International Journal of Toxicology http://ijt.sagepub.com/
Toxicity Study of Cerium Oxide Nanoparticles in Human Neuroblastoma Cells Monika Kumari, Shailendra Pratap Singh, Srinivas Chinde, Mohammed Fazlur Rahman, Mohammed Mahboob and Paramjit Grover International Journal of Toxicology published online 6 February 2014 DOI: 10.1177/1091581814522305 The online version of this article can be found at: http://ijt.sagepub.com/content/early/2014/02/06/1091581814522305
Published by: http://www.sagepublications.com
On behalf of:
American College of Toxicology
Additional services and information for International Journal of Toxicology can be found at: Email Alerts: http://ijt.sagepub.com/cgi/alerts Subscriptions: http://ijt.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav
>> OnlineFirst Version of Record - Feb 6, 2014 What is This?
Downloaded from ijt.sagepub.com at Scientific library of Moscow State University on February 13, 2014
Article
Toxicity Study of Cerium Oxide Nanoparticles in Human Neuroblastoma Cells
International Journal of Toxicology 1-12 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1091581814522305 ijt.sagepub.com
Monika Kumari1,2, Shailendra Pratap Singh1, Srinivas Chinde1,2, Mohammed Fazlur Rahman1, Mohammed Mahboob1, and Paramjit Grover1
Abstract The present study consisted of cytotoxic, genotoxic, and oxidative stress responses of human neuroblastoma cell line (IMR32) following exposure to different doses of cerium oxide nanoparticles (CeO2 NPs; nanoceria) and its microparticles (MPs) for 24 hours. Cytotoxicity was evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and lactate dehydrogenase assays whereas genotoxicity was assessed using the cytokinesis-block micronucleus and comet assays. A battery of assays including lipid peroxidation, reactive oxygen species (ROS), hydrogen peroxide, reduced glutathione, nitric oxide, glutathione reductase, glutathione peroxidase, superoxide dismutase, catalase, and glutathione S-transferase were performed to test the hypothesis that ROS was responsible for the toxicity of nanoceria. The results showed that nanosized CeO2 was more toxic than cerium oxide MPs. Hence, further study on safety evaluation of CeO2 NPs on other models is recommended. Keywords cerium oxide nanoparticles, human neuroblastoma cells, cytotoxicity, oxidative stress, genotoxicity
Introduction The application of nanomaterials has had a great impact on biomedical science and engineering in last few decades because of its novel characteristics. The unique physical and chemical properties of nanoparticles (NPs) due to their small size, chemical composition, structure, and large surface area have resulted in their incorporation into thousands of commercial products. Increased use of nanotechnology enhances the risk of exposure to NPs. Hence, the routes of entry, interaction with cells and tissues, molecular mechanisms of cytotoxicity, and different effects on biological systems relative to the composition, size, and shape of emerging nanomaterials need to be well understood.1,2 Cerium is a lanthanide series rare earth element that can exist either as a free metal or as a cycle between the cerium (III) and the cerium (IV) oxidation states.3 Cerium oxide NPs (CeO2 NPs; nanoceria) consist of a cerium core surrounded by oxygen lattice. The CeO2 NPs have shown many promising applications because of their high performance as an oxygen buffer and active support for noble metals in catalysis, which relies on an efficient supply of lattice oxygen at reaction sites governed by oxygen vacancy formation.4 Reactions involving redox cycles between the Ce3þ and Ce4þ oxidation state allow nanoceria to react catalytically with free radicals and reactive oxygen species (ROS) often produced during inflammatory cascades. The unique properties of CeO2 NPs have resulted in widespread use in the treatment of medical disorders caused
by oxidative species.5 Moreover, nanoceria is commonly used as polishing agent,6 ultraviolet-absorbing compound in sunscreen,7 electrolyte in solid oxide fuel cells,8 as a fuel additive to promote combustion, and as a subcatalyst for automotive exhaust cleaning.9,10 These applications of engineered CeO2 NPs may increase the risk of exposure to humans and the environment. Therefore, human health risk assessment of CeO2 NPs is important. Neuronal cells are of special interest to evaluate the NP-induced toxicity. The use of NPs has been explored for neuroimaging strategy due to its optical and electrical functionalities. 11 Hence, this study evaluated the cytotoxicity, genotoxicity, and oxidative stress caused by CeO2 NPs in human neuroblastoma (NB) cell line (IMR32). As the physical and chemical properties of NPs can vary significantly from those of their bulk counterparts, CeO2 microparticles (MPs) were used to compare the size effect. The CeO2 NPs were characterized by transmission electron microscopy (TEM),
1
Toxicology Unit, Biology Division, Indian Institute of Chemical Technology, Hyderabad, Andhra Pradesh, India 2 Department of Genetics, Osmania University, Osmania University Main Road, Hyderabad, Andhra Pradesh, India Corresponding Author: Paramjit Grover, Toxicology Unit, Biology Division, Indian Institute of Chemical Technology, Hyderabad, Andhra Pradesh 500 007, India. Email: [email protected]; [email protected]
Downloaded from ijt.sagepub.com at Scientific library of Moscow State University on February 13, 2014
2
International Journal of Toxicology
dynamic light scattering (DLS), and laser Doppler velocimetry (LDV) studies to characterize the size, mean hydrodynamic diameter, and z potential of CeO 2 NPs, respectively. Cytotoxicity was evaluated by the formazan reduction 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. There is evidence of both induction and mitigation of oxidative stress by CeO2 NPs in both in vivo and in vitro in reports. Owing to their lesser size, CeO2 NPs were found to be more toxic than equimolar bulk CeO 2 in Caenorhabditis elegans and showed dose-dependent growth inhibition.12 In a study with male Sprague Dawley rats, oxidative damage of protein in liver and spleen was reported and it was suggested that the ceria NPs toxicity was time dependent with respect to peripheral organs and this effect may be related to the oxidative state of the ceria NPs.13 Further, CeO2 NPs were shown to induce apoptosis and autophagy in human peripheral blood monocytes at relatively low doses.14 When the human lung adenocarcinoma (A549) cell line was exposed to various concentrations of 20 nm CeO2 NPs, a dose- and time-dependent alteration was observed in indicators of oxidative stress and cytotoxicity. 15 In addition, human lung epithelial cells (BEAS-2B) exposed to CeO2 NPs showed an increase in the expression of oxidative stress-related genes, including catalase (CAT), glutathione S-transferase (GST), heme oxygenase 1, and thioredoxin reductase.16 In contrast, CeO2 NPs suppressed ROS production and induced cellular resistance to an exogenous source of oxidative stress in BEAS-2B and RAW 264.7 cells.17 Further, CeO2 NPs were reported to be neuroprotective to the cells derived from rodent nervous system (HT22 cell line) through the reduction in endogenous ROS induced by glutamate.18 Moreover, Niu et al19 suggested that nanoceria partially prevented heart dysfunction through inhibition of the myocardial oxidative stress, endoplasmic reticulum stress, and inflammatory processes in monocyte chemoattractant protein (MCP) 1 transgenic mice (MCP mice) that normally exhibit progressive heart damage. Administration of CeO2 NPs to mice with induced liver toxicity showed therapeutic property of reducing oxidative stress by decreasing ROS.20 Therefore, in the present study, lipid peroxidation (LPO), ROS, hydrogen peroxide (H2O2), reduced glutathione (GSH), lactate dehydrogenase (LDH), nitric oxide (NO), glutathione reductase (GR), glutathione peroxidase (GPx), superoxide dismutase (SOD), CAT, and GST estimations were carried out. The in vitro micronucleus (MN) test has become a standard cytogenetic test for genotoxicity testing. It is simple to score and accurate and applicable in different cell types.21 The cytokinesis-block MN (CBMN) assay is the preferred method for measuring micronuclei (MNi) in cultured cell lines because scoring is specifically restricted to binucleated cells that have undergone 1 cell division.22 At the same time, CBMN assay is a good approach to evaluate other damage events including nucleoplasmic bridges, a biomarker of DNA misrepair and/or telomere end fusions and nuclear buds (NBUDs), a biomarker of elimination of amplified DNA and/or DNA repair complexes, and the number of apoptotic and necrotic cells.23
The single-cell gel electrophoresis or comet assay is important to assess the DNA damaging potential of these particles because of rapid and sensitive detection of DNA damage in individual cells.24 Hence, the CBMN and comet assays were additionally performed in the present study.
