuccinate, sodium salt, quercetin] with anionic surfactant (AOT), and silver ions on injected mice (BALB/c line) in vivo has been reported. Toxic effects (12–24 h death) at two maximum doses of silver nanoparticles (5 and 3.3 mg ml 1) were observed. The LD50/30 values for silver nanoparticles and AOT were 3 mg ml 1 and 30 mmol ml 1, respectively. The frequencies of abnormal sperm heads were similar for silver nanoparticles or AOT, but both were significantly higher than those found with silver ions, and in the control mice group. Comet assay showed an increase in the DNA percentage in the comet tail in spleen cells after the injection of silver nanoparticles and AOT in concentrations of half of LD50/30 values. Tail DNA% was found to be 32.8 and 26.3% (170 mg ml 1), respectively, against the value of 16.2% observed for the untreated control group. The authors suggested that the genotoxic effects of the silver nanoparticles were associated with the presence of AOT rather than the particles themselves (Ordzhonikidze et al., 2009). In another study commercial silver nanoparticles (Sigma; sizes 40–100 nm) were filtered and sonicated in the presence of bovine serum albumin. These nanoparticles showed cytotoxicity to cultures of mouse peritoneal macrophage cell line (RAW264.7; 80% survival at 1.6 mg ml 1 at 24 h and 50% at 96 h) by increasing



871

polysaccharide-coated and uncoated silver nanoparticles induced cell death (annexin V protein expression). In a cytotoxicity study with MTT assay, mammalian cells were incubated with coated and uncoated silver nanoparticles. The authors reported that, after 24 h of incubation, cells treated with both coated and uncoated silver nanoparticles at 50 mg ml 1 showed a 50% survival. After 48 h of incubation, cell survival in the presence of uncoated nanoparticles was maintained at 50%; however, for coated silver nanoparticles the cell survival decreased to 20%. Moreover, cells incubated with coated nanoparticles exhibited more severe cell damage compared with cells incubated with uncoated silver nanoparticles. The authors suggested that polysaccharide-coated particles were more individually distributed while agglomeration of the uncoated particles limits the surface area availability and access to membrane bound organelles (Ahmed et al., 2008). In a similar work, the deleterious effects of capped silver nanoparticles were investigated in zebrafish embryos (Danio rerio; AshaRani et al., 2008). Silver nanoparticles, in sizes of 5–20 nm, were synthesized by reduction with sodium borohydride, and they were capped with starch and bovine serum albumin. Following incubation of zebrafish embryos with capped (starch and bovine serum albumin) and uncapped silver nanoparticles, the authors evaluated the toxicological endpoints, such as mortality, hatching, pericardial edema and heart rate. In general, the LD50 was observed between 25 and 50 mg ml 1, and linearity in mortality was not observed at 100 mg ml 1. In addition, no changes were observed in the heart rate and hatching delay in treated embryos up to a dose of 25 mg ml 1. However, the authors reported a decrease in these two biomarkers with an increase in silver nanoparticle concentration over 100 mg ml 1. These observed effects were found to be more significant with the embryos treated with silver nanoparticles capped with serum albumin compared with nanoparticles capped with starch. Besides these observations, at 100 mg ml 1, the phenotypes exhibited abnormal body axes, twisted notochord, slow blood flow, pericardial edema and cardiac arrhythmia. By microscopic techniques the silver nanoparticles were found to be distributed in the brain, heart, yolk and blood of embryos. An increase of apoptosis in the embryos was also observed. The presence of nanoparticles was found to be evident inside the nucleus cells, which led to alterations and breaks in DNA. The authors concluded that silver nanoparticles induced a dose-dependent toxicity, which hindered normal embryo development (AshaRani et al., 2008). A continuous study by AshaRani et al. (2011) was carried out using zebrafish embryos incubated with silver nanoparticles to evaluate their developmental effects. Silver nanoparticles (size 5–35 nm, synthesized by borohydride reduction using polyvinyl alcohol as a capping agent) were used in different concentrations (5–100 mg ml 1). The authors observed that the addition of silver nanoparticles to embryos resulted in a concentration-dependent increase in mortality rate. In fact, silver nanoparticles induced hatching delays, as well as a concentration-dependent drop in heart rate, touch response and axis curvatures (100 mg ml 1). Silver nanoparticles also induced other significant phenotypic changes starting from 25 mg ml 1, and increased silver nanoparticles concentrations led to phenotypic changes, such as pericardial effusion, abnormal cardiac morphology, circulatory defects and absence or malformation of the eyes. Silver nanoparticles in concentrations of 25 and 50 mg ml 1 were taken up by the embryos (AshaRani et al., 2011).

