Published: March 23, 2011

r 2011 American Chemical Society 3725 dx.doi.org/10.1021/es103309n | Environ. Sci. Technol. 2011, 45, 3725–3730

ARTICLE

pubs.acs.org/est

Nano-CeO2 Exhibits Adverse Effects at Environmental RelevantConcentrationsHaifeng Zhang,† Xiao He,† Zhiyong Zhang,* Peng Zhang, Yuanyuan Li, Yuhui Ma, Yashu Kuang,Yuliang Zhao, and Zhifang Chai

Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Key Laboratory of Nuclear Analytical Techniques, Institute ofHigh Energy Physics, Chinese Academy of Sciences, Beijing 100049, China

bS Supporting Information

’ INTRODUCTION

With the rapid development of nanotechnology and its broadapplications, a wide variety of engineered nanoparticles (NPs)are now used in commodities, pharmaceutics, cosmetics, biome-dical products, and industries. As a result, NPs are increasinglybeing released into the environment. Because of their uniqueproperties, such as smaller size, larger specific surface area, andgreater reactivity, NPs have raised concerns about the potentialhealth and environmental risks.1,2

The toxicity of NPs to the lower and micro- organisms hascaused many concerns because these organisms are abundant inecosystems, and such investigations have dual meanings: theexperimental results using these organisms contribute not only tothe understanding of the underlying mechanisms for nanotoxi-city, but also to the assessment of ecotoxicity of the specialnanomaterials.3�5 Caenorhabditis elegans (C. elegans), a free-living nematode in soil and fed with bacteria, is a simple multi-cellular eukaryote whose genome is completely sequenced.3 Theproperties of short life cycle, small size, transparency of thebody, and ease of cultivation make it a good animal model fortoxicological study and environmental evaluation. Those inves-tigations of NPs using C. elegans mainly focused on the ecotoxi-city with the toxic end points, such as body length, reproduction,behaviors, and metabolism. Previous reports have shown that NPsexposure could cause harmful effects onC. elegans, especially on the

reproductive capability.4�7 C. elegans is sensitive to oxidativestress, so it is also an ideal model organism for those materialswith redox capability. Kim et al. reported that the nano-Pt is moreeffective in antiaging properties than a well-known SOD/catalasemimetic EUK-8 by reducing the accumulation of lipofuscin andROS under oxidative stress conditions.8 We believe the applica-tion of C. elegans as a model organism in nanotoxicology wouldbe further extended, since NPs-induced oxidative damage isfrequently involved in the mechanisms for nanotoxicity.

Cerium dioxide is an important rare-earth oxide, and thesynthesis and functional research of its nanoparticles (nano-CeO2)have been a hot topic in a variety of fields recently, due to theirwidespread applications.9�11 Therefore, the toxicological and envir-onmental effects of nano-CeO2 have attracted more and moreattention. Nevertheless, the toxicological definition for nano-CeO2

is still quite controversial. It was reported that nano-CeO2 had theSOD mimetic activity and its catalytic rate constant exceeds that ofenzyme SOD.12 Several other papers also showed that CeO2

nanoparticles had neuroprotective,13,14 cardioprotective,15 and radio-protective16 effects in biological systems. Generally, nano-CeO2

Received: September 30, 2010Accepted: March 8, 2011Revised: March 4, 2011

ABSTRACT: Ceria nanoparticles (nano-CeO2), due to theirwidespread applications, have attracted a lot of concern abouttheir toxic effects on both human health and the environment.The present work aimed to evaluate the in vivo effects of nano-CeO2 (8.5 nm) on Caenorhabditis elegans (C. elegans) atenvironmental relevant concentrations (molar concentrationsranging from 1 nM to 100 nM). The results indicate that nano-CeO2 could induce ROS accumulation and oxidative damage inC. elegans, and finally lead to a decreased lifespan. The mostsurprising thing is that the mean lifespan of nematodes wassignificantly decreased by 12% even at the exposure level of 1nM (p < 0.01). In vitro tests suggest that the ability of nano-CeO2 to catalyze ROS generation was involved in the mechanism for its toxicity to C. elegans. To our best knowledge, this is the firstcase in which nanoparticles exhibit adverse effects on organisms at such low concentrations (1nM-100 nM). So, our findings indicatethe importance of nanotoxicological investigations at environmentally relevant concentrations and will attract more attentions onthe risks of NPs exposure.