Materials and Methods Particles and Chemicals Cerium oxide NPs (CeO2 < 25 nm, CAS No. 1306-38-3) and CeO2 MPs (CeO2, 99.9%, 13.0) and then electrophoresis was performed at 25 V adjusted at 300 mA for 20 minutes. The slides were neutralized twice in 0.4 mol/L Tris buffer, pH 7.5, for 5 minutes and once in absolute methanol for 5 minutes. Coded slides were scored after staining with ethidium bromide (20 mg/mL) using a fluorescence microscope (Olympus, Shinjuku-ku, Tokyo, Japan) with a blue (488 nm) excitation filter and yellow (515 nm) emission (barrier) filter at 400 magnification. A total of 150 randomly selected cells per sample (3 replicates, each with 50 cells per slide) were used to measure the amount of DNA damage and expressed as percentage of DNA in the comet tail. Quantification of DNA breakage was realized using a Comet Image Analysis System, version Komet 5.5 (single-cell gel electrophoresis analysis company, Andor Technology 2005; Kinetic Imaging Ltd, Nottingham, United Kingdom).
except where it is differently indicated. Multiple pairwise comparisons were done using the Dunnett multiple comparison posttest to verify the significance of positive response. Statistical analyses were performed using GraphPad Instat Prism 3 Software package for windows (GraphPad Software, Inc, La Jolla, California). The statistical significance for all tests was set at P < 0.05.
Results Characterization The mean size of CeO2 NPs and CeO2 MPs was calculated using TEM by measuring over 100 particles in random fields. The size obtained for CeO2 NPs and CeO2 MPs was 25 + 1.512 nm (Figure 1A) and 3.01 + 1.023 mm (Figure 1B), respectively. In DMEM, an average hydrodynamic diameter of CeO2 NPs was 269.7 + 27.398 nm using DLS, revealing the tendency of agglomeration in DMEM suspension. z potential and electrophoretic mobility of CeO2 NPs in DMEM were determined by LDV and found to be 7.74 mV and 1.24 mm cm/s V, respectively, at pH 7.4. The size and charge of CeO2 NPs in DMEM using TEM, DLS, and LDV, respectively, are presented in Table 1. In culture medium, NPs showed a slight increase in the size with a concomitant decrease in the z potential.
Cytotoxicity Assay A significant dose-dependent decrease in mitochondrial function was observed after IMR32 cells were exposed to the CeO2 NPs. The cell viability after 24 hours incubation with CeO2 NPs and MPs at concentrations 10, 20, 50, 100, and 200 mg/ mL is shown in Figure 2A. The inhibitory concentration 50 (IC50) calculated for CeO2 NPS was 1.09 mg/mL and for CeO2 MPs was 1.64 mg/mL using probit analysis. The concentrations lower than that of IC50 of CeO2 NPs and MPs were used in this study, which was in order to avoid the physical hindrance due to overaccumulation of the particles in the culture medium. The cytotoxicity due to loss in cell viability was obvious at 200, 100, and 50 mg/mL of CeO2 NPs and found to be 76.40%, 77.49% (P < 0.01), and 78.68% (P < 0.05), respectively, as compared to control (100%) while CeO2 MPs did not significantly decrease the cell viability. Exposure to CeO 2 NPs for 24 hours resulted in a concentration-dependent increase in LDH leakage and exhibited a significant (P < 0.01) cytotoxicity at 100 and 200 mg/mL (Figure 2B). The evident difference between CeO2 NPs and MPs was noted, as CeO2 MPs did not induce a significant change.
Intracellular Release of ROS and H2O2
Statistical Analysis The statistically significant changes between treated and control groups were analyzed by 1-way analysis of variance. All results are expressed as mean and standard deviation (SD; mean + SD) of the mean for 3 replicates of experiments,
Reactive oxygen specieslevels were quantified to examine the involvement of oxidative stress in CeO2 NPs (Figure 3A). It was noted that CeO2 NPs induced a significant 1.61- and 2.44-fold increase at 100 and 200 mg/mL, respectively, in ROS levels, whereas CeO2 MPs did not show a significant change
Downloaded from ijt.sagepub.com at Scientific library of Moscow State University on February 13, 2014
6
International Journal of Toxicology
Figure 1. Transmission electron microscopy image of cerium oxide nanoparticles (CeO2 NPs; A) and CeO2 microparticles (MPs; B) in MilliQ water. Table 1. Characterization of Cerium Oxide Nanoparticles (CeO2 NPs) and CeO2 Microparticles (MPs).a DLS Particles CeO2 NPs CeO2 MPs
LDV
Size using TEM
Average diameter, nm
PDI
z potential, mV
Electrophoretic mobility, mm cm/s V
pH
25 + 1.512 nm 3.01 + 1.023 mm
269.7 + 27.398 ND
0.436 ND
7.74 ND
1.25 ND
7.4 7.4
Abbreviations: PDI, polydispersity index, DLS, dynamic light scattering, LDV, laser Doppler velocimetry; DMEM, Dulbecco modified Eagle medium; ND, not detectable; TEM, transmission electron microscopy. a CeO2 NPs and MPs at the concentration of 40 mg/mL were dispersed in DMEM medium, mixing was done via probe sonication for 10 minutes just before estimations.
relative to control. Therefore, the production of ROS in IMR32 cells incubated with CeO2 NPs was much higher than the corresponding MP-exposed cells. Cerium oxide NPs significantly increased intracellular H2O2 within IMR32 cells at concentrations of 100 (P < 0.05) and 200 mg/mL (P < 0.01) in comparison to control (Figure 3B). There was no significant induction in the H2O2 levels in cells treated with CeO2 MPs.
Glutathione Content
Nitric Oxide Production
Activity of Enzymes Associated With Oxidative Stress
Nitric oxide production in IMR32 cells following 24 hours exposure to CeO2 NPs at 0 to 200 mg/mL is shown in Figure 3C. The NO levels were significantly (P < 0.01) increased at 100 to 200 mg/mL in relation to control. However, in CeO2 MP-exposed cells, there was no significant alteration.
Cerium oxideNP-exposed cells at concentrations of 100 and 200 mg/mL revealed significant reduction in SOD (Figure 3F) and CAT activity (Figure 3G) when compared to control at P < 0.05 and P < 0.01, respectively. Likewise, GR activity in IMR32 cells increased significantly (P < 0.01) in dosedependent manner (Figure 3H) when incubated with CeO2 NPs at the concentrations of 100 and 200 mg/mL. However, the cells exposed to CeO2 MPs did not exhibit any significant change in the activities of these enzymes. Further, CeO2 NPs exposure for 24 hours did not cause any significant changes in the GPx and GST activity in the IMR32 cells when compared to control and same result was found with CeO2 MPs (data not shown).
Lipid Peroxidation Assay Lipid peroxidation assay was performed to determine the MDA levels in the IMR32 cell suspension after 24 hours treatment with CeO2 NPs and MPs. A significant increase (P < 0.01) in MDA level was observed at 200 mg/mL of CeO2 NPs (Figure 3D).
IMR32 cells exposed to CeO 2 NPs showed a dosedependent depletion in GSH levels. The exposure concentrations of 100 and 200 mg/mL exhibited statistically significant (P < 0.01) depletion of 79% and 76%, respectively, after 24 hours (Figure 3E).
Downloaded from ijt.sagepub.com at Scientific library of Moscow State University on February 13, 2014
Kumari et al
7
Figure 2. Effects of CeO2 NPs and CeO2 MPs on mitochondrial function (A) and LDH leakage (B) in human neuroblastoma IMR32 cells. Cells were treated with different concentration of CeO2 NPs and MPs for 24 hours. At the end of incubation, mitochondrial function was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay and LDH leakage by LDH assay. Control cells cultured in NP- and MP-free media were run in parallel to treatment groups. Treatment group included NP- and MPtreated cells. Bars indicate the standard deviation (SD) from 3 replicates. The data are represented as mean + SD. Significantly different from control at aP < 0.05 and bP < 0.01. CeO2 NP indicates cerium oxide nanoparticle; LDH, lactate dehydrogenase; MP, microparticle.
Cytokinesis-Block MN Assay
Discussion
Chromosomal damage induced by CeO2 NPs was evaluated using the CBMN assay, in which cell division is blocked to allow the counting of once-divided binucleated cells. In the untreated cells, MN frequency was 2 per 1000 binucleated cells (Figure 4A). At dose levels of 100 and 200 mg/mL of CeO2 NPs, the number of MN formed per 1000 binucleated cells were 10.67 + 2.08 and 16 + 2, which was significant at P < 0.05 and P < 0.01, respectively (Figure 4A). The cell proliferation was assessed during the CBMN assay by the calculation of CBPI. Cell proliferation index was reduced significantly at CeO2 NPs doses of 200 mg/mL (1.30 + 0.02), 100 mg/mL (1.31 + 0.04), and 50 mg/mL (1.36 + 0.02) in comparison to control (1.45 + 0.03; Figure 4B). Therefore, there was a dose-dependent increase in MN frequency whereas the cell proliferation index decreased as the dose increased after CeO2 exposure. Moreover, mono-, bi-, and multinucleated cells (Figure 5A) along with an insignificant number of necrotic cells (Figure 5B), apoptotic cells (Figure 5C), and NBUDs (Figure 5D) were observed along with MN in binucleated cells (Figure 5E-H).