R. de Lima et al.

872

the sub G1 fraction (cellular apoptosis). Further, it was observed that the G1 fraction changed from 48.4% (control) to 56.1% (1.6 mg ml 1), while the G2 fraction decreased in concentration from 3.3% (negative control) to 0.0% (1.6 mg ml 1), probably owing to inhibition of DNA synthesis. To investigate the correlation between oxidative stress and cytotoxicity induced by silver nanoparticles, the intracellular glutathione (GSH) concentration was measured, since GSH acts as antioxidant to the reactive oxygen species in the body. Intracellular GSH was decreased to 81.4% in the control group by 1.6 mg ml 1 concentration. In order to investigate the relation between inflammation and cytotoxicity caused by silver nanoparticles, the concentration of secreted nitric oxide (NO) was measured, since NO acts as a second messenger in inflammatory signaling. The detected level of NO was found to be 2.3 0.6 mmol l 1 in the control group, and 4.6 0.9 mmol l 1 in the cultured cells group treated with silver nanoparticles at 1.6 mg ml 1 concentration. These results together show that silver nanoparticles influenced significantly the intracellular GSH level and NO secretion. In addition, increased TNF-a in protein and gene levels and increased gene expression of matrix metalloproteinases (MMP-3, MMP-11, and MMP-19) were found. The silver nanoparticles were observed in the cytosol of the activated cells and the culture medium, contrary to the damaged cells. The authors suggested that silver nanoparticles phagocyted by culture of RAW cells induced cytotoxicity to the cells by ionization of the particles. Phagocytosis of silver nanoparticles induced release of TNF-a, causing damage to cell membranes, and apoptosis. The authors suggest that, inside the cells, ionization of silver nanoparticles is responsible for the observed toxic effects, as probably expressed by the Trojan horse-type mechanism (Park et al., 2010). Glycolipid-conjugated silver nanoparticles (sizes 15–20 nm) were biosynthesized by sophorolipid produced by Candida bombicola and oleic acid, and tested for their cytotoxicity using the MTT assay. These nanoparticles were found to be cytocompatible up to 13 mg ml 1 of silver nanoparticle concentrations in the human hepatocellular cell line (HepG2), with a cell survival of 58%. The effect of these silver nanoparticles on the DNA of HepG2 cells was determined by comet assay. The authors reported that, either for cells treated with up to 13 mg ml 1 of silver nanoparticles or for untreated cells, a similar extent of percentage tail DNA was observed, suggesting nongenotoxic effects of silver nanoparticles at these concentrations (Singh et al., 2010). In another study, cyto-, geno- and photo-toxicity of surface coating silver nanomaterials with different shapes were investigated in human skin HaCaT keratinocytes. Silver nanospheres (30 nm diameter prepared by citrate reduction method) and silver nanoprisms [30 nm diameter prepared using a two-step seed mediated procedure using polyvinylpyrrolidone (PVP), in which the first step involved citrate/borohydride and the second step involved citrate/ascorbic acid in PVP solutions] were studied. It was found that the citrate-coated colloidal silver nanoparticles at the concentration of 100 mg ml 1 were not geno-, cyto- and phototoxic. However, silver nitrate was genotoxic even at 10 mg ml 1. The citrate-coated powder form of silver nanoparticles was also found to be toxic. Other silver nanoparticle sizes between 20 and 80 nm were tested and they did not have toxicity. Therefore, the authors suggested that coating silver nanoparticles with a biodegradable polymer prevents the toxicity of the powder (Lu et al., 2010). In a similar study, a commercial silver nanoparticle powder was capped with 0.2% PVP by plasma-enhanced chemical vapor deposition, leading to PVP-coated silver nanoparticles at a size of 78 nm. The toxicity of these nanoparticles was evaluated in the human