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were reported to exhibit positive effects in most of the previouswork, and were regarded as a promising biomaterial for biome-dical applications.17 However, there were still a few paperssuggested nano-CeO2 exposure would lead to oxidative stressand DNA damage, though the mechanisms in some casesremained unknown.18�21 It should be noted that the concentra-tions of nano-CeO2 in these studies mentioned above weremostly at levels varying from μM to mM, much higher than therelevant concentrations of NPs in the environment, which meansthat the results of many of the currently published studies maynot represent the impacts in the real environment.22,23

The aim of the present study was to evaluate the potentialtoxicological effects of nano-CeO2 at environmental relevantconcentrations (1�100 nM), using C. elegans as test organism.We chose lifespan as the end point and tried to illustrate theoxidative damage induced by nano-CeO2 exposure at levelsranging from 1 nM to 100 nM.

’MATERIALS AND METHODS

1. Preparation of CeO2 Nanoparticles. Nano-CeO2 (withmean sizeof 8.5(1.5 nm)was synthesizedby aprecipitationmethod24,25 with some modifications. The details of preparation and chara-cterization are described in the Supporting Information (SI).2. C. elegans and Growth Conditions. The wild type N2

strain was maintained at 20 �C, and age-synchronous populationswere prepared according to the standard procedures.26 Escherichiacoli (E. coli) OP50 suspensions were spread on the nematode

growth media (NGM) agar to form bacterial lawn. The C. elegansarrested in the L1 larval stage in the absence of food were thentransferred onto the E. coli lawn and cultured at 20 �C in darkness.3. Lifespan Assay. Newly hatched L1 larvae (t = 0) were

transferred to E. coli lawn with or without nano-CeO2 (molarconcentrations: 0, 1 nM, 5 nM, 10 nM, and 100 nM). They weretransferred to new plates each day during the reproduction. Oncereproduction ceased, the nematodes were transferred to newplates every 3 days and examined daily for live and dead.Nematodes were scored as dead when they did not respond tomechanical stimulation. The nematodes crawling to the edge ofthe plates and hatching in the body were excluded from analysis.4. Thermotolerance. Thermotolerance assays were per-

formed as described by Lithgow.27 Three-day old hermaphro-dites were transferred into 35 �C. At the indicated time point(after 10 h), live nematodes were counted.5. Oxidative Stress Resistance. Juglone was chosen as an

oxidative stressor and the test was performed as previouslydescribed.28 Briefly, 3-day old nematodes were placed in NGMplates containing 600 μM juglone (Sigma, St. Louis, MO)immediately after the plates were prepared, and incubated at20 �C. Survival was monitored each hour.6. Fluorescence Microscopy. Lipofuscin, an endogenous

autofluorescent biomarker of aging, is a ROS derived productwhich accumulates progressively in C. elegans as a result of agingand oxidative degeneration of cellular components. The lipofuscinand ROS were determined using fluorescence methods (SI).29

Figure 1. Effects of nano-CeO2 on lifespan of N2 C. elegans. (A) The lifespan curves; (B) mean lifespan. The experiment was repeated at least 3 timeswith n = 40�60 worms each. Data are expressed as Mean ( SEM. * represents p < 0.05; ** represents p < 0.01, compared with control.

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7. In Vitro Test on Radical Production. To detect whethernano-CeO2would catalyze a Fenton-like reaction in the presenceof hydrogen peroxide, 2,20-azinobis-(3-ethylbenzthiazoline-6-sulfonicacid) (ABTS) was used to follow the production of the freeradical as described by Heckert et al.30 (SI).Statistical Analysis. The lipofuscin and ROS levels of C.

elegans were calculated as the average pixel intensity of eachworm using Matlab software. To prepare survival curves, mor-tality data were subjected to Kaplan�Meier survival analysis.Log-rank test was used to compare the statistical significance ofthe mean lifespan between nano-CeO2 treated populations anduntreated controls (CT). Other data were determined employ-ing one-way ANOVA followed by post hoc Tukey’s test orNonparametric analysis followed by Mann�Whitney U testwhere appropriate. All statistical analyses were conducted usingStatistical Packages for the Social Sciences (SPSS) Version 15.0.

’RESULTS AND DISCUSSION

We studied the consequences of nano-CeO2 exposure usingC.elegans as the test organism. First, the effects of nano-CeO2

exposure on the growth of E. coliOP50 were examined since foodabundance would influence the lifespan of nematodes.31 Ourdata showed there was no measurable disturbance to the growthof E. coli in the presence of the growthmedium (SI), which was inaccordance with the previous report.32 For the lifespan assay,newly hatched C. elegans were introduced on a solid nematodegrowth medium (NGM) in the presence of OP50 and nano-CeO2.The results indicated that nano-CeO2 exposure had adverse effectson the lifespan of C. elegans at all tested concentrations (p < 0.01,

Figure 1). What surprised us most was that the mean lifespan ofnematodes was significantly decreased by 12% even at theexposure level of 1 nM; this trend became more obvious in thenematodes exposed to higher concentrations of nano-CeO2.

