Chemosphere 85 (2011) 3442

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Chemosphere
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Differential toxicity of silver and titanium dioxide nanoparticles on Drosophila melanogaster development, reproductive effort, and viability: Size, coatings and antioxidants matter
Ryan Posgai a, Caitlin B. Cipolla-McCulloch a, Kyle R. Murphy a, Saber M. Hussain b, John J. Rowe a, Mark G. Nielsen a,
a b

Department of Biology, Center for Tissue Regeneration and Engineering, University of Dayton, Dayton, OH 45469, USA Applied Biotechnology Branch, Human Effectiveness Directorate, Air Force Research Laboratory, WrightPatterson Air Force Base, Dayton, OH 45433, USA

a r t i c l e

i n f o

a b s t r a c t
Silver and titanium dioxide nanoparticles are known to induce oxidative stress in vitro and in vivo. Here we test if they impact development, mating success, and survivorship in Drosophila melanogaster, and if so, if these effects are reversible by antioxidants. Ingestion of nanotitanium dioxide during the larval stage of the life cycle showed no effects on development or survivorship, up to doses of 200 lg mL1. Conversely, ingestion of nanosilver had major dose, size, and coating-dependent effects on each of these aspects of life history. Each of these effects was partially or fully reversible by vitamin C. Larvae growing on nanosilver supplemented with vitamin C showed a greater than twofold increase in survivorship compared to ies reared on nanosilver alone, and a threefold increase in mating success. Vitamin C also rescued cuticular and pigmentation defects in nanosilver fed ies. Biochemical assays of superoxide dismutase and glutathione show these markers respond to nanotitanium dioxide and nanosilver induced oxidative stress, and this response is reduced by vitamin C. These results indicate that life history effects of nanosilver ingestion result from oxidative stress, and suggest antioxidants as a potential remediation for nanosilver toxicity. Conversely, the lack of nanotitanium dioxide life history toxicity shows that oxidative stress does not necessarily result in whole organism effects, and argues that nanoparticle toxicity needs to be examined at different levels of biological organization. 2011 Elsevier Ltd. All rights reserved.

Article history: Received 18 February 2011 Received in revised form 25 May 2011 Accepted 5 June 2011 Available online 5 July 2011 Keywords: Silver nanoparticles Titanium dioxide nanoparticles Nanoparticle coating Oxidative stress Drosophila Vitamin C

1. Introduction Nanoparticle-based technologies are a rapidly growing facet of our industrial, medical and environmental economies. Their toxicological properties, different from bulk form, have been subject to intensive research (Oberdrster et al., 2005; Nel et al., 2006; Rivera Gil et al., 2010). Silver and titanium dioxide nanoparticles, used in clothing, food industry, paints, cosmetics, electronics, coatings and medical products, are of particular concern because they are two of the fastest growing product categories in the nanotechnology industry (Cheng et al., 2004; Cohen et al., 2007; Lee et al., 2007; Vigneshwaran et al., 2007). Oxidative stress is an emerging, general mechanism underlying nanoparticle toxicity (Nel et al., 2006; Mocan et al., 2010). Previous work in our lab (Ahamed et al., 2010) nds that nanosilver ingestion in Drosophila larvae activates oxidative stress pathways, including superoxide dismutase (SOD), catalase, caspase-3 and caspase-9, glutathione and malondialdehyde (MDA), a product of lipid
Corresponding author. Tel.: +1 937 229 2587; fax: +1 937 229 2021.
E-mail address: Mark.Nielsen@notes.udayton.edu (M.G. Nielsen). 0045-6535/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2011.06.040

peroxidation. Titanium dioxide is best known for reactive oxygen species (ROS) production via photoactivation (Hirakawa et al., 2004), however, studies nd ROS production from both rutile and anatase in the absence of photoactivation (Gurr et al., 2005; Braydich-Stolle et al., 2009). Yet to be determined is whether oxidative stress has effects on the whole organism, in terms of development, reproduction, and survival. The lowest level of cellular response to oxidative stress entails the induction of antioxidant molecules and detoxication enzymes via Nrf-2 activation (Xiao et al., 2003). Thus, acute exposures may have only transient cellular effects that do not impact the health of the organism. Conversely, sustained ROS production can lead to inammation, circulatory problems, and DNA damage that could ultimately impact the life history of the organism through effects on development, viability, and/or reproductive effort. There are few in vivo studies on life history effects of chronic titanium dioxide and silver nanoparticle exposure, almost all in aquatic organisms (reviewed in Menard et al., 2011). Such studies are needed, both to determine the potential for life history toxicity in mammalian models, and the potential for environmental damage through their extensive commercial production and consumer