lung carcinoma cell line (A549), and compared with silver ions. The results showed a dose-dependent cellular toxicity of PVPcapped silver nanoparticles compared with silver ions as evaluated by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT assay) (IC50 of 12.5 and 5 mg ml 1 for silver nanoparticles and silver ions, respectively) and the annexin V/propidium iodide (at 10 mg ml 1, 34% live cells, 6% apoptosis, 60% necrosis) assay. Moreover, an attenuation of cytotoxicity of both compounds in the presence of the antioxidant N-acetyl-cysteine was reported. This fact correlated with the reactive oxygen species levels and mitochondrial damage or early apoptosis. Indeed, at 10 mg ml 1 concentration level, in the absence of cysteine derivative, silver nanoparticles and silver ions suppressed the mitochondrial activity by 40 and 80%, respectively. In the presence of N-acetyl-cysteine, the suppressions were similar in both cases (20%). At 1 mg ml 1 concentration, no significant effect was observed. The authors suggested that silver nanoparticles mediated reactive oxygen species-induced genotoxicity (Foldbjerg et al., 2011). Finally, cytotoxicity (trypan blue exclusion test and fluoresceindiacetate test) and DNA damage (comet assay and chromosomal aberration test on impairment) of silver nanoparticles (from Sigma, sonicated and capped with bovine serum albumin in sizes of 25–67 nm) were studied in human mesenchymal stem cells (hMSCs). Silver nanoparticles found in the cytoplasm were also monitored. Cytotoxic effects (around IC50 of 10 mg ml 1 at times of 1, 3 and 24 h), comet assay and chromosomal aberration tests indicated DNA damage (at 0.1 mg ml 1). Cytotoxic parameters such as released IL-6, IL-8 and VEGF were found to be increased at the dose of 1 mg ml 1 of silver nanoparticles, indicating hMSC activation, while at a dose of 10 mg ml 1 of nanoparticles, secreted levels of IL-6 and VEGF were found to be decreased. The authors demonstrated the cyto- and genotoxic potential of silver nanoparticles on hMSCs, under the studied conditions (Hackenberg et al., 2011). Therefore, taking all these studies together, silver nanoparticles can lead to geno- and cyto-toxicity in a dose-dependent manner. Moreover the chemical nature of the capping agent on the nanoparticle surface was found to be very important, since some capping agents are more toxic than others. Capped Silver Nanoparticles: Biogenic Nanoparticles Protein capped silver nanoparticles (24–55 nm) acting on Allium cepa at 10 mg ml 1 showed no genotoxic effects. Genotoxicity was only observed at 20–80 mg ml 1 of nanoparticles, and it was prevented by the presence of antioxidants (Panda et al., 2011). Similarly, no genotoxicity was observed owing the presence of protein capped silver nanoparticles (25–45 nm) at a level of 50 mg ml 1 acting on human lymphocytes. Only higher doses of nanoparticles (over 100–400 mg ml 1) showed increased DNA damage (Sarkar et al., 2011). Protein Capped Biogenic Synthesis Silver nanoparticles (size 24–55 nm, synthesized biogenically using the broth of aromatic spath of male inflorescence of screw pine, Pandanus odorifer) and commercial silver nanoparticles (from Sigma, size 70 nm) were incubated with Allium cepa. Up to 10 mg ml 1 of nanoparticles, no effects on genotoxicity were observed for both types of nanoparticles. The evaluation of biomarkers, including the generation of reactive oxygen species (ROS; O2 . and H2O2), cell death, mitotic index, micronucleus, mitotic aberrations and DNA damage (evaluated by the comet


J. Appl. Toxicol. 2012; 32: 867–879

Genotoxicity of silver nanoparticles assay at 20–80 mg ml 1) showed that biogenic silver nanoparticles and commercial silver nanoparticles from Sigma exhibited similar biological effects. Both nanoparticles (biogenic and commercial) caused lesser cytotoxicity and greater genotoxicity compared with silver ions alone. It has been reported that the potential to induce cell death in root tissue of A. cepa of different chemical forms of silver follows the order: silver ions > colloidal silver chloride > commercial silver nanoparticles from Sigma biogenic silver nanoparticles. Cell death and DNA damage induced by biogenic silver nanoparticles were prevented by both Tiron and dimethyl thiourea, which scavenge O2 and H2O2, respectively. The authors suggested a role for ROS in silver nanoparticle-induced cell death and DNA damage (Panda et al., 2011). In another work, the toxicity of biogenic silver nanoparticles produced by Alternaria alternate, capped with protein, and in sizes of 25–45 nm, was evaluated for DNA damage in human lymphocytes using comet assay. The trypan blue dye exclusion method showed no significant changes in cellular viability on exposed cells compared with untreated control cells (up to 400 mg ml 1). The in vitro treatment of lymphocytes using comet assay for DNA damage evaluation showed that, up to 50 mg ml 1 of nanoparticles, no DNA damage was observed. However, over 100 mg ml 1, with the increase in the concentration of silver nanoparticles an increase in DNA damage was observed up to 300 mg ml 1, as represented in terms of percentage of DNA in the tail and olive tail moment test. The values of comet parameters were ~5-fold higher in the positive control (100 mmol l 1 methyl methanesulphonate) compared with the lowest treatment dose (Sarkar et al., 2011).