In order to validate the lifespan results, formation of lipofuscinin nematodes was analyzed. Lipofuscin, an endogenous brown-yellow, autofluorescent pigment, is a ROS derived product which

Figure 2. Effects of nano-CeO2 treatments on the accumulation of lipofuscin in age-synchronizedN2worms of Day 7. (A) The intensity of fluorescenceis expressed asMean( SEM, relative to the control (n = 30�50). ** p < 0.01 compared with control; (B�F) representative fluorescent images of wormstreated with 0, 1, 5, 10, and 100 nM nano-CeO2, respectively.

Figure 3. Effects of nano-CeO2 treatments on thermotolerance. N2worms were incubated at 20 �C in the presence of nano-CeO2 from L1larvae and the survival rate was calculated after exposed to 35 �C for 10 h.Data are expressed as Mean ( SEM (n = 400�600). ** p < 0.01compared with control.

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accumulates progressively in many organisms (including C.elegans) as a result of aging and oxidative degeneration of cellularcomponents.8,33 Data shown in Figure 2 demonstrated thatnano-CeO2 exposure could significantly augment the generationand accumulation of lipofuscin in nematodes (p < 0.01) and thefluorescence in C. elegans exposed to 100 nM increased by 85%versus control group. These results were consistent with thelifespan analysis above, suggesting that aging was accelerated inC. elegans due to the intensified oxidative processes induced bynano-CeO2 exposure.

The sensitivities of nano-CeO2 pretreated nematodes tooxidative stress were evaluated in the following investigation.The intrinsic stress resistance of nematodes was assessed byrecording the survival of C. elegans at 35 �C (thermotolerance)after pretreatment with nano-CeO2 for 72 h. The results showedthat the exposure significantly impaired the thermotolerance ofC. elegans in a dose-dependent manner (p < 0.01, Figure 3). Thepretreatment with nano-CeO2 will also weaken the tolerance tojuglone, an extrinsic free radical generator (p < 0.01, Figure 4). Afurther investigation indicated that C. elegans pretreated withnano-CeO2 could generate more ROS under juglone treatment(Figure 5). The intracellular accumulations of ROS induced byjuglone were negative correlated with the survival of C. elegans(p < 0.05) and doubled in the nano-CeO2 pretreated nematodesexcept the 1 nM group. These results suggested that nano-CeO2

exposure would lead to an increasing susceptibility ofC. elegans tothe oxidative stress, both intrinsic and extrinsic.

Previous research revealed an extremely good correlationbetween the level of oxidative damage and the rate of aging ofcells, tissues, and individuals,33,34 so we presumed that oxidativedamage induced by ROS accumulation was involved in theadverse effects of nano-CeO2 exposure on C. elegans. There are

already some reports indicate nano-CeO2 exposure can causeoxidative stress in rats,18 cell culture models,19�21 as well as abacterial model.32 According to the free radical theory, cellularand organismal aging is the consequence from excessive genera-tion of ROS, despite the inherent cellular defenses.35,36 Inorganisms, ROS are produced at the respiratory chain in themitochondria by the incomplete reduction of oxygen and as side-products of cellular reactions.37 In addition to this endogenousformation, ROS generation could also be trigged by a number ofexternal agents like drugs, radiation, UV, heat, and environmentaltoxin.36 In some previous reports, nano-CeO2 was regarded as anexogenous source of ROS for cells or organisms.20,21 Heckert andhis colleagues reported that Ce3þ was capable of redox-cyclingwith peroxide to generate damaging oxygen radicals.30 Theyfurther presumed that Ce3þ accompanied by the oxygen vacancyon particle surface was the most likely active sites for ROSgeneration in the nanotoxicological studies. But meanwhile, oneof their other papers proposed that the surface oxidation state ofnano-CeO2 played an integral role in the SODmimetic activity ofnano-CeO2 and that ability of nano-CeO2 to scavenge super-oxide was directly related to Ce3þ concentrations at the surface

Figure 4. Effects of nano-CeO2 on juglone-induced oxidative stress. N2worms were pretreated with different concentrations of nano-CeO2

from L1 larvae and then incubated with 600 uM juglone at 20 �C onDay3 (n = 90�200). The mean survival time (B) was calculated from thesurvival curves (A) and expressed asMean( SEM. ** p < 0.01 comparedwith control.

Figure 5. Effects of nano-CeO2 on ROS accumulation in C. elegansunder juglone-induced oxidative stress. The average intensity is ex-pressed as Mean ( SEM, relative to the control. ** p < 0.01 comparedwith control.

Figure 6. Radical produced by 100 μM nano-CeO2 in the presence ofH2O2. The radical production was captured with ABTS and the form ofcation radical (ABTSþ 3 ) was spectrophotometrically determined at405 nm. The radical production catalyzed by 24 nM Ce3þ (equal tothe concentration of Ce3þ ions in the nano-CeO2 suspension) was alsodetermined.