Wide application of nanoceria in different sectors of human welfare and its scanty data on toxicity prompted us to investigate the cellular response of CeO2 NPs and its bulk analog. The findings of the present study demonstrated significant ROS generation at 100 and 200 mg/mL doses of CeO2 NPs in human NB cells. Further, the alterations in ROS production and various oxidative stress-related indicators were concentration and size dependent. Moreover, the CeO2 NPs showed DNA damaging potential at these 2 higher doses. The correlation between ROS generation and DNA damage indicated that CeO2 NPs can lead to oxidative stress and could cause DNA damage and cell death. The physicochemical characterization is mandatory for the toxicity study of NPs. Therefore, the size analysis of CeO2 NPs was done by TEM and DLS. The size obtained from TEM analysis was 25 nm whereas the mean hydrodynamic size obtained from DLS was 269.7 nm. This difference in size could be due to difference in the principles used for the measurement (ie, TEM and DLS). The TEM provides the direct measurement of particle size, distribution, and morphology by image analysis in dry state while DLS measures the size distribution in the aqueous state, which is usually larger than the TEM diameter. The increased diameter may be due to agglomeration in aqueous medium, which could be due to physicochemical interactions between NPs. Therefore, in vitro testing with homogeneous NPs suspension is a challenging task and for that proper sonication is essential. The polydispersity index (PDI) is a measure of the heterogeneity of molecules in a mixture. The CeO2 NPs were found to be unstable in DMEM and instantly forming agglomerates
DNA Damage Comet assay of CeO2 NP- and MP-treated cells showed a concentration-dependent increase in the percentage of tail DNA (Figure 6) when compared to control, which indicates the extent of DNA damage. However, only the highest dose of 200 mg/mL depicted significant (P < 0.01) increases in the percentage of tail DNA (17.53 + 2.83).
Downloaded from ijt.sagepub.com at Scientific library of Moscow State University on February 13, 2014
8
International Journal of Toxicology
Figure 3. Effects of CeO2 NPs and CeO2 MPs on (A) intracellular reactive oxygen species (ROS), (B) hydrogen peroxide (H2O2), (C) nitric oxide (NO), (D) lipid peroxidation (LPO), (E) reduced glutathione (GSH), (F) superoxide dismutase (SOD), (G) catalase, and (H) glutathione reductase (GR). All assays were performed on the culture medium after 24 hours incubation with CeO2 NPs and MPs at the concentrations of 10, 20, 50, 100, and 200 mg/mL. The control group was treated with media only. Bars indicate the standard deviation (SD) from 3 replicates. The data are represented as mean + SD. Significantly different from control at aP < 0.05 and bP < 0.01. CeO2 NP indicates cerium oxide nanoparticle; MP, microparticle. Downloaded from ijt.sagepub.com at Scientific library of Moscow State University on February 13, 2014
Kumari et al
9
Figure 4. The genotoxicity of cerium oxide nanoparticles (CeO2 NPs) and CeO2 microparticles (MPs) using in vitro micronucleus test. A, Frequency of micronucleus in binucleated cells and (B) cytokinesis-block proliferation index (CBPI). The control group was treated with media only, cyclophosphamide (CP) was used as positive control. Bars indicate the standard deviation (SD) from 3 replicates. The data are represented as mean + SD. Significantly different from control at aP < 0.05 and bP < 0.01.
Figure 5. Photomicrographs of the IMR32 cells scored in the cytokinesis-block micronucleus (CBMN) assay. A, Mononucleated cell, binucleated cell, and multinucleated cell, (B) necrotic cells, (C) apoptotic cells, (D) binucleated cells containing nuclear buds (indicated by arrow), and (E-H) binucleated cells containing a micronucleus (indicated by arrow).
(PDI ¼ 0.436) and showed incipient instability due to slightly negative surface charge (z ¼ 7.74). This negative charge may be because of adsorption of serum proteins.38
Viability assays are vital steps in toxicology evaluation that explain the cellular response to a toxicant and they give information on cell death, survival, and metabolic activities.
Downloaded from ijt.sagepub.com at Scientific library of Moscow State University on February 13, 2014
10
International Journal of Toxicology
Figure 6. Mean percentage of tail DNA in IMR32 cells after 24 hours exposure of different doses of cerium oxide nanoparticles (CeO2 NPs) and microparticles (MPs). Bars indicate the standard deviation (SD) from 3 replicates. The data are represented as mean + SD. Significantly different from control at aP < 0.05 and bP < 0.01.
Both the MTT and the LDH assays are crucial for cytotoxicity study. Our data demonstrated dose-dependent cytotoxicity on exposure to CeO2 NPs with significant decrease in cell viability and increase in LDH release at higher doses. The significant loss in cell viability was observed at 100 to 200 mg/mL whereas weak but significant toxicity was evident at 50 mg/mL. The LDH release assay indicated that 25 nm CeO2 NPs induced a loss in cell membrane integrity and cell death of IMR32 cells at higher doses of 100 to 200 mg/mL but not in lower doses. In affirmation of our finding, it was shown that 30 nm CeO2 NPs did not cause significant decrease in the viability of cultured BEAS-2B cells up to 40 mg/mL after 24 hours exposure.16 In the same study, no significant cytotoxicity was shown either in rat cardiomyocytes (H9C2) or in human brain fibroblast cells (T98G) upon exposure to 5 mg/mL dose of 30 nm CeO2.16 However, even a 10.5 mg/mL dose of 20 nm CeO2 NPs significantly induced the LDH activity in A549 cells after 75 hours exposure. 15 Hence, these studies suggested differential sensitivity of the cells toward nanoceria. Further, duration of exposure also had an important role in induction of toxicity. The interactions between 2 commercial CeO2 NPs and A549 cells were investigated and weak cytotoxicity was observed only at the highest concentration of 200 mg/mL. 38 Size-dependent toxicity of CeO2 NPs was quite evident in the present study. Rosenkranz et al39 also reported the size, concentration, and time-dependent cytotoxicity of CeO2 particles with H4IIE rat hepatoma cells and rainbow trout-derived RTG2 cells. Further, CeO2 NPs caused membrane damage and inhibited colony formation in the long term but with different degrees depending on the cell lines.40 The oxidative stress induced in IMR32 cells upon 24 hours exposure of CeO2 NPs was reflected in the ROS, H2O2, and NO production, MDA levels, GSH content, and SOD, GR, and CAT activity in the present study. In our study, CeO2 NPs exposure induced a significant increase in the production of ROS, H2O2, NO, MDA, and SOD whereas a significant decrease in GSH content, CAT, and GR activity at the higher dose levels was
observed. The ROS are produced in many processes in humans which include atheroma, asthma, joint disease, aging, and cancer.41 Zhang et al42 studied the mechanism of toxicity of nanoceria of 8.5 nm size in C. elegans and suggested that its ability to catalyze the ROS generation was involved in the induction of toxicity at environmentally relevant concentrations. The NO and ROS are specialized chemical mediators produced in an active program during the resolution of inflammation. One of the repercussions of elevated oxidative stress is the production of MDA, an indicator of oxidative membrane damage. A study has revealed that CeO2 NPs exposure aggravated LPO and oxidative stress in A549 cells.15 Cheng et al43 concluded from their study that CeO2 NP-induced oxidative stress can lead to cytotoxicity in human hepatoma SMMC7721 cells after finding significant increase in the production of ROS and MDA and significant reduction in the activity of SOD, GPx, and CAT. The GSH is a ubiquitous sulfhydrylcontaining molecule in cells which is responsible for maintaining cellular oxidation–reduction homeostasis.44 Similarly, copper oxide (CuO) NPs induced LPO and loss in cell viability at higher concentrations (400 mg/mL) after 24 hours of treatment with mouse NB cell line (Neuro 2A).45 The toxicity study of different NPs in rat liver-derived cell line (BRL 3A) after 24 hours exposure showed that the alterations in mitochondrial function, LDH activity, GSH levels, and ROS were the basis for toxicity evaluation of NPs.46 A combination of CBMN and in vitro comet assays are excellent for interpreting the results from in vitro genotoxic hazard assessment of compounds. 47 The MN assay with cytokinesis facilitates the possibility of measuring important biomarker of DNA damage, cytostasis, and cytotoxicity, which can only be measured in once-divided binucleated cells.23 A comet-like tail implies the presence of a damaged DNA strand that lags behind when electrophoresis is done with an intact nucleus. The length of tail increases with the extent of DNA damage. DNA damage by CeO2 NPs was studied using CBMN and comet assay. There was common pattern of dosedependent increase in MN frequency and DNA damage. However, significant genotoxicity was observed only at highest dose (200 mg/mL) of CeO2 NPs. Results of some investigations are in accordance with our findings. Perreault et al45 reported significant increase in MN frequency at 12.5 mg/L CuO NPs after 24 hours treatment in Neuro 2A cells. The induction of chromosome damage and DNA lesion related to oxidative stress was observed in vitro in human dermal fibroblasts using CBMN and comet assay on exposure to the 7-nm CeO2 NPs at very low doses indicating the possible nanotoxocity and size effect of CeO2 NPs.48 Moreover, toxicity was observed within human hepatic carcinoma cells (HepG2), human colon carcinoma (CaCo2), and A549 cells when exposed to ceria NPs over a concentration range of 0.5 to 5000 mg/mL for 10 days and comet assay along with a cytotoxicity test were performed.49 In the same study, 24 hours exposure of ceria NPs to various cell lines did not induce DNA damage.49 These differences might be due to the difference in cell type and culture conditions.50 In NPs, toxicity ROS generation has been proposed as a possible
Downloaded from ijt.sagepub.com at Scientific library of Moscow State University on February 13, 2014
Kumari et al
11
mechanism involved51 and ROS play a major role in the genotoxicity.52 The data from the present study suggested that CeO 2 NPs induce ROS and oxidative stress leading to genotoxicity in IMR32 cell at higher doses.