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use. Drosophila melanogaster provides a relevant model for investigating human health, as counterparts of genes responsible for more than 700 different human genetic diseases including neurological, immunological, cardiovascular, auditory, visual, developmental and metabolic disorders are found in Drosophila (Reiter et al., 2001; Koh et al., 2006; Wolf et al., 2006; Khurana et al., 2006). Moreover, the Nrf-2 gene and its function are conserved in Drosophila, making it specically relevant to model oxidative stress responses (Sykiotis and Bohmann, 2008). The cost effectiveness, experimental exibility, and short generation time of Drosophila permit rapid in vivo assessment of the vast number of nanoparticles being produced, including life history effects of chronic exposure. We nd that nanosilver ingestion had negative, dose-dependent effects on survivorship, mating success and development, with smaller, uncoated particles showing the greatest toxicity. Conversely, titanium dioxide had no effect on y life history. Vitamin C ingestion partially or fully reversed each of these life history effects of nanosilver ingestion, and reduced the response of oxidative stress markers SOD and glutathione (GSH). While antioxidant reversal of nanoparticle induced stress has been observed in vitro (Kim et al., 2009; Akhtar et al., 2010; Sharma et al., 2010; Foldbjerg et al., 2011), this is the rst in vivo study that shows nanoparticle toxicity can be alleviated by antioxidants. The in vivo reversal of each facet of nanosilver toxicity indicates that the primary mechanism of nanosilver toxicity is oxidative stress. The lack of nanotitanium dioxide life history toxicity provides a caution that biochemical toxicity does not necessarily extrapolate to whole organism toxicity.

2. Experimental procedures 2.1. Nanoparticles used in this study Uncoated and poly-saccharide-coated 10 nm and 60 nm silver particles were generously provided by Dr. Dan Goia (Clarkson University Center for Advanced Materials Processing, Potsdam, NY). The coated silver nanoparticles were synthesized by the reduction of silver ions in a solution of a poly-saccharide (acacia gum), which leads to surface coating. Nanoparticles were diluted from bulk concentrations to 1 mg mL1 working stock concentrations and sonicated for 1 min at 3540 W to aid in mixing and forming a homogeneous dispersion.

2.2. Nanoparticle characterization Transmission electron microscopy (TEM) characterization was performed to determine nanoparticle size and morphology using a Hitachi H-7600 tungsten-tip instrument at an accelerating voltage of 100 kV. Nanoparticles were examined after deposition of nanoparticles suspensions onto carbon lm-coated Cu TEM grids. The AMT software for the digital TEM camera was calibrated for size measurements of the nanoparticles. Information on mean size and SD was calculated using point to point method (Murdock et al., 2008). Dynamic light scattering (DLS) and laser Doppler velocimetry (LDV) were used for characterization of hydrodynamic size and zeta potential (f) of nanoparticles suspended in H2O, performed

Fig. 1. Characterization of silver nanoparticles. (A and B) Represents the TEM characterization of 10 nm uncoated Ag NPs. A total of 190 silver nanoparticles (Ag NPs) were measured by TEM for size distribution. (A) Depicts the morphology of silver nanoparticles and (B) represents frequency of size distribution of Ag NPs. TEM mean SD of Ag NPs was 11.92 3.86 nm. (C and D) Depicts DLS and LDV characterization of 10 nm uncoated Ag NPs. (C) Represents the Ag NPs diameter in water suspension (19.39 nm) and (D) depicts the zeta potential (37.1 mV).