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of the particle.38 These findings suggested that its redox reactivityendows nano-CeO2 with both abilities to scavenge radicals andto generate ROS, just as two sides of the coin. A similar view wasreported by Auffan et al.19

However, until now, there are few direct evidence to provethat ROS are produced when cerium in particles reversiblyalternate between Ce3þ and Ce4þ states. ROS generation andoxidative stress is the best developed paradigm to explain thetoxic effects of NPs, even those with no redox surface activities;1

cellular ROS accumulation and oxidative damage is one of themost frequent phenomena under NPs exposure, which might bea consequence from immune response, especially when the NPsdose is quite high. So, further study is needed to demonstratewhether the surface reactivity of nano-CeO2 could trigger thegeneration of ROS.

Here, we tried to prove the above hypothesis in a more directway. ABTS was used to capture the hydroxyl radical generated bynano-CeO2 according to Heckert’s method.30 The results in-dicated that ABTSþ 3 production accumulated in a time depen-dent manner over a 10min incubation at 25 �C (Figure 6). Nano-CeO2was an insolublemetal oxide,18,24 and only 0.024( 0.001%of the total cerium remained as Ce3þ ions in our nano-CeO2

suspension. The role of Ce3þ ions in the radical production wasdiscerned and the contribution was negligible. This result con-firmed the hypothesis that nano-CeO2 has Fenton-like reactivityin the presence of peroxides. Seen from the TEM image, nano-CeO2 could be uptaken by C. elegans and found in the intestinallumen (SI). The growth of E. coliOP50 under aerobic conditionswould generate a small amount of H2O2 as a byproduct,

39 and aportion of H2O2 would enter into nematodes during fooduptaking. Besides, nano-CeO2 in the intestinal lumen wassurrounded by bacterial residuals which would generate diet-ary-derived oxidants like peroxidized lipids.40 Since there wereperoxides, the results of in vitro test suggested that nano-CeO2

could act as an exogenous source of ROS for C. elegans.The above results as a whole indicated that nano-CeO2 could

induce ROS accumulation and oxidative damage in C. elegans, andfinally shorten the lifespan. These findings could also explain thecyto- and genotoxicity of nano-CeO2 mentioned in the previouswork. Moreover, these data were available for assessment of theecotoxicity of nano-CeO2. Nevertheless, toxicity of nano-CeO2 isquite unexpected at exposure concentrations of nM level, evenwhenthe nano-CeO2 mediated ROS generation and the sensitivity of C.elegans to oxidative stress are taken into account. Maybe, there aresome other stories underlying the unprecedented nanotoxicityfound in the present work, beyond themechanism involving surfaceredox reactivity; or the toxicity of nano-CeO2 is augmented by somefactors during interaction process.

To avoid misleading information on toxicity of NPs, toxico-logical study of NPs should be performed in an environmentalrelevant mode, using appropriate concentrations and naturalexposure routes.41 Nowadays, the typical concentrations of NPsused in toxicological experiments were mostly at levels varyingfrom μM to mM, much higher than the relevant concentrationsof NPs in the environment.22,23 However, the exposure concen-trations we chose in the present work ranged from 1 nM to 100nM, very closing to the environmental relevant concentration.23

Besides, the exposure design is quite close to the nature mode:C.elegans was cultured on solid medium, and the nano-CeO2 wasmixed with bacteria. Therefore, our findings are helpful to betterunderstand the toxicity of nano-CeO2, the mechanism, and theinteractions with biological systems and the environment.

Basing on the previous results, the risk rankings for nanoscaleCeO2 were of relatively low concern.23 However, our datarevealed the adverse effects of nano-CeO2 on lower organismsat environmental relevant concentrations. To our best knowl-edge, this is the first case in which nanoparticles exhibit adverseeffects on organisms at such low concentrations. The presentwork indicates the importance of nanotoxicological investiga-tions at environmentally relevant concentrations and calls formore attention to the potential environmental and health risks ofNPs exposure.

’ASSOCIATED CONTENT

bS Supporting Information. More details on experimentalsetup, particle characterization and toxicity studies. This materialis available free of charge via the Internet at http://pubs.acs.org.

’AUTHOR INFORMATION

Corresponding Author*Phone: þ86 10 88233215; fax: þ86 10 88235294; e-mail:zhangzhy@ihep.ac.cn.

Author Contributions†These authors contributed equally to this work.

’ACKNOWLEDGMENT

This work is financially supported by the Ministry of Scienceand Technology of China (grant No. 2011CB933400), theNational Natural Science Foundation of China (Grant No.10905062, 10875136, 11005118), and the Knowledge Innova-tion Program of the Chinese Academy of Sciences.

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