Conclusion Overall, the results obtained in this study revealed that CeO2 NPs have size- and dose-dependent toxicity in the tested cell line whereas CeO 2 MPs did not induced any significant changes in the exposed cells. Moreover, CeO2 NPs did not induce toxicity below 100 mg/mL concentration and IMR32 cells were found to be less sensitive to CeO2 NPs. The cytotoxicity observed in the present study was in concurrence with oxidative stress and genotoxicity induced by CeO2 NPs. This investigation provides a better understanding of CeO2 NPs toxicity in IMR32 cells. However, further investigations are warranted on in vivo system in order to achieve a firm conclusion regarding the toxicity of CeO2 NPs and to get clear picture of its toxicokinetics and tolerance in the system. Acknowledgments Monika Kumari (SRF) is grateful to the University Grant Commission (UGC), India, and Shailendra Pratap Singh (SRF) is grateful to Indian Council of Medical Research (ICMR), India, for the award of a fellowship. The authors express their sincere thanks to the Director, IICT, Hyderabad, India, for providing facilities for this study.
Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by Asian Office of Aerospace Research and Development (AOARD), Japan under the Grant no. FA2386-11-1-4085.
References 1. Medina C, Santos-Martinez MJ, Radomski A, Corrigan OI, Radomski MW. Nanoparticles: pharmacological and toxicological significance. Br J Pharmacol. 2007;150(5):552-558. 2. Bucchianico SD, Fabbrizi MR, Misra SK, et al. Multiple cytotoxic and genotoxic effects induced in vitro by differently shaped copper oxide nanomaterials. Mutagenesis. 2013;28(3): 287-299. 3. Hu Z, Haneklaus S, Sparovek G, Schnug E. Rare earth elements in soils. Commun Soil Sci Plan. 2006;37(9-10):1381-1420. 4. Esch F, Fabris S, Zhou L, et al. Electron localization determines defect formation on ceria substrates. Science. 2005;309(5735): 752-755. 5. Wason MS, Zhao J. Cerium oxide nanoparticles: potential applications for cancer and other diseases. Am J Transl Res. 2013;5(2): 126-131.
6. Yu T, Park YI, Kang MC, et al. Large-scale synthesis of water dispersible ceria nano crystals by a simple sol–gel process and their use as a chemical mechanical planarization slurry. Eur J Inorg Chem. 2008;2008(6):855-858. 7. Wu W, Li S, Liao S, Xiang F, Wu X. Preparation of new sunscreen materials Ce1xZnxO2x via solid-state reaction at room temperature and study on their properties. Rare Metals. 2010;29(2):149-153. 8. Joo JH, Choi GM. Micro-solid oxide fuel cell using thick-film ceria. Solid State Ion. 2009;180(11-13):839-842. 9. Park B, Donaldson K, Duffin R, et al. Hazard and risk assessment of a nanoparticulate cerium oxide-based diesel fuel additive—a case study. Inhal Toxicol. 2008;20(6):547-566. 10. Masui T, Ozaki T, Machida K, Adachi G. Preparation of ceriazirconia sub-catalysts for automotive exhaust cleaning. J Alloys Compd. 2000;303-304(1):49-55. 11. Jan E, Byrne SJ, Cuddihy M, et al. High-content screening as a universal tool for fingerprinting of cytotoxicity of nanoparticles. ACS Nano. 2008;2(5):928-938. 12. Arnold MC, Badireddy AR, Wiesner MR, Di Giulio RT, Meyer JN. Cerium oxide nanoparticles are more toxic than equimolar bulk cerium oxide in Caenorhabditis elegans . Arch Environ Contam Toxicol. 2013;65(2):224-233. 13. Yokel RA, Au TC, MacPhail R, et al. Distribution, elimination, and biopersistance to 90 days of a systemically introduced 30 nm ceriaengineered nanomaterial in rats. Toxicol Sci. 2012;127(1):256-268. 14. Hussain S, Al-Nsour F, Rice AB, et al. Cerium dioxide nanoparticles induce apoptosis and autophagy in human peripheral blood monocytes. ACS Nano. 2012;6(7):5820-5829. 15. Lin W, Huang YW, Zhou XD, Ma Y. Toxicity of cerium oxide nanoparticles in human lung cancer cells. Int J Toxicol. 2006; 25(6):451-457. 16. Park EJ, Choi J, Park YK, Park K. Oxidative stress induced by cerium oxide nanoparticles in cultured BEAS-2B cells. Toxicology. 2008;245(1-2):90-100. 17. Xia T, Kovochich M, Liong M, et al. Comparison of the mechanism of toxicity of zinc oxide and cerium oxide nanoparticles based on dissolution and oxidative stress properties. ACS Nano. 2008; 2(10):2121-2134. 18. Schubert D, Dargusch R, Raitano J, Chan SW. Cerium and yttrium oxide nanoparticles are neuroprotective. Biochem Biophys Res Commun. 2006;342(1):86-91. 19. Niu JL, Azfer A, Rogers LM, Wang X, Kolattukudy PE. Cardioprotective effects of cerium oxide nanoparticles in a transgenic murine model of cardiomyopathy. Cardiovasc Res. 2007;73(3):549-559. 20. Hirst SM, Karakoti A, Singh S, et al. Bio-distribution and in vivo antioxidant effects of cerium oxide nanoparticles in mice. Environ Toxicol. 2013;28(2):107-118. 21. Kirsch-Volders M, Decordier I, Elhajouji A, Plas G, Aardema MJ, Fenech M. In vitro genotoxicity testing using the micronucleus assay in cell lines, human lymphocytes and 3D human skin models. Mutagenesis. 2011;26(1):177-184. 22. Fenech M. Cytokinesis-block micronucleus assay evolves into a ‘‘cytome’’ assay of chromosomal instability, mitotic dysfunction and cell death. Mutat Res. 2006;600(1-2):58-66. 23. Fenech M. Cytokinesis-block micronucleus cytome assay. Nat Protoc. 2007;2(5):1084-1104.
Downloaded from ijt.sagepub.com at Scientific library of Moscow State University on February 13, 2014
12
International Journal of Toxicology
24. Olive PL, Banath JP. The comet assay: a method to measure DNA damage in individual cells. Nat Protoc. 2006;1(1):23-29. 25. Hansen MB, Nielsen SE, Berg K. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J Immunol Methods. 1989;119(2):203-210. 26. Royall JA, Ischiropoulos H. Evaluation of 2,7-dichlorofluorescein and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells. Arch Biochem Biophys. 1993; 302(2):348-352. 27. Lee JY, Park W, Yi DK. Immunostimulatory effects of gold nanorod and silica-coated gold nanorod on RAW 264.7 mouse macrophages. Toxicol Lett. 2012;209(1):51-57. 28. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem. 1976;72:248-254. 29. Wills ED. Lipid peroxide formation in microsomes. Relation of hydroxylation to lipid peroxide formation. Biochem J. 1969; 113(2):333-341. 30. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959;82(1):70-77. 31. Marklund S, Marklund G. Involvement of superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem. 1974;47(3):469-474. 32. Aebi H. Catalase. In: Packer L, ed. Methods in Enzymology. Vol 105. Orlando, FL: Academic pres; 1984:121-126. 33. Carlberg I, Mannervik B. Glutathione reductase. Methods Enzymol. 1985;113:484-490. 34. Floche L, Gunzler WA. Assay of glutathione peroxidase. In: Packer L, ed. Methods Enzymol. Vol 105. New York: Academic Press; 1984:114-121. 35. Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferase. The first enzymatic step in mercapturic acid formation. J Biol Chem. 1974;249(22):7130-7139. 36. Organisation for Economic Co-operation and Development. Guideline for Testing of Chemicals: In Vitro Mammalian Cell Micronucleus Test (MNvit) (487). Paris: Organization for Economic Co-operation and Development; 2007. 37. Tice RR, Agurell E, Anderson D, et al. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen. 2000;35(3):206-221. 38. Zhou X, Wang B, Chen Y, Mao Z, Gao C. Uptake of cerium oxide nanoparticles and their influences on functions of A549 cells. J Nanosci Nanotechnol. 2013;13(1):204-215.