Effects of Silver Nanoparticles Administrated In Vitro and In Vivo Culture Cells

J. Appl. Toxicol. 2012; 32: 867–879

It was observed that the genotoxicity of silver nanoparticles alone (sizes 20–50 nm, synthesized by citrate reduction) was weak on calf thymus DNA (ctDNA); however, in the presence of detergent (cetylpyridine bromide), the nanoparticles showed significant genotoxicity, mainly owing to the presence of the surfactant (Chi et al., 2009). This result is important from an environmental viewpoint, since considerable amounts of silver nanoparticles are eliminated to water (rivers, oceans) and probably readily interact with surfactant present in the water, leading to a strong genotoxicity owing to the formation of silver nanoparticle–detergent interactions.

In Vivo Assays Another tendency observed from the recent literature related to in vivo studies is that mice were found to be more sensitive than fish to capped silver nanoparticles. For instance, silver nanoparticles capped with starch (sizes 8–15 nm), or capped with bovine serum albumin (sizes 10–20 nm; AshaRani et al., 2008) or with polyvinyl alcohol (sizes 5–35 nm; Asharani et al., 2011) showed no genotoxicity on zebrafish embryos up to nanoparticle concentrations of 25 mg ml 1. However, an increase in the nanoparticle concentration to >100 mg ml 1 led to genotoxicity, which was found to be more significant for albumin-capped silver nanoparticles than for other capped nanoparticles, with a concomitant increase in apoptosis in the embryos and abnormalities. In the case of mice injected with silver nanoparticles capped with anionic surfactant (sizes 3–15 nm) an LD50 value of 3 mg ml 1 and high frequencies of abnormal sperm heads, as well as DNA damage, were observed after the injection of silver nanoparticles in concentrations of half of the LE50/30 values. Apparently, the anionic surfactant was found to be more genotoxic than uncapped silver nanoparticles; however, at low nanoparticle dose, such as 1 mg ml 1, this effect was very discrete (Ordzhonikidze et al., 2009). Johnston et al. (2010) stated that there is limited information available related to pulmonary toxicity of silver nanoparticles (Sung et al., 2009; Hyun et al., 2008; Ji et al., 2007; Table 3). The pro-inflammatory and oxidative potentials of silver nanoparticles within the lung are known to drive nanoparticle toxicity, and so the applicability of silver nanoparticles requires more investigation. Apparently the exposure time to nanoparticles, their concentrations, routes of applications and particle sizes are of paramount importance to define nanoparticle toxicity. Silver nanoparticle translocation from the exposure site of application and their potential accumulation within several secondary targets, including liver, spleen and brain, following pulmonary exposure, were reported. Johnston et al. (2010) also reported that, at the present moment, it is not possible to state that the silver content of any cell distribution is due to silver nanoparticles or to silver ions, since the methods used do not discriminate between them. In biological systems both forms can exist owing to degradation and/or metabolism of nanoparticles. Therefore, further studies are required in this field. Another interesting use of silver nanoparticles is dermatological applications, such as wound dressings containing silver nanoparticles. The same nanoparticles can have different biological behaviors in wounded skin compared with normal skin. It is important to define clearly the hazards associated with dermal exposure to silver nanoparticles, since the liver has been



873

No genotoxicity effects were observed for different human culture cells owing to incubation with up to 10 mg ml 1 of capped silver nanoparticles (diameters of 6–80 nm; Hussain et al., 2005; AshaRani et al., 2008; Singh et al., 2010; Lu et al., 2010; Foldbjerg et al., 2011). However, some cases described in the literature, e.g. culture of human mesenchymal stem cells incubated with 0.1 mg ml 1 of albumin-capped silver nanoparticles (average size 46 nm; Hackenberg et al., 2011) or of normal human lung fibroblast cells, or human glioblastoma cells starch incubated with capped silver nanoparticles (sizes 6–20 nm; AshaRani et al., 2008), showed genotoxicity up to nanoparticle doses of 50 mg ml 1. Albumin-capped silver nanoparticles were reported to be more genotoxic than polysaccharide-capped ones. Indeed, silver nanoparticles capped with albumin (size 70 nm) were found to be more genotoxic on a mouse peritoneal macrophage cell line (genotoxicity at around 2 mg ml 1; Park et al., 2010) compared with silver nanoparticles capped with polysaccharides (size 25 nm) on mouse embryonic stem and fibroblasts, which exhibited genotoxicity at 50 mg ml 1 (Ahmed et al., 2008). In addition, from the reviewed literature, there is a tendency for human culture cells treated with capped silver nanoparticles to be less sensitive than mouse culture cells, independent of the kind of capped silver nanoparticles, with some exceptions. This tendency should be investigated in further studies.