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on a Malvern Instruments Zetasizer Nano-ZS instrument as described by Murdock et al. (2008). 2.3. Fly husbandry OreRS ies were obtained from the Bloomington Stock Center, Bloomington, IN and reared on standard cornmealmaltyeast medium (Bloomington Stock Center recipe) at 25 C. Nanoparticles and vitamin C were added to the media while cooling and decanted into 60 mm 15 mm petri plates for larval feeding experiments. Control plates consisted of 20 mL standard Drosophila cornmeal media. In treatment lines, standard medium was supplemented with nanoparticles resulting in nal suspensions from 5 lg to 200 lg nanoparticles per mL of medium, depending on the experiment. In antioxidant experiments, medium was supplemented with ascorbic acid (vitamin C) or vitamin C palmitate (a lipid-soluble vitamin C) to a nal concentration of 50 mM. 2.4. Survivorship assay Drosophila embryos were laid over a 2 h time period on control medium, 50 of which were collected and moved to control or treatment plates for the assay. Seven plates of 50 embryos each were used for each treatment. Percent survivorship was calculated as the number of embryos that pupated divided by the total number of embryos. The time from rst larval instar hatch to pupation was also recorded. Adult phenotypes were scored following eclosion for cuticle maturation and melanization, two features found to vary with nanosilver dose.

2.5. Mating success assay Virgin male and female ies that survived ingestion of 60 nm uncoated silver during the larval stage were mated in pairs. Twenty pairs were used for each treatment. The number of successful matings (those resulting in offspring) and number of progeny produced were recorded and compared to control lines (mated pairs not exposed to nanosilver during larval development).

2.6. Biochemical assays 2.6.1. Reagents Reduced nicotinamide adenine dinucleotide (NADH), reduced glutathione (GSH), 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB), nitrobluetetrazolium (NBT), and phenazine methosulphate (PMS) were purchased from SigmaAldrich, MO. All other chemicals used were of the highest purity available from commercial sources.

2.6.2. Preparation of crude extracts For glutathione and superoxide dismutase assays, 4 d old third instar larvae were exposed for 24 h on control, 50 lg mL1 Ag NP, 50 lg mL1 TiO2, or 50 lg mL1 Ag NP supplemented with 50 nM vitamin C. Treated larvae were homogenized in 0.1 M sodium phosphate buffer (pH 7.4) containing 0.15 M KCl. Following centrifugation (2300g for 15 min at 4 C), total protein of the supernatant was determined using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL). The supernatant (crude extract) was maintained on ice for use in biochemical assays.

Fig. 2. Characterization of silver nanoparticles. (A and B) Represents the TEM characterization of 60 nm uncoated Ag NPs. A total of 130 silver nanoparticles were measured by TEM for size distribution. (A) Depicts the morphology of silver nanoparticles and (B) represents frequency of size distribution of Ag NPs. TEM mean SD of Ag NPs was 60.27 11.74 nm. (C and D) Depicts DLS and LDV characterization of 60 nm uncoated Ag NPs. (C) Represents the Ag NPs diameter in water suspension (192 nm) and (D) depicts the zeta potential (29.4 mV).

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2.6.3. Glutathione GSH level was quantied using Ellmans reagent (Ellman, 1959). Briey, a mixture of 0.25 mL of 1 mg mL1 crude extract and 0.9 mL of 5% TCA was centrifuged (2300g for 15 min at 37 C). Then 0.5 mL of supernatant added into 1.5 mL of 0.01% DTBN and the reaction was monitored at 412 nm. The amount of GSH was expressed in terms of nmol mg1 protein. 2.6.4. Superoxide dismutase SOD activity was determined employing a modied version (Kakkar et al., 1984) of the method described earlier (Nishikimi et al., 1972). Briey, at room temperature the reaction was initiated by adding 0.2 mL of a 780 lM NADH solution into a reaction mixture containing 1.25 mL of 0.052 M sodium pyrophosphate buffer (pH 8.0), 0.1 mL of 186 lM PMS, 0.3 mL of 300 lM NBT, 0.1 mL of distilled water and 50 lL of 1 mg mL1 crude extract (total volume of 2.0 mL). The reaction was stopped after 1.5 min at room temperature by adding 1.0 mL acetic acid and then 4.0 mL of n-butanol. Following centrifugation (2300g for 15 min at room temperature) the color intensity of chromogen in butanol was measured at 560 nm. A reaction mixture devoid of enzyme served as a control. One unit of enzyme activity is dened as the enzyme concentration required to inhibit chromogen production by 50% in 1 min at room temperature and specic activity expressed as units min1 mg1 protein. 2.7. Statistical analysis The mean and standard deviation of percent survivorship, percent mating success, and time to pupation were calculated and sta-