39. Rosenkranz P, Fernandez-Cruz ML, Conde E, et al. Effects of cerium oxide nanoparticles to fish and mammalian cell lines: an assessment of cytotoxicity and methodology. Toxicol In Vitro. 2012;26(6):888-896. 40. Kim IS, Baek M, Choi SJ. Comparative cytotoxicity of Al2O3, CeO2, TiO2 and ZnO nanoparticles to human lung cells. J Nanosci Nanotechnol. 2010;10(5):3453-3458. 41. Afonso V, Champy R, Mitrovic D, Collin P, Lomri A. Reactive oxygen species and superoxide dismutases: role in joint diseases. Joint Bone Spine. 2007;74(4):324-329. 42. Zhang H, He X, Zhang Z, et al. Nano-CeO2 exhibits adverse effects at environmental relevant concentrations. Environ Sci Technol. 2011;45(8):3725-3730. 43. Cheng G, Guo W, Han L, et al. Cerium oxide nanoparticles induce cytotoxicity in human hepatoma SMMC-7721 cells via oxidative stress and the activation of MAPK signaling pathways. Toxicol In Vitro. 2013;27(3):1082-1088. 44. Sies H. Glutathione and its role in cellular functions. Free Radic Biol Med. 1999;27(9-10):916-921. 45. Perreault F, Melegari SP, da Costa CH, Rossetto ALOF, Popovic R, Matias WG. Genotoxic effects of copper oxide nanoparticles in Neuro 2A cell cultures. Sci Total Environ. 2012;441:117-124. 46. Hussain SM, Hess KL, Gearhart JM, Geiss KT, Schlager JJ. In vitro toxicity of nanoparticles in BRL 3A rat liver cells. Toxicol In Vitro. 2005;19(7):975-983. 47. Kimura A, Miyata A, Honma M. A combination of in vitro comet assay and micronucleus test using human lymphoblastoid TK6 cells. Mutagenesis. 2013;28(5):583-590. 48. Auffan M, Rose J, Orsiere T, et al. CeO2 nanoparticles induce DNA damage towards human dermal fibroblasts in vitro . Nanotoxicology. 2009;3(2):161-171. 49. Marzi LD, Monaco A, Lapuente JD, et al. Cytotoxicity and genotoxicity of ceria nanoparticles on different cell lines in vitro . Int J Mol Sci. 2013;14(2):3065-3077. 50. Lanone S, Rogerieux F, Geys J, et al. Comparative toxicity of 24 manufactured nanoparticles in human alveolar epithelial and macrophage cell lines. Part Fibre Toxicol. 2009;6:14. 51. Gurr JR, Wang AS, Chen CH, Jan KY. Ultrafine titanium dioxide particles in the absence of photoactivation can induce oxidative damage to human bronchial epithelial cells. Toxicology. 2005; 213(1-2):66-73. 52. Schins RPF, Knaapen AdM. Genotoxicity of poorly soluble particles. Inhal Toxicol. 2007;19(s 1):189-198.
Downloaded from ijt.sagepub.com at Scientific library of Moscow State University on February 13, 2014
Toxicity Study of Cerium Oxide Nanoparticles in Human Neuroblastoma Cells Monika Kumari, Shailendra Pratap Singh, Srinivas Chinde, Mohammed Fazlur Rahman, Mohammed Mahboob and Paramjit Grover International Journal of Toxicology published online 6 February 2014 DOI: 10.1177/1091581814522305 The online version of this article can be found at: http://ijt.sagepub.com/content/early/2014/02/06/1091581814522305
Published by: http://www.sagepublications.com
On behalf of:
American College of Toxicology
Additional services and information for International Journal of Toxicology can be found at: Email Alerts: http://ijt.sagepub.com/cgi/alerts Subscriptions: http://ijt.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav
>> OnlineFirst Version of Record - Feb 6, 2014 What is This?
Downloaded from ijt.sagepub.com at Scientific library of Moscow State University on February 13, 2014
Article
Toxicity Study of Cerium Oxide Nanoparticles in Human Neuroblastoma Cells
International Journal of Toxicology 1-12 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1091581814522305 ijt.sagepub.com
Monika Kumari1,2, Shailendra Pratap Singh1, Srinivas Chinde1,2, Mohammed Fazlur Rahman1, Mohammed Mahboob1, and Paramjit Grover1
Abstract The present study consisted of cytotoxic, genotoxic, and oxidative stress responses of human neuroblastoma cell line (IMR32) following exposure to different doses of cerium oxide nanoparticles (CeO2 NPs; nanoceria) and its microparticles (MPs) for 24 hours. Cytotoxicity was evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and lactate dehydrogenase assays whereas genotoxicity was assessed using the cytokinesis-block micronucleus and comet assays. A battery of assays including lipid peroxidation, reactive oxygen species (ROS), hydrogen peroxide, reduced glutathione, nitric oxide, glutathione reductase, glutathione peroxidase, superoxide dismutase, catalase, and glutathione S-transferase were performed to test the hypothesis that ROS was responsible for the toxicity of nanoceria. The results showed that nanosized CeO2 was more toxic than cerium oxide MPs. Hence, further study on safety evaluation of CeO2 NPs on other models is recommended. Keywords cerium oxide nanoparticles, human neuroblastoma cells, cytotoxicity, oxidative stress, genotoxicity
Introduction The application of nanomaterials has had a great impact on biomedical science and engineering in last few decades because of its novel characteristics. The unique physical and chemical properties of nanoparticles (NPs) due to their small size, chemical composition, structure, and large surface area have resulted in their incorporation into thousands of commercial products. Increased use of nanotechnology enhances the risk of exposure to NPs. Hence, the routes of entry, interaction with cells and tissues, molecular mechanisms of cytotoxicity, and different effects on biological systems relative to the composition, size, and shape of emerging nanomaterials need to be well understood.1,2 Cerium is a lanthanide series rare earth element that can exist either as a free metal or as a cycle between the cerium (III) and the cerium (IV) oxidation states.3 Cerium oxide NPs (CeO2 NPs; nanoceria) consist of a cerium core surrounded by oxygen lattice. The CeO2 NPs have shown many promising applications because of their high performance as an oxygen buffer and active support for noble metals in catalysis, which relies on an efficient supply of lattice oxygen at reaction sites governed by oxygen vacancy formation.4 Reactions involving redox cycles between the Ce3þ and Ce4þ oxidation state allow nanoceria to react catalytically with free radicals and reactive oxygen species (ROS) often produced during inflammatory cascades. The unique properties of CeO2 NPs have resulted in widespread use in the treatment of medical disorders caused
by oxidative species.5 Moreover, nanoceria is commonly used as polishing agent,6 ultraviolet-absorbing compound in sunscreen,7 electrolyte in solid oxide fuel cells,8 as a fuel additive to promote combustion, and as a subcatalyst for automotive exhaust cleaning.9,10 These applications of engineered CeO2 NPs may increase the risk of exposure to humans and the environment. Therefore, human health risk assessment of CeO2 NPs is important. Neuronal cells are of special interest to evaluate the NP-induced toxicity. The use of NPs has been explored for neuroimaging strategy due to its optical and electrical functionalities. 11 Hence, this study evaluated the cytotoxicity, genotoxicity, and oxidative stress caused by CeO2 NPs in human neuroblastoma (NB) cell line (IMR32). As the physical and chemical properties of NPs can vary significantly from those of their bulk counterparts, CeO2 microparticles (MPs) were used to compare the size effect. The CeO2 NPs were characterized by transmission electron microscopy (TEM),
1
Toxicology Unit, Biology Division, Indian Institute of Chemical Technology, Hyderabad, Andhra Pradesh, India 2 Department of Genetics, Osmania University, Osmania University Main Road, Hyderabad, Andhra Pradesh, India Corresponding Author: Paramjit Grover, Toxicology Unit, Biology Division, Indian Institute of Chemical Technology, Hyderabad, Andhra Pradesh 500 007, India. Email: [email protected]; [email protected]
Downloaded from ijt.sagepub.com at Scientific library of Moscow State University on February 13, 2014
2
International Journal of Toxicology
dynamic light scattering (DLS), and laser Doppler velocimetry (LDV) studies to characterize the size, mean hydrodynamic diameter, and z potential of CeO 2 NPs, respectively. Cytotoxicity was evaluated by the formazan reduction 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. There is evidence of both induction and mitigation of oxidative stress by CeO2 NPs in both in vivo and in vitro in reports. Owing to their lesser size, CeO2 NPs were found to be more toxic than equimolar bulk CeO 2 in Caenorhabditis elegans and showed dose-dependent growth inhibition.12 In a study with male Sprague Dawley rats, oxidative damage of protein in liver and spleen was reported and it was suggested that the ceria NPs toxicity was time dependent with respect to peripheral organs and this effect may be related to the oxidative state of the ceria NPs.13 Further, CeO2 NPs were shown to induce apoptosis and autophagy in human peripheral blood monocytes at relatively low doses.14 When the human lung adenocarcinoma (A549) cell line was exposed to various concentrations of 20 nm CeO2 NPs, a dose- and time-dependent alteration was observed in indicators of oxidative stress and cytotoxicity. 15 In addition, human lung epithelial cells (BEAS-2B) exposed to CeO2 NPs showed an increase in the expression of oxidative stress-related genes, including catalase (CAT), glutathione S-transferase (GST), heme oxygenase 1, and thioredoxin reductase.16 In contrast, CeO2 NPs suppressed ROS production and induced cellular resistance to an exogenous source of oxidative stress in BEAS-2B and RAW 264.7 cells.17 Further, CeO2 NPs were reported to be neuroprotective to the cells derived from rodent nervous system (HT22 cell line) through the reduction in endogenous ROS induced by glutamate.18 Moreover, Niu et al19 suggested that nanoceria partially prevented heart dysfunction through inhibition of the myocardial oxidative stress, endoplasmic reticulum stress, and inflammatory processes in monocyte chemoattractant protein (MCP) 1 transgenic mice (MCP mice) that normally exhibit progressive heart damage. Administration of CeO2 NPs to mice with induced liver toxicity showed therapeutic property of reducing oxidative stress by decreasing ROS.20 Therefore, in the present study, lipid peroxidation (LPO), ROS, hydrogen peroxide (H2O2), reduced glutathione (GSH), lactate dehydrogenase (LDH), nitric oxide (NO), glutathione reductase (GR), glutathione peroxidase (GPx), superoxide dismutase (SOD), CAT, and GST estimations were carried out. The in vitro micronucleus (MN) test has become a standard cytogenetic test for genotoxicity testing. It is simple to score and accurate and applicable in different cell types.21 The cytokinesis-block MN (CBMN) assay is the preferred method for measuring micronuclei (MNi) in cultured cell lines because scoring is specifically restricted to binucleated cells that have undergone 1 cell division.22 At the same time, CBMN assay is a good approach to evaluate other damage events including nucleoplasmic bridges, a biomarker of DNA misrepair and/or telomere end fusions and nuclear buds (NBUDs), a biomarker of elimination of amplified DNA and/or DNA repair complexes, and the number of apoptotic and necrotic cells.23
The single-cell gel electrophoresis or comet assay is important to assess the DNA damaging potential of these particles because of rapid and sensitive detection of DNA damage in individual cells.24 Hence, the CBMN and comet assays were additionally performed in the present study.