Calf Thymus DNA

874


Allium cepa 5000 cells OLHN12 (medaka fish) 100 metaphases Primary trout hepatocytes n = 3, each treatment Caco-2 and C3A n = 3, each treatment BRL 3A rat liver cells

Plantae/Liliopsida

RAW264.7 mouse peritoneal macrophage

mES mouse embryonic stem cells

MEF mouse embryonic fibroblasts

Vertebrate/Chordata/ mammalian



Vertebrate/Chordata/ mammalian Vertebrate/Chordata/ mammalian

Vertebrate/ Chordata/fish

Vertebrate/ Chordata/fish

Allium cepa 5000 cells

No organism Simulation in vitro (CPB) Allium cepa 5000 cells

Cell

Plantae/Liliopsida

Plantae/Liliopsida

No organism

Classification


25

25

Reduction of silver ions by solution of polysaccharides from acacia gum

Reduction of silver ions by solution of polysaccharides from acacia gum

Reduction of silver ions by solution of polysaccharides from acacia gum Commercial, Sigma

25

Commercial, Sigma

Citrate method

Commercial, Sigma

Biogenically male inflorescence of screw pine, Pandanus odorifer

Commercial, Sigma

Commercial, Sigma

40–100

Cytotoxicity (LC50) Genotoxicity

Hussain et al., 2005

Continues

1.6 mg ml 1, 96 h Park et al., 2010 An increased TNF-a in protein and gene levels, and increased gene expression of matrix metalloproteinases (MMP-3, MMP-11, and MMP-19) were found 50 mg ml 1 50 mg ml 1 Ahmed et al., 2008 This concentration both upregulated the cell cycle checkpoint protein p53 and DNA damage repair proteins Rad51 and phosphorylated -H2AX expression 50 mg ml 1 50 mg ml 1 Ahmed et al., 2008 This concentration both upregulated the cell cycle checkpoint protein p53 and DNA damage repair proteins Rad51 and phosphorylated -H2AX expression

1

Gaiser et al., 2012

C3A = LC50 of 50 mg ml 1–1 Particles were observed in the cells 50 mg ml 1

10 mg ml

Gaiser et al., 2012

Wise et al., 2010

Panda et al., 2011

Panda et al., 2011

Kumari et al., 2009

Chi et al., 2009

Reference

Significant toxicity from 500 mg ml 1 1.000 mg ml 1 did not cause significant lactate dehydrogenase release

100 mg ml 1 20-30% mitotic index causing chromatin bridge, stickiness, disturbed metaphase, multiple chromosomal breaks and cell disintegration ROS-induced cell death and DNA damage doses 20 mg ml 1 Up to 10 mg ml 1 no genotoxic Concentrations 80 mg l 1 were cytotoxic, which was evident from cell death as well as MI Up to 10 mg ml 1 no effects on genotoxicity Comet assay 20–40 mg ml 1. DNA damage doses 10 mg ml 1 1.3 mg ml 1 1.2 mg ml 1 – aneuplidy 15.8%

1

Citrate method 3.3 mg ml silver nanoparticles and 6.0 mg ml 1 CPB cetylpyridine bromide

Synthesis

35

35

20–30

70

24–55

AgNP-Sigma > AgNP-P (biogenic), showing that the least toxic form of silver was capped silver nanoparticles. In addition, it must be noted that nanoparticle size is an important parameter to determine particle toxicity. However, this topic needs more studies to be performed. In this context, there is the need to elaborate standard protocols to carefully analyze nanoparticles toxicity in order to decrease the possible discrepancies related to final conclusions. Thus, this present review illustrates the difficulties of correlating genotoxicity evaluation in cell cultures, organisms or in animals, since the experiments are carried out in different strains or animals treated with different uncapped or capped silver nanoparticles, and also owing to the use of different nanoparticle concentrations. Even so, from the revised literature it is possible to conclude that the most resistant organism to genotoxicity is human cell cultures. The in vivo experiments with mice and humans also demonstrate this fact. Overall, biogenic silver nanoparticles appeared to be less genotoxic than other chemically synthesized silver nanoparticles, and it seems that smaller particles present higher toxicity compared with bigger nanoparticles (Ordzhonikidze et al., 2009; Kim et al., 2011). However, more studies are needed to better understand the genotoxicity mechanisms, as well as the correlations between nanoparticles and their impact on the human health and the environment. Acknowledgments Support from FAPESP, CNPq, and INOMAT (MCT/CNPq) is acknowledged.