tistically analyzed using an exact binomial test (Goldstein, 1954). Best-t lines for each treatment were generated using linear regression, and used to determine LD50 (survivorship) or ED50 (mating success). LD50 and ED50 values and biochemical assays were compared to controls using Students t-test (a = 0.05). 3. Results 3.1. Nanoparticle characterization Silver 10 nm coated particles were previously characterized by DLS and TEM (Ahamed et al., 2010) and show good dispersion and uniform size. TEM analyses show that all nanoparticles tested are uniform in size (Figs. 14). DLS and zeta potential analyses indicate the particle suspensions are relatively stable and form agglomerates in water. 3.2. Survivorship is reduced and developmental time extended by nanosilver ingestion Nanosilver ingestion had major, dose-dependent effects on survivorship to pupal stage (Fig. 5). Uncoated particles were more toxic than coated particles, and smaller particles were more toxic than larger particles. Time to pupation was slowed by nanosilver ingestion in a dose-dependent manner (Fig. 6). This effect plateaus around 180 h, apparently extension of the pre-adult lifecycle is not viable beyond this time under our experimental conditions. Neither survivorship nor time to pupation was affected by nanotitanium dioxide (rutile) ingestion at doses up to 200 lg mL1.

Fig. 3. Characterization of silver nanoparticles. (A and B) Represents the TEM characterization of 60 nm poly-saccharide coated Ag NPs. A total of 139 silver nanoparticles were measured by TEM for size distribution. (A) Depicts the morphology of silver nanoparticles and (B) represents frequency of size distribution of Ag NPs. TEM mean SD of Ag NPs was 66.54 15.7 nm. (C and D) Depicts DLS and LDV characterization of 60 nm poly-saccharide coated Ag NPs. (C) Represents the Ag NPs diameter in water suspension (93 nm) and (D) depicts the zeta potential (32.7 mV).

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Fig. 4. Characterization of titanium dioxide nanoparticles. (A and B) Represents the TEM characterization of titanium oxide NPs. A total of 200 titanium dioxide NPs were measured by TEM for size distribution. (A) Depicts the morphology of titanium dioxide NPs and (B) represents frequency of size distribution of titanium dioxide NPs. TEM mean SD of titanium dioxide NPs length was 38.22 12.14 nm and width was 13.53 3.54 nm. (C and D) Depicts DLS and LDV characterization of titanium dioxide NPs. (C) Represents the titanium dioxide NPs diameter in water suspension (357 nm) and (D) depicts the zeta potential (25.5 mV).

Fig. 5. Mean and SD percent survivorship for ies fed different concentrations of nanotitanium dioxide and nanosilver during larval development. Treatment line survivorship is normalized to control lines, which are set at 100% survivorship. LD50 determined from best-t line for each treatment generated by linear regression (LD50 lg mL1, R2): 10 nm Ag coated (32.4, 0.96), 10 nm Ag uncoated (31.0, 0.95), 60 nm Ag coated (47.4, 0.98), 60 nm Ag uncoated (31.2, 0.96). These LD50 values are statistically different from each other (t-test, p < 0.05) except 10 nm coated vs. 60 nm uncoated (p = 0.10). Nanosilver ingestion had dose-, size-, and coating-dependent effects on survivorship. Conversely, nanotitanium dioxide had no effect on survivorship (LD50 763 lg mL1, R2 = 0.99) at doses up to 200 lg mL1 (data point not shown). N = 350 embryos/treatment.

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3.3. Mating success is reduced by nanosilver ingestion Nanosilver ingestion during the larval stage reduced adult mating success (Fig. 7). Lower doses of nanosilver were sufcient to disrupt reproduction compared to viability; ED50 (a dose that results in 50% viable matings compared to control) for uncoated 60 nm silver was 19.6 lg mL1 (R2 = 0.90), about 2/3 that of LD50 for 60 nm uncoated Ag. Many nanosilver-treated ies did not produce offspring, and those that did averaged fewer than half the progeny of control lines. 3.4. Nanosilver ingestion impairs adult cuticle development and melanization Nanosilver ingestion during the larval stage resulted in cuticular and melanization defects in adults (Fig. 8). Flies that survived nanosilver ingestion had a soft, unpigmented cuticle. No such ef-

fect was observed in nanotitanium dioxide-fed ies. As epidermal pigments are secreted by the cuticle (Wright, 1987), the cuticle defect is likely the root cause of these phenotypes. 3.5. Vitamin C ingestion reverses toxic effects of silver ingestion Previous research nds that nanosilver ingestion causes oxidative stress (Ahamed et al., 2010). This suggests the potential for toxicity reversal through antioxidant treatment, which we verify here. Larvae growing on 30 lg mL1 60 nm uncoated silver supplemented with 50 mM vitamin C or vitamin C palmitate showed greater than twofold survivorship and threefold mating success compared to ies reared on nanosilver alone (Fig. 9). Time to pupation was also reduced signicantly (t-test), from 158.3 14.2 for nanosilver alone, to 151.0 18.7 (Ha: vitamin C < Ag; p = 0.003) and 150.0 14.53 (Ha: vit. C palmitate < Ag; p < 0.001). Nanosilver