Materials and Methods Particles and Chemicals Cerium oxide NPs (CeO2 < 25 nm, CAS No. 1306-38-3) and CeO2 MPs (CeO2, 99.9%, 13.0) and then electrophoresis was performed at 25 V adjusted at 300 mA for 20 minutes. The slides were neutralized twice in 0.4 mol/L Tris buffer, pH 7.5, for 5 minutes and once in absolute methanol for 5 minutes. Coded slides were scored after staining with ethidium bromide (20 mg/mL) using a fluorescence microscope (Olympus, Shinjuku-ku, Tokyo, Japan) with a blue (488 nm) excitation filter and yellow (515 nm) emission (barrier) filter at 400 magnification. A total of 150 randomly selected cells per sample (3 replicates, each with 50 cells per slide) were used to measure the amount of DNA damage and expressed as percentage of DNA in the comet tail. Quantification of DNA breakage was realized using a Comet Image Analysis System, version Komet 5.5 (single-cell gel electrophoresis analysis company, Andor Technology 2005; Kinetic Imaging Ltd, Nottingham, United Kingdom).
except where it is differently indicated. Multiple pairwise comparisons were done using the Dunnett multiple comparison posttest to verify the significance of positive response. Statistical analyses were performed using GraphPad Instat Prism 3 Software package for windows (GraphPad Software, Inc, La Jolla, California). The statistical significance for all tests was set at P < 0.05.
Results Characterization The mean size of CeO2 NPs and CeO2 MPs was calculated using TEM by measuring over 100 particles in random fields. The size obtained for CeO2 NPs and CeO2 MPs was 25 + 1.512 nm (Figure 1A) and 3.01 + 1.023 mm (Figure 1B), respectively. In DMEM, an average hydrodynamic diameter of CeO2 NPs was 269.7 + 27.398 nm using DLS, revealing the tendency of agglomeration in DMEM suspension. z potential and electrophoretic mobility of CeO2 NPs in DMEM were determined by LDV and found to be 7.74 mV and 1.24 mm cm/s V, respectively, at pH 7.4. The size and charge of CeO2 NPs in DMEM using TEM, DLS, and LDV, respectively, are presented in Table 1. In culture medium, NPs showed a slight increase in the size with a concomitant decrease in the z potential.
Cytotoxicity Assay A significant dose-dependent decrease in mitochondrial function was observed after IMR32 cells were exposed to the CeO2 NPs. The cell viability after 24 hours incubation with CeO2 NPs and MPs at concentrations 10, 20, 50, 100, and 200 mg/ mL is shown in Figure 2A. The inhibitory concentration 50 (IC50) calculated for CeO2 NPS was 1.09 mg/mL and for CeO2 MPs was 1.64 mg/mL using probit analysis. The concentrations lower than that of IC50 of CeO2 NPs and MPs were used in this study, which was in order to avoid the physical hindrance due to overaccumulation of the particles in the culture medium. The cytotoxicity due to loss in cell viability was obvious at 200, 100, and 50 mg/mL of CeO2 NPs and found to be 76.40%, 77.49% (P < 0.01), and 78.68% (P < 0.05), respectively, as compared to control (100%) while CeO2 MPs did not significantly decrease the cell viability. Exposure to CeO 2 NPs for 24 hours resulted in a concentration-dependent increase in LDH leakage and exhibited a significant (P < 0.01) cytotoxicity at 100 and 200 mg/mL (Figure 2B). The evident difference between CeO2 NPs and MPs was noted, as CeO2 MPs did not induce a significant change.
Intracellular Release of ROS and H2O2
Statistical Analysis The statistically significant changes between treated and control groups were analyzed by 1-way analysis of variance. All results are expressed as mean and standard deviation (SD; mean + SD) of the mean for 3 replicates of experiments,
Reactive oxygen specieslevels were quantified to examine the involvement of oxidative stress in CeO2 NPs (Figure 3A). It was noted that CeO2 NPs induced a significant 1.61- and 2.44-fold increase at 100 and 200 mg/mL, respectively, in ROS levels, whereas CeO2 MPs did not show a significant change
Downloaded from ijt.sagepub.com at Scientific library of Moscow State University on February 13, 2014
6
International Journal of Toxicology
Figure 1. Transmission electron microscopy image of cerium oxide nanoparticles (CeO2 NPs; A) and CeO2 microparticles (MPs; B) in MilliQ water. Table 1. Characterization of Cerium Oxide Nanoparticles (CeO2 NPs) and CeO2 Microparticles (MPs).a DLS Particles CeO2 NPs CeO2 MPs
LDV
Size using TEM
Average diameter, nm
PDI
z potential, mV
Electrophoretic mobility, mm cm/s V
pH
25 + 1.512 nm 3.01 + 1.023 mm
269.7 + 27.398 ND
0.436 ND
7.74 ND
1.25 ND
7.4 7.4
Abbreviations: PDI, polydispersity index, DLS, dynamic light scattering, LDV, laser Doppler velocimetry; DMEM, Dulbecco modified Eagle medium; ND, not detectable; TEM, transmission electron microscopy. a CeO2 NPs and MPs at the concentration of 40 mg/mL were dispersed in DMEM medium, mixing was done via probe sonication for 10 minutes just before estimations.
relative to control. Therefore, the production of ROS in IMR32 cells incubated with CeO2 NPs was much higher than the corresponding MP-exposed cells. Cerium oxide NPs significantly increased intracellular H2O2 within IMR32 cells at concentrations of 100 (P < 0.05) and 200 mg/mL (P < 0.01) in comparison to control (Figure 3B). There was no significant induction in the H2O2 levels in cells treated with CeO2 MPs.
Glutathione Content
Nitric Oxide Production
Activity of Enzymes Associated With Oxidative Stress
Nitric oxide production in IMR32 cells following 24 hours exposure to CeO2 NPs at 0 to 200 mg/mL is shown in Figure 3C. The NO levels were significantly (P < 0.01) increased at 100 to 200 mg/mL in relation to control. However, in CeO2 MP-exposed cells, there was no significant alteration.
Cerium oxideNP-exposed cells at concentrations of 100 and 200 mg/mL revealed significant reduction in SOD (Figure 3F) and CAT activity (Figure 3G) when compared to control at P < 0.05 and P < 0.01, respectively. Likewise, GR activity in IMR32 cells increased significantly (P < 0.01) in dosedependent manner (Figure 3H) when incubated with CeO2 NPs at the concentrations of 100 and 200 mg/mL. However, the cells exposed to CeO2 MPs did not exhibit any significant change in the activities of these enzymes. Further, CeO2 NPs exposure for 24 hours did not cause any significant changes in the GPx and GST activity in the IMR32 cells when compared to control and same result was found with CeO2 MPs (data not shown).
Lipid Peroxidation Assay Lipid peroxidation assay was performed to determine the MDA levels in the IMR32 cell suspension after 24 hours treatment with CeO2 NPs and MPs. A significant increase (P < 0.01) in MDA level was observed at 200 mg/mL of CeO2 NPs (Figure 3D).
IMR32 cells exposed to CeO 2 NPs showed a dosedependent depletion in GSH levels. The exposure concentrations of 100 and 200 mg/mL exhibited statistically significant (P < 0.01) depletion of 79% and 76%, respectively, after 24 hours (Figure 3E).