References

878

Ahmed M, Karns M, Goodson M, Rowe J, Hussain SM, Schlager JJ, Hong Y. 2008. DNA damage response to different surface chemistry of silver nanoparticles in mammalian cells. Toxicol. Appl. Pharmacol. 233: 404–410. Arora S, Jain J, Rajwade JM, Paknikar KM. 2008. Cellular responses induced by silver nanoparticles: in vitro studies. Toxicol. Lett. 79: 93–100. AshaRani PV, Wu YL, Gong Z, Valiyaveettil S. 2008. Toxicity of silver nanoparticles in zebrafish models. Nanotechnology 19: 255102. AshaRani PV, Mun GLK, Hande MP, Valiyaveettil S. 2009. Cytotoxicity and genotoxicity of silver nanoparticles in human cells. ACS Nano 3: 279–290. AshaRani PV, Lianwu Y, Gong Z, Valiyaveettil S. 2011. Comparison of the toxicity of silver, gold and platinum nanoparticles in developing zebrafish embryos. Nanotoxicology 5: 43–54. Brayner R 2008. The toxicological impact of nanoparticles. Nanotoday 3: 48–55. Brown DM, Wilson MR, MacNee W, Stone V, Donaldson K. 2001. Sizedependent proinflammatory effects of ultrafine polystyrene particles: a role for surface area and oxidative stress in the enhanced activity of ultrafines. Toxicol. Appl. Pharmacol. 175: 191–199. Cha K, Hong HW, Choi YG, Lee MJ, Park JH, Chae HK, Ryu G, Myung H. 2008. Comparison of acute responses of mice livers to short-term


exposure to nano-sized or micro-sized silver particles. Biotechnol. Lett. 30: 1893–1899. Chi Z, Li R, Zhao L, Qin P, Pan X, Sun F, Hao X. 2009. A new strategy to probe the genotoxicity of silver nanoparticles combined with cetylpyridine bromide. Spectrochim. Acta, Part A 72: 577–581. Donaldson K, Stone V, MacNee W. 1999. The toxicology of ultrafine particles. In Particulate Matter Properties and Effects Upon Health, Maynard LA, Howards CA (eds). Bios Scientific: Oxford; 115–127. Duffin R, Tran L, Brown D, Stone V, Donaldson K. 2007. Proinflammogenic effects of low-toxicity and metal nanoparticles in vivo and in vitro: highlighting the role of particle surface area and surface reactivity. Inhal. Toxicol. 19: 849–856. Durán N, Marcato PD, De Conti R, Alves OL, Costa FTM, Brocchi M. 2010. Potential use of silver nanoparticles on pathogenic bacteria, their toxicity and possible mechanism of actionteria, their toxicity and possible mechanisms of action. J. Braz. Chem. Soc. 21: 949–959. Foldbjerg R, Dang DA, Autrup H. 2011. Cytotoxicity and genotoxicity of silver nanoparticles in the human lung cancer cell line, A549. Arch. Toxicol. 85: 743–750. Gaiser BK, Biswas A, Rosenkranz P, Jepson MA, Lead JR, Stone V, Tyler CR, Fernandes TF. 2011. Effects of silver and cerium dioxide micro- and nano-sized particles on Daphnia magna. J. Environ. Monit. 13: 1227–1235. Gaiser BK, Fernandes TF, Jepson MA, Lead JR, Tyler CH, Baalousha M, Biswas A, Britton GJ, Coles PA, Johnston BD, Ju-Nam Y, Rosenkranz P, Scown TM, Stone V. 2012. Environ. Toxicol. Chem 31: 144–154. Griffitt RJ, Luo J, Gao J, Bonzongo J-C, Barber DS. 2008. Effects of particle composition and species on toxicity of metallic nanomaterials in aquatic organisms. Environ. Toxicol. Chem. 27: 1972–1978. Griffitt RJ, Hyndman K, Denslow ND, Barber DS. 2009. Comparison of molecular and histological changes in zebrafish gills exposed to metallic nanoparticles. Toxicol. Sci. 107: 404–415. Gurr JR, Wang AS, Chen CH, Jan KY. 2005. Ultrafine titanium dioxide particles in the absence of photoactivation can induce oxidative damage to human bronchial epithelial cells. Toxicology 213: 66–73. Hackenberg S, Scherzed A, Kessler M, Hummel S, Technau A, Froelich K, Ginzkey C, Koehler C, Hagen R, Kleinsasser N. 2011. Silver nanoparticles: evaluation of DNA damage, toxicity and functional impairment in human mesenchymal stem cells. Toxicol. Lett. 201: 27–33. Hood E. 2004. Nanotechnology, diving into the unknown. Environ. Health Perspect. 112: A747–A749. Hussain SM, Hess KL, Gearhart JM, Geiss KT, Schlager JJ. 2005. In vitro toxicity of nanoparticles in BRL 3A rat liver cells. Toxicol. In Vitro 19: 975–983. Hyun JS, Lee BS, Ryu HY, Sung JH, Chung KH, Yu IJ. 2008. Effects of repeated silver nanoparticles exposure on the histological structure and mucins of nasal respiratory mucosa in rats. Toxicol. Lett. 182: 24–28. Ji JH, Jung JH, Kim SS, Yoon JU, Park JD, Choi BS, Chung YH, Kwon IH, Jeong J, Han BS, Shin JH, Sung JH, Song KS, Yu IJ. 2007. Twentyeight-day inhalation toxicity study of silver nanoparticles in Sprague–Dawley rats. Inhal. Toxicol. 19: 857–871. Johnston HJ, Hutchison G, Christensen FM, Peters S, Hankin S, Stone V. 2010. A review of the in vivo and in vitro toxicity of silver and gold particulates: particle attributes and biological mechanisms responsible for the observed toxicity. Crit. Rev. Toxicol. 40: 328–346. Ju-Nam Y, Lead JR. 2008. Manufactured nanoparticles: an overview of their chemistry, interactions and potential environmental implications. Sci. Total Environ. 400: 396–414. Karlsson LH, Gustafsson J, Cronholm P, Moller L. 2009. Size-dependent toxicity of metal oxide particles. A comparison between nano- and micrometer size. Toxicol. Lett. 188: 112–118. Kim JS, Sung JH, Ji JH, Song KS, Lee JH, Kang CS, Yu IJ. 2011. In vivo genotoxicity of silver nanoparticles after 90-day silver nanoparticle inhalation exposure. Saf. Health Work 2: 34–38. Kim S, Choi JE, Cho J, Chung KH, Park K, Yi J, Ryu DY. 2009a. Oxidative stress-dependent toxicity of silver nanoparticles in human hepatoma cells. Toxicol. In Vitro 23: 1076–1084. Kim Y, Suh HS, Cha HJ, Kim SH, Jeong KS, Kim DH. 2009b. A case of generalized argyria after ingestion of colloidal silver solution. Am. J. Ind. Med. 52: 246–250. Kim YS, Kim JS, Cho HS, Rha DS, Kim JM, Park JD, Choi BS, Lim R, Chang HK, Chung YH, Kwon IH, Han BS, Yu IJ. 2008. Twenty-eight-day oral toxicity, genotoxicity, and gender-related tissue distribution of silver nanoparticles in Sprague-Dawley rats. Inhal. Toxicol. 20: 575–583.