Fig. 6. Mean and SD time from larval hatch to pupation for ies surviving different doses of nanotitanium dioxide and nanosilver during larval development.

Fig. 7. Mean and SD mating success for adult ies surviving different doses of nanosilver during larval development. Treatment line mating success is normalized to control lines, which are set at 100% mating success. ED50 = 19.6 lg mL1, as determined from best t line generated by linear regression (R2 = 0.90). N = 20 mated pairs/treatment.

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Negative control

30g/mL Ag uncoated

30g/mL Ag uncoated +50mM VitC

30g/mL Ag uncoated +50mM VitC Palmitate

Fig. 8. Larval nanosilver ingestion shows dose-dependent effects on adult melanization and cuticular development. The y in Panel B was fed 30 lg mL1 Ag uncoated during larval development, and shows a soft adult cuticle lacking pigmentation compared to the control in Panel A. The cuticlar and melanization phenotype are rescued by coingestion with vitamin C (Panel C, 30 lg mL1 Ag uncoated + 50 mM vitamin C) and vitamin C palmitate (Panel D, 30 lg mL1 Ag uncoated + 50 mM vitamin C palmitate).

Fig. 9. The toxic effects of nanosilver ingestion on survivorship and reproductive effort are reversed by vitamin C. Mean and SD percent survivorship and mating pair success are presented for larvae reared on standard medium (negative control), 30 lg mL1 60 nm uncoated nanosilver, 30 lg mL1 60 nm uncoated nanosilver + 50 mM vitamin C, and 30 lg mL1 60 nm uncoated nanosilver + 50 mM vitamin C palmitate. Survivorship comparisons between control and each treatment line are signicantly different (ttest, p < 0.01). Survivorship comparisons between nanosilver and nanosilver + vitamin C treatments are signicantly different (t-test, p < 0.001). Mating success comparisons between control and each treatment line are signicantly different (t-test, p < 0.01), with the exception of vitamin C (t-test, p = 0.34). Mating success comparisons between nanosilver and nanosilver + vitamin C treatments are signicantly different (t-test, p < 0.001).

Table 1 Biochemical assays. Treatment SOD assay Units (min mg1 protein) Control TiO2 Ag Ag + vit. C 4.97 6.01 5.77 5.28 Ha, p Ha: TiO2 > C p = 0.0371 Ha: Ag > C p = 0.0004 Ha: Ag + vit. C > C p = 0.0539 Ha: Ag + vit. C < Ag p = 0.0243 GSH assay Nanomole GSH (mg1 protein) 130.28 147.25 30.30 59.99 H a, p Ha: TiO2 < C p = 0.9087 Ha: Ag < C p = 0.0004 Ha: Ag + vit. C < C p = 0.0856 Ha: Ag + vit. C < Ag p = 0.1930

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induced cuticular and melanization defects were also reversed by co-ingestion of vitamin C (Fig. 8c and d). Larvae growing on 30 lg mL1 60 nm uncoated silver supplemented with 50 mM vitamin C showed reduced levels of superoxide dismutase (SOD) and increased, though not statistically signicant, levels of glutathione (GSH) compared to ies reared on nanosilver alone (Table 1). This provides a biochemical mechanism whereby vitamin C ingestion can alleviate the effects on nanosilver on the life history traits examined here. Nanotitanium dioxide had comparable effects to nanosilver on SOD activity; but unlike nanosilver, had no effect on GSH levels.