Downloaded from ijt.sagepub.com at Scientific library of Moscow State University on February 13, 2014
Kumari et al
7
Figure 2. Effects of CeO2 NPs and CeO2 MPs on mitochondrial function (A) and LDH leakage (B) in human neuroblastoma IMR32 cells. Cells were treated with different concentration of CeO2 NPs and MPs for 24 hours. At the end of incubation, mitochondrial function was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay and LDH leakage by LDH assay. Control cells cultured in NP- and MP-free media were run in parallel to treatment groups. Treatment group included NP- and MPtreated cells. Bars indicate the standard deviation (SD) from 3 replicates. The data are represented as mean + SD. Significantly different from control at aP < 0.05 and bP < 0.01. CeO2 NP indicates cerium oxide nanoparticle; LDH, lactate dehydrogenase; MP, microparticle.
Cytokinesis-Block MN Assay
Discussion
Chromosomal damage induced by CeO2 NPs was evaluated using the CBMN assay, in which cell division is blocked to allow the counting of once-divided binucleated cells. In the untreated cells, MN frequency was 2 per 1000 binucleated cells (Figure 4A). At dose levels of 100 and 200 mg/mL of CeO2 NPs, the number of MN formed per 1000 binucleated cells were 10.67 + 2.08 and 16 + 2, which was significant at P < 0.05 and P < 0.01, respectively (Figure 4A). The cell proliferation was assessed during the CBMN assay by the calculation of CBPI. Cell proliferation index was reduced significantly at CeO2 NPs doses of 200 mg/mL (1.30 + 0.02), 100 mg/mL (1.31 + 0.04), and 50 mg/mL (1.36 + 0.02) in comparison to control (1.45 + 0.03; Figure 4B). Therefore, there was a dose-dependent increase in MN frequency whereas the cell proliferation index decreased as the dose increased after CeO2 exposure. Moreover, mono-, bi-, and multinucleated cells (Figure 5A) along with an insignificant number of necrotic cells (Figure 5B), apoptotic cells (Figure 5C), and NBUDs (Figure 5D) were observed along with MN in binucleated cells (Figure 5E-H).
Wide application of nanoceria in different sectors of human welfare and its scanty data on toxicity prompted us to investigate the cellular response of CeO2 NPs and its bulk analog. The findings of the present study demonstrated significant ROS generation at 100 and 200 mg/mL doses of CeO2 NPs in human NB cells. Further, the alterations in ROS production and various oxidative stress-related indicators were concentration and size dependent. Moreover, the CeO2 NPs showed DNA damaging potential at these 2 higher doses. The correlation between ROS generation and DNA damage indicated that CeO2 NPs can lead to oxidative stress and could cause DNA damage and cell death. The physicochemical characterization is mandatory for the toxicity study of NPs. Therefore, the size analysis of CeO2 NPs was done by TEM and DLS. The size obtained from TEM analysis was 25 nm whereas the mean hydrodynamic size obtained from DLS was 269.7 nm. This difference in size could be due to difference in the principles used for the measurement (ie, TEM and DLS). The TEM provides the direct measurement of particle size, distribution, and morphology by image analysis in dry state while DLS measures the size distribution in the aqueous state, which is usually larger than the TEM diameter. The increased diameter may be due to agglomeration in aqueous medium, which could be due to physicochemical interactions between NPs. Therefore, in vitro testing with homogeneous NPs suspension is a challenging task and for that proper sonication is essential. The polydispersity index (PDI) is a measure of the heterogeneity of molecules in a mixture. The CeO2 NPs were found to be unstable in DMEM and instantly forming agglomerates
DNA Damage Comet assay of CeO2 NP- and MP-treated cells showed a concentration-dependent increase in the percentage of tail DNA (Figure 6) when compared to control, which indicates the extent of DNA damage. However, only the highest dose of 200 mg/mL depicted significant (P < 0.01) increases in the percentage of tail DNA (17.53 + 2.83).
Downloaded from ijt.sagepub.com at Scientific library of Moscow State University on February 13, 2014
8
International Journal of Toxicology
Figure 3. Effects of CeO2 NPs and CeO2 MPs on (A) intracellular reactive oxygen species (ROS), (B) hydrogen peroxide (H2O2), (C) nitric oxide (NO), (D) lipid peroxidation (LPO), (E) reduced glutathione (GSH), (F) superoxide dismutase (SOD), (G) catalase, and (H) glutathione reductase (GR). All assays were performed on the culture medium after 24 hours incubation with CeO2 NPs and MPs at the concentrations of 10, 20, 50, 100, and 200 mg/mL. The control group was treated with media only. Bars indicate the standard deviation (SD) from 3 replicates. The data are represented as mean + SD. Significantly different from control at aP < 0.05 and bP < 0.01. CeO2 NP indicates cerium oxide nanoparticle; MP, microparticle. Downloaded from ijt.sagepub.com at Scientific library of Moscow State University on February 13, 2014
Kumari et al
9
Figure 4. The genotoxicity of cerium oxide nanoparticles (CeO2 NPs) and CeO2 microparticles (MPs) using in vitro micronucleus test. A, Frequency of micronucleus in binucleated cells and (B) cytokinesis-block proliferation index (CBPI). The control group was treated with media only, cyclophosphamide (CP) was used as positive control. Bars indicate the standard deviation (SD) from 3 replicates. The data are represented as mean + SD. Significantly different from control at aP < 0.05 and bP < 0.01.
Figure 5. Photomicrographs of the IMR32 cells scored in the cytokinesis-block micronucleus (CBMN) assay. A, Mononucleated cell, binucleated cell, and multinucleated cell, (B) necrotic cells, (C) apoptotic cells, (D) binucleated cells containing nuclear buds (indicated by arrow), and (E-H) binucleated cells containing a micronucleus (indicated by arrow).
(PDI ¼ 0.436) and showed incipient instability due to slightly negative surface charge (z ¼ 7.74). This negative charge may be because of adsorption of serum proteins.38
Viability assays are vital steps in toxicology evaluation that explain the cellular response to a toxicant and they give information on cell death, survival, and metabolic activities.
Downloaded from ijt.sagepub.com at Scientific library of Moscow State University on February 13, 2014
10
International Journal of Toxicology
Figure 6. Mean percentage of tail DNA in IMR32 cells after 24 hours exposure of different doses of cerium oxide nanoparticles (CeO2 NPs) and microparticles (MPs). Bars indicate the standard deviation (SD) from 3 replicates. The data are represented as mean + SD. Significantly different from control at aP < 0.05 and bP < 0.01.