J. Appl. Toxicol. 2012; 32: 867–879

Genotoxicity of silver nanoparticles Kumari M, Mukherjee A, Chandrasekaran N. 2009. Genotoxicity of silver nanoparticles in Allium cepa. Sci. Total Environ. 407: 5243–5246. Lewinski N, Colvin V, Drezedk R. 2008. Cytotoxicity of nanoparticles. Small 4: 26–49. Lima R, Feitosa L, Pereira AES, Moura MR, Aouada FA, Mattoso LHC, Fraceto LF. 2010. Evaluation of the genotoxicity of chitosan nanoparticles for use in food packaging films. J. Food Sci. 75: 89–96. Lu W, Senapati D, Wang S, Tovmachenko O, Singh AK, Yu H, Ray HPC. 2010. Effect of surface coating on the toxicity of silver nanomaterials on human skin keratinocytes. Chem. Phys. Lett. 487: 92–96. Nair PMG, Park SY, Lee SW, Choi J. 2011. Differential expression of ribosomal protein gene, gonadotrophin releasing hormone gene and Balbiani ring protein gene in silver nanoparticles exposed Chironomus riparius. Aquat. Toxicol. 101: 31–37. Nel A, Xia T, Madler L, Li N. 2006. Toxic potential of materials at the nanolevel. Science 311: 622–627. Ng CT, Li JJ, Bay BH, Yung LYL. 2010. Current studies into the genotoxic effects of nanomaterials. J. Nucleic Acid. ID 947859, 2010: 12; doi: 10.4061/2010/947859. Oberdörster E. 2004. Manufactured nanomaterials (fullerenes, C60) induce oxidative stress in the brain of juvenile Largemouth Bass. Environ. Health Perspect. 112: 1058–1062. Oberdörster G, Oberdörster E, Oberdörster J. 2005. Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ. Health Perspect. 113: 823–839. Ordzhonikidze CG, Ramaiyya LK, Egorova EM, Rubanovich AV. 2009. Genotoxic effects of silver nanoparticles on mice in vivo. Acta Naturae (Russia) 3: 99–101. Panda KK, Achary VMM, Krishnaveni R, Padhi BK, Sarangi SN, Sahu SN, Panda BB. 2011. In vitro biosynthesis and genotoxicity bioassay of silver nanoparticles using plants. Toxicol. In Vitro 25: 1097–1105. Park AEJ, Yi J, Kim Y, Choi K, Park K. 2010. Silver nanoparticles induce cytotoxicity by a Trojan-horse type mechanism. Toxicol. In Vitro 24: 872–878. Park BSY, Choi J. 2010. Geno- and ecotoxicity evaluation of silver nanoparticles in freshwater crustacean Daphnia magna. Environ. Eng. Res. 15: 23–27. Rahman MF, Wang J, Patterson TA, Saini UT, Robinson BL, Newport GD, Murdock RC, Schlager JJ, Hussain SM, Ali SF. 2009. Expression of genes related to oxidative stress in the mouse brain after exposure to silver-25 nanoparticles. Toxicol. Lett. 187: 15–21. Rico CM, Majumdar S, Duarte-Gardea M, Peralta-Videa JR, Gardea-Torresdey JL. 2011. Interaction of nanoparticles with edible plants and their possible implications in the food chain. J. Agric. Food Chem. 59: 3485–3498. Samberg ME, Oldenburg SJ, Monteiro-Riviere NA. 2010. Evaluation of silver nanoparticle toxicity in skin in vivo and keratinocytes in vitro. Environ. Health Perspect. 118: 407–413.