4. Discussion We show that nanoparticle induced oxidative stress can result in severe life history effects. However, as in the case of TiO2, oxidative stress does not necessarily affect life history. Both biochemical and life history studies are needed to fully characterize nanoparticle toxicity. Both nanotitanium dioxide and nanosilver have been shown to activate oxidative stress, DNA, and mitochondrial damage biochemical pathways (Hirakawa et al., 2004; Hussain et al., 2005; Ahamed et al., 2008, 2010; Jemec et al., 2008; Wang et al., 2008; Sharma, 2009; Jin et al., in press). We nd that these activations are not necessarily toxic to the whole organism. Nanosilver has major, negative effects on each facet of y life history assayed in this study. Conversely, nanotitanium dioxide had no effect on survivorship, developmental rate, or adult cuticle phenotype. This shows that nanoparticles must be assayed at different levels of biological organization to determine their full toxicity prole. Differences in silver and titanium dioxide life history toxicity may simply result from nanosilver showing greater biochemical toxicity. Although the ranges of particle variables relevant to toxicity in the aforementioned studies (different crystal structures, sizes, and shapes) complicate comparisons between nanosilver and nanotitanium dioxide, nanosilver has been shown to induce greater levels of oxidative stress than nanotitanium dioxide, including in a head-to-head study of silver and titanium dioxide nanoparticles comparable to those used in this study (Hussain et al., 2005). While nanotitanium dioxide and nanosilver had comparable effects on SOD levels, nanosilver had a much greater impact on GSH levels than nanotitanium dioxide. GSH is the rst line of defense against oxidative stress, its depletion in nanosilver-fed ies could underlie its greater life history toxicity. Each facet of nanosilver toxicity is partially or wholly remediated by vitamin C. This indicates that oxidative stress is the major contributor to nanosilver toxicity at each level of organization. In Drosophila, oxidative stress pathways have dual function, they remediate oxidative stress but are also targets of the hormone ecdysone, which directs progression through larval stages of molting and pupal metamorphosis. Their activation in response to oxidative stress could disrupt the timing of these lifecycle events, a possible mechanism underlying the developmental phenotypes observed in treated ies. Such dual roles must be considered in evaluating biochemical toxicity in mammalian models. Moreover, oxidative stress may not be the only mechanism underlying nanoparticle toxicity, it is also possible that other mechanisms additional to oxidative stress are important to nanoparticle life history toxicity and underlie the different toxicity of nanotitanium vs. nanosilver. Both aqueous and lipid-soluble forms of vitamin C reversed the effects of nanosilver ingestion. Two mechanisms through which vitamin C can act to reverse nanosilver toxicity are: (1) directly through reduction of reactive oxygen species (superoxide ion O2 , hydroxyl radical OH, and/or hydrogen peroxide H2O2) and/or (2)