Sarkar J, Chattopadhyay D, Patra S, Deo SS, Sinha S, Ghosh M, Mukherjee A, Acharya K. 2011. Alternaria alternata mediated synthesis of protein capped silver nanoparticles and their genotoxic activity. Dig. J. Nanomat. Biostruct. 6: 563–573. Seabra AB, Durán N. 2010. Nitric oxide-releasing vehicles for biomedical applications. J. Mater. Chem. 20: 1624–1637. Shrivastava S, Bera T, Singh SK, Singh G, Ramachandrarao P, Dash D. 2009. Characterization of antiplatelet properties of silver nanoparticles. ACS Nano 3: 1357–1364. Sing S, Patel P, Jaiswal S, Prabhune AA, Ramana CV, Prasad BLV. 2009. A direct method for the preparation of glycolipid–metal nanoparticle conjugates: sophorolipids as reducing and capping agents for the synthesis of water re-dispersible silver nanoparticles and their antibacterial activity. New J. Chem. 33: 646–652. Singh S, D’Britto V, Prabhune AA, Ramana CV, Dhawan A, Prasad BLV. 2010. Cytotoxic and genotoxic assessment of glycolipidreduced and -capped gold and silver nanoparticlesw. New J. Chem. 34: 294–301. Sung JH, Ji JH, Park JD, Yoon JU, Kim DS, Jeon KS, Song MY, Jeong J, Han BS, Han JH, Chung YH, Chang HK, Lee JH, Cho MH, Kelman BJ, Yu IJ. 2009. Subchronic inhalation toxicity of silver nanoparticles. Toxicol. Sci. 108: 452–461. Takenaka S, Karg E, Roth C, Schulz H, Ziesenis A, Heinzmann U, Schramel P, Heyder J. 2001. Pulmonary and systemic distribution of inhaled ultrafine silver particles in rats. Environ. Health Perspect. 109: 547–551. Tian J, Wong KK, Ho CM, Lok CN, Yu WY, Che CM, Chiu JF, Tam PK. 2007. Topical delivery of silver nanoparticles promotes wound healing. Chem. Med. Chem. 2: 129–136. Trop M, Novak M, Rodl S, Hellbom B, Kroell W, Goessler W. 2006. Silver coated ressing Acticoat caused raised liver enzymes and argyria-like symptoms in burn patient. J. Trauma 60: 648–652. Vlachou E, Chipp E, Shale E, Wilson YT, Papini R, Moiemen NS. 2007. The safety of nanocrystalline silver dressings on burns: a study of systemic silver absorption. Burns 33: 979–985. Wadhera A, Fung M. 2005. Systemic argyria associated with ingestion of coloidal silver. Dermatol. Online J. 11: 12. Available at: http://derma tology. cdlib.org Wijnhoven SWP, Peijnenburg WJGM, Herberts CA, Hagens WI, Oomen AG, Heugens EHW, Roszek B, Bisschops J, Gosens I, Van De Meent D, Dekkers S, De Jong WH, van Zijverden M, Sips AJAM, Geertsma RE. 2009. Nano-silver – a review of available data and knowledge gaps in human and environmental risk assessment. Nanotoxicology 3: 109–138. Wise Sr JP, Goodale BC, Wise SS, Craig GA, Pongan AF, Walter RB, Thompson D, Ng AK, Aboueissa AM, Mitani H, Spalding MJ, Mason MD. 2010. Silver nanospheres are cytotoxic and genotoxic to fish cells. Aquat. Toxicol. 97: 34–41.

879

J. Appl. Toxicol. 2012; 32: 867–879