through the chelation of silver ions released by silver nanoparticles. Foldbjerg et al. (2011) show in vitro reduction of ROS in human lung cancer cell lines exposed to nanosilver when cells are pretreated with the antioxidant, N-acetyl-cysteine, however, this could result from antioxidant effects on either the nanosilver particle or the ion. Preliminary results in our lab nd that the polysaccharide-coated nanosilver tested here release silver ions at a greater rate than uncoated nanosilver. Yet uncoated silver particles had greater toxicity than the same size coated particles, indicating silver ion release at most makes a small contribution to toxicity. A direct comparison of the efcacy of vitamin C on coated vs. uncoated nanosilver, with a silver ion control would help determine the mechanism of vitamin C reversal of nanosilver toxicity, and more generally nanosilver toxicity. In addition to coating, size and agglomeration state are also relevant to toxicity. Though coated particles show less agglomeration in our DLS measurements, resulting in a relatively higher reactive surface area for the generation of ROS, they are less toxic than the same sized uncoated particle. The nature of the surface is clearly more relevant to toxicity than the amount of surface area available. Although the smaller 10 nm silver particles showed somewhat greater toxicity than equivalently coated 60 nm particles, the smaller particles provide six times more reactive surface area per unit mass, a much greater difference than found in their toxicity, in particular as smaller particles show less agglomeration in DLS measurements. The interaction between coating, size and agglomeration state in toxicity is clearly complex and in need of further study. The lack of 1:1 correspondence between biochemical and life history aspects of nanoparticle toxicity strongly indicates a need to research the impact of biochemical stress on the whole organism, in chronic toxicity models capable of testing for life history effects. Studies that focus on the relationship between in vitro and in vivo effects of oxidative stress may determine threshold values of biochemical ROS generation that result in life history toxicity, which would be relevant to setting exposure standards in industrial and commercial use. Acknowledgments This work was funded by National Science Foundation Grant CBET-0833953, and the Department of Defense Consortium Research Fellows Program. The work was conducted under a CRADA agreement with WPAFB in a shared UD/AFRL laboratory. The authors would also like to thank Mr. Timothy Gorey and Mr. Andrew Lewis from the University of Dayton for help with nanoparticle characterization and statistics respectively. References
Ahamed, M., Karns, M., Goodson, M., Rowe, J., Hussain, S., Schlager, J., Hong, Y., 2008. DNA damage response to different surface chemistry of silver nanoparticles in mammalian cells. Toxicol. Appl. Pharmacol. 233, 404410. Ahamed, M., Posgai, R., Gorey, T.J., Nielsen, M., Hussain, S.M., Rowe, J.J., 2010. Silver nanoparticles induced heat shock protein 70, oxidative stress and apoptosis in Drosophila melanogaster. Toxicol. Appl. Pharmacol. 242, 263269. Akhtar, M.J., Kumar, S., Murthy, R.C., Ashquin, M., Khan, M.I., Patil, G., Ahmad, I., 2010. The primary role of iron-mediated lipid peroxidation in the differential cytotoxicity caused by two varieties of talc nanoparticles on A549 cells and lipid peroxidation inhibitory effect exerted by ascorbic acid. Toxicol. in Vitro 24 (4), 11391147. Braydich-Stolle, L., Schaeublin, N.M., Murdock, R.C., Jiang, J., Biswas, P., Schlager, J.J., Hussain, S.M., 2009. Crystal structure mediates mode of cell death in TiO2 nanotoxicity. J. Nanopart. Res. 11 (6), 13611374. Cheng, D., Yang, J., Zhao, Y., 2004. Antibacterial materials of silver nanoparticles application in medical appliances and appliances for daily use. Chin. Med. Equip. J. 4, 2632. Cohen, M.S., Stern, J.M., Vanni, A.J., Kelley, R.S., Baumgart, E., Field, D., Libertino, J.A., Summerhayes, I.C., 2007. In vitro analysis of a nanocrystalline silver-coated surgical mesh. Surg. Infect. 8, 397403. Ellman, G.l., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 7077.

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R. Posgai et al. / Chemosphere 85 (2011) 3442 Nel, A., Xia, T., Madler, L., Li, N., 2006. Toxic potential of materials at the nanolevel. Science 311 (5761), 622627. Nishikimi, M., Rao, N.A., Yagi, K., 1972. The occurrence of superoxide anion in the reaction of reduced phenazine methosulphate and molecular oxygen. Biochem. Biophys. Res. Commun. 48, 849851. Oberdrster, G., Oberdrster, E., Oberdrster, J., 2005. Nanotoxicology: an emerging discipline evolving from studies of ultrane particles. Environ. Health Perspect. 113, 823839. Reiter, L.T., Potocki, L., Chien, S., Gribskov, M., Bier, E., 2001. A systematic analysis of human disease-associated gene sequences in Drosophila melanogaster. Genome Res. 11, 11141125. Rivera Gil, P., Oberdrster, G., Elder, A., Puntes, V., Parak, W.J., 2010. Correlating physico-chemical with toxicological properties of nanoparticles: the present and the future. ACS Nano 4 (10), 55275531. Sharma, V.K., 2009. Aggregation and toxicity of titanium dioxide nanoparticles in aquatic environment a review. J. Environ. Sci. Health, Part A: Toxic/Hazard. Subst. Environ. Eng. 44 (14), 14851495. Sharma, H.S., Sharma, A., Hussain, S., Schlager, J., Sjquist, P.O., Muresanu, D., 2010. A new antioxidant compound H-290/51 attenuates nanoparticle induced neurotoxicity and enhances neurorepair in hyperthermia. Acta Neurochir. Suppl. 106, 351357. Sykiotis, G.P., Bohmann, D., 2008. Keapl/Nrf2 signaling regulates oxidative stress tolerance and lifespan in Drosophila. Dev. Cell 14, 7685. Vigneshwaran, N., Kathe, A.A., Varadarajan, P.V., Nachane, R.P., Balasubramanya, R.H.J., 2007. Functional nishing of cotton fabrics using silver nanoparticles. Nanosci. Nanotechnol. 7, 18931897. Wang, J., Chen, C., Liu, Y., Jiao, F., Li, W., Lao, F., Li, Y., Li, B., Ge, C., Zhou, G., Gao, Y., Zhao, Y., Chai, Z., 2008. Potential neurological lesion after nasal instillation of TiO(2) nanoparticles in the anatase and rutile crystal phases. Toxicol. Lett. 183 (13), 7280. Wolf, M.J., Amrein, H., Izatt, J.A., Choma, M.A., Reedy, M.C., Rockman, H.A., 2006. Drosophila as a model for the identication of genes causing adult human heart disease. PNAS 103, 13941399. Wright, T.R.F., 1987. The genetics of biogenic amine metabolism, sclerotization, and melanization in Drosophila melanogaster. Adv. Genet. 24, 127222. Xiao, G.G., Wang, M., Li, N., Loo, J.A., Nel, A.E., 2003. Use of proteomics to demonstrate a hierarchical oxidative stress response to diesel exhaust particle chemicals in a macrophage cell line. J. Biol. Chem. 278 (50), 5078150790.

Foldbjerg, R., Dang, D.A., Autrup, H., 2011. Cytotoxicity and genotoxicity of silver nanoparticles in the human lung cancer cell line. Arch. Toxicol. 85 (7), 743750. Goldstein, A., 1954. Biostatistics. MacMillan Co., New York. Gurr, J.R., Wang, A.S.S., Chen, C.H., Jan, K.Y., 2005. Ultrane titanium dioxide particles in the absence of photoactivation can induce oxidative damage to human bronchial epithelial cells. Toxicology 213, 6673. Hirakawa, K., Mori, M., Yoshida, M., Oikawa, S., Kawanishi, S., 2004. Photo-irradiated titanium dioxide catalyzes site specic DNA damage via generation of hydrogen peroxide. Free Radical Res. 38 (5), 439447. Hussain, S.M., Hess, K.L., Gearhart, J.M., Geiss, K.T., Schlager, J.J., 2005. In vitro toxicity of nanoparticles in BRL 3A rat liver cells. Toxicol. in Vitro 19, 975983. , K., Tisler, T., 2008. Effects of ingested Jemec, A., Drobne, D., Remskar, M., Sepcic nano-sized titanium dioxide on terrestrial isopods (Porcellio scaber). Environ. Toxicol. Chem. 27 (9), 19041914. Jin, C., Tang, Y., Yang, F.G., Li, X.L., Xu, S., Fan, X.Y., Huang, Y.Y., Yang, Y.J., in press. Cellular toxicity of TiO(2) nanoparticles in anatase and rutile crystal phase. Biol. Trace Elem. Res. Kakkar, P.S., Das, B., Viswanathan, P.N., 1984. A modied spectrophotometric assay of superoxide dismutase. Ind. J. Biochem. Biophys. 21, 130132. Kim, S., Choi, J.E., Choi, J., Chung, K.H., Park, K., Yi, J., Ryu, D.Y., 2009. Oxidative stress-dependent toxicity of silver nanoparticles in human hepatoma cells. Toxicol. in Vitro 23 (6), 10761084. Koh, K., Evans, J.M., Hendricks, J.C., Sehagal, A., 2006. A Drosophila model for ageassociated changes in sleep: wake cycles. Proc. Natl. Acad. Sci. U. S. A. 103, 1384313847. Khurana, V., Lu, Y., Steinhilb, M.L., Oldham, S., Shulman, J.M., Feany, M.B., 2006. TORmediated cell-cycle activation causes neurodegeneration in a Drosophila tauopathy model. Curr. Biol. 16, 230241. Lee, H.Y., Park, H.K., Lee, Y.M., Kim, K., Park, S.B., 2007. A practical procedure for producing silver nanocoated fabric and its antibacterial evaluation for biomedical applications. Chem. Commun. 28, 29592961. Menard, A., Drobne, D., Jemec, A., 2011. Ecotoxicity of nanosized TiO2. Review of in vivo data. Environ. Pollut. 159 (3), 677684. Mocan, T., Clichici, S., Agos ton-Coldea, L., Mocan, L., Simon, S., Ilie, I.R., Biris , A.R., Mures an, A., 2010. Implications of oxidative stress mechanisms in toxicity of nanoparticles (review). Acta Physiol. Hung. 97 (3), 247255. Murdock, R.C., Braydich-Stolle, L., Schrand, A.M., Schlager, J.J., Hussain, S.M., 2008. Characterization of nanomaterial dispersion in solution prior to in vitro exposure using dynamic light scattering technique. Toxicol. Sci. 101 (2), 239 253.