Both the MTT and the LDH assays are crucial for cytotoxicity study. Our data demonstrated dose-dependent cytotoxicity on exposure to CeO2 NPs with significant decrease in cell viability and increase in LDH release at higher doses. The significant loss in cell viability was observed at 100 to 200 mg/mL whereas weak but significant toxicity was evident at 50 mg/mL. The LDH release assay indicated that 25 nm CeO2 NPs induced a loss in cell membrane integrity and cell death of IMR32 cells at higher doses of 100 to 200 mg/mL but not in lower doses. In affirmation of our finding, it was shown that 30 nm CeO2 NPs did not cause significant decrease in the viability of cultured BEAS-2B cells up to 40 mg/mL after 24 hours exposure.16 In the same study, no significant cytotoxicity was shown either in rat cardiomyocytes (H9C2) or in human brain fibroblast cells (T98G) upon exposure to 5 mg/mL dose of 30 nm CeO2.16 However, even a 10.5 mg/mL dose of 20 nm CeO2 NPs significantly induced the LDH activity in A549 cells after 75 hours exposure. 15 Hence, these studies suggested differential sensitivity of the cells toward nanoceria. Further, duration of exposure also had an important role in induction of toxicity. The interactions between 2 commercial CeO2 NPs and A549 cells were investigated and weak cytotoxicity was observed only at the highest concentration of 200 mg/mL. 38 Size-dependent toxicity of CeO2 NPs was quite evident in the present study. Rosenkranz et al39 also reported the size, concentration, and time-dependent cytotoxicity of CeO2 particles with H4IIE rat hepatoma cells and rainbow trout-derived RTG2 cells. Further, CeO2 NPs caused membrane damage and inhibited colony formation in the long term but with different degrees depending on the cell lines.40 The oxidative stress induced in IMR32 cells upon 24 hours exposure of CeO2 NPs was reflected in the ROS, H2O2, and NO production, MDA levels, GSH content, and SOD, GR, and CAT activity in the present study. In our study, CeO2 NPs exposure induced a significant increase in the production of ROS, H2O2, NO, MDA, and SOD whereas a significant decrease in GSH content, CAT, and GR activity at the higher dose levels was
observed. The ROS are produced in many processes in humans which include atheroma, asthma, joint disease, aging, and cancer.41 Zhang et al42 studied the mechanism of toxicity of nanoceria of 8.5 nm size in C. elegans and suggested that its ability to catalyze the ROS generation was involved in the induction of toxicity at environmentally relevant concentrations. The NO and ROS are specialized chemical mediators produced in an active program during the resolution of inflammation. One of the repercussions of elevated oxidative stress is the production of MDA, an indicator of oxidative membrane damage. A study has revealed that CeO2 NPs exposure aggravated LPO and oxidative stress in A549 cells.15 Cheng et al43 concluded from their study that CeO2 NP-induced oxidative stress can lead to cytotoxicity in human hepatoma SMMC7721 cells after finding significant increase in the production of ROS and MDA and significant reduction in the activity of SOD, GPx, and CAT. The GSH is a ubiquitous sulfhydrylcontaining molecule in cells which is responsible for maintaining cellular oxidation–reduction homeostasis.44 Similarly, copper oxide (CuO) NPs induced LPO and loss in cell viability at higher concentrations (400 mg/mL) after 24 hours of treatment with mouse NB cell line (Neuro 2A).45 The toxicity study of different NPs in rat liver-derived cell line (BRL 3A) after 24 hours exposure showed that the alterations in mitochondrial function, LDH activity, GSH levels, and ROS were the basis for toxicity evaluation of NPs.46 A combination of CBMN and in vitro comet assays are excellent for interpreting the results from in vitro genotoxic hazard assessment of compounds. 47 The MN assay with cytokinesis facilitates the possibility of measuring important biomarker of DNA damage, cytostasis, and cytotoxicity, which can only be measured in once-divided binucleated cells.23 A comet-like tail implies the presence of a damaged DNA strand that lags behind when electrophoresis is done with an intact nucleus. The length of tail increases with the extent of DNA damage. DNA damage by CeO2 NPs was studied using CBMN and comet assay. There was common pattern of dosedependent increase in MN frequency and DNA damage. However, significant genotoxicity was observed only at highest dose (200 mg/mL) of CeO2 NPs. Results of some investigations are in accordance with our findings. Perreault et al45 reported significant increase in MN frequency at 12.5 mg/L CuO NPs after 24 hours treatment in Neuro 2A cells. The induction of chromosome damage and DNA lesion related to oxidative stress was observed in vitro in human dermal fibroblasts using CBMN and comet assay on exposure to the 7-nm CeO2 NPs at very low doses indicating the possible nanotoxocity and size effect of CeO2 NPs.48 Moreover, toxicity was observed within human hepatic carcinoma cells (HepG2), human colon carcinoma (CaCo2), and A549 cells when exposed to ceria NPs over a concentration range of 0.5 to 5000 mg/mL for 10 days and comet assay along with a cytotoxicity test were performed.49 In the same study, 24 hours exposure of ceria NPs to various cell lines did not induce DNA damage.49 These differences might be due to the difference in cell type and culture conditions.50 In NPs, toxicity ROS generation has been proposed as a possible
Downloaded from ijt.sagepub.com at Scientific library of Moscow State University on February 13, 2014
Kumari et al
11
mechanism involved51 and ROS play a major role in the genotoxicity.52 The data from the present study suggested that CeO 2 NPs induce ROS and oxidative stress leading to genotoxicity in IMR32 cell at higher doses.
Conclusion Overall, the results obtained in this study revealed that CeO2 NPs have size- and dose-dependent toxicity in the tested cell line whereas CeO 2 MPs did not induced any significant changes in the exposed cells. Moreover, CeO2 NPs did not induce toxicity below 100 mg/mL concentration and IMR32 cells were found to be less sensitive to CeO2 NPs. The cytotoxicity observed in the present study was in concurrence with oxidative stress and genotoxicity induced by CeO2 NPs. This investigation provides a better understanding of CeO2 NPs toxicity in IMR32 cells. However, further investigations are warranted on in vivo system in order to achieve a firm conclusion regarding the toxicity of CeO2 NPs and to get clear picture of its toxicokinetics and tolerance in the system. Acknowledgments Monika Kumari (SRF) is grateful to the University Grant Commission (UGC), India, and Shailendra Pratap Singh (SRF) is grateful to Indian Council of Medical Research (ICMR), India, for the award of a fellowship. The authors express their sincere thanks to the Director, IICT, Hyderabad, India, for providing facilities for this study.
Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by Asian Office of Aerospace Research and Development (AOARD), Japan under the Grant no. FA2386-11-1-4085.
References 1. Medina C, Santos-Martinez MJ, Radomski A, Corrigan OI, Radomski MW. Nanoparticles: pharmacological and toxicological significance. Br J Pharmacol. 2007;150(5):552-558. 2. Bucchianico SD, Fabbrizi MR, Misra SK, et al. Multiple cytotoxic and genotoxic effects induced in vitro by differently shaped copper oxide nanomaterials. Mutagenesis. 2013;28(3): 287-299. 3. Hu Z, Haneklaus S, Sparovek G, Schnug E. Rare earth elements in soils. Commun Soil Sci Plan. 2006;37(9-10):1381-1420. 4. Esch F, Fabris S, Zhou L, et al. Electron localization determines defect formation on ceria substrates. Science. 2005;309(5735): 752-755. 5. Wason MS, Zhao J. Cerium oxide nanoparticles: potential applications for cancer and other diseases. Am J Transl Res. 2013;5(2): 126-131.
6. Yu T, Park YI, Kang MC, et al. Large-scale synthesis of water dispersible ceria nano crystals by a simple sol–gel process and their use as a chemical mechanical planarization slurry. Eur J Inorg Chem. 2008;2008(6):855-858. 7. Wu W, Li S, Liao S, Xiang F, Wu X. Preparation of new sunscreen materials Ce1xZnxO2x via solid-state reaction at room temperature and study on their properties. Rare Metals. 2010;29(2):149-153. 8. Joo JH, Choi GM. Micro-solid oxide fuel cell using thick-film ceria. Solid State Ion. 2009;180(11-13):839-842. 9. Park B, Donaldson K, Duffin R, et al. Hazard and risk assessment of a nanoparticulate cerium oxide-based diesel fuel additive—a case study. Inhal Toxicol. 2008;20(6):547-566. 10. Masui T, Ozaki T, Machida K, Adachi G. Preparation of ceriazirconia sub-catalysts for automotive exhaust cleaning. J Alloys Compd. 2000;303-304(1):49-55. 11. Jan E, Byrne SJ, Cuddihy M, et al. High-content screening as a universal tool for fingerprinting of cytotoxicity of nanoparticles. ACS Nano. 2008;2(5):928-938. 12. Arnold MC, Badireddy AR, Wiesner MR, Di Giulio RT, Meyer JN. Cerium oxide nanoparticles are more toxic than equimolar bulk cerium oxide in Caenorhabditis elegans . Arch Environ Contam Toxicol. 2013;65(2):224-233. 13. Yokel RA, Au TC, MacPhail R, et al. Distribution, elimination, and biopersistance to 90 days of a systemically introduced 30 nm ceriaengineered nanomaterial in rats. Toxicol Sci. 2012;127(1):256-268. 14. Hussain S, Al-Nsour F, Rice AB, et al. Cerium dioxide nanoparticles induce apoptosis and autophagy in human peripheral blood monocytes. ACS Nano. 2012;6(7):5820-5829. 15. Lin W, Huang YW, Zhou XD, Ma Y. Toxicity of cerium oxide nanoparticles in human lung cancer cells. Int J Toxicol. 2006; 25(6):451-457. 16. Park EJ, Choi J, Park YK, Park K. Oxidative stress induced by cerium oxide nanoparticles in cultured BEAS-2B cells. Toxicology. 2008;245(1-2):90-100. 17. Xia T, Kovochich M, Liong M, et al. Comparison of the mechanism of toxicity of zinc oxide and cerium oxide nanoparticles based on dissolution and oxidative stress properties. ACS Nano. 2008; 2(10):2121-2134. 18. Schubert D, Dargusch R, Raitano J, Chan SW. Cerium and yttrium oxide nanoparticles are neuroprotective. Biochem Biophys Res Commun. 2006;342(1):86-91. 19. Niu JL, Azfer A, Rogers LM, Wang X, Kolattukudy PE. Cardioprotective effects of cerium oxide nanoparticles in a transgenic murine model of cardiomyopathy. Cardiovasc Res. 2007;73(3):549-559. 20. Hirst SM, Karakoti A, Singh S, et al. Bio-distribution and in vivo antioxidant effects of cerium oxide nanoparticles in mice. Environ Toxicol. 2013;28(2):107-118. 21. Kirsch-Volders M, Decordier I, Elhajouji A, Plas G, Aardema MJ, Fenech M. In vitro genotoxicity testing using the micronucleus assay in cell lines, human lymphocytes and 3D human skin models. Mutagenesis. 2011;26(1):177-184. 22. Fenech M. Cytokinesis-block micronucleus assay evolves into a ‘‘cytome’’ assay of chromosomal instability, mitotic dysfunction and cell death. Mutat Res. 2006;600(1-2):58-66. 23. Fenech M. Cytokinesis-block micronucleus cytome assay. Nat Protoc. 2007;2(5):1084-1104.
Downloaded from ijt.sagepub.com at Scientific library of Moscow State University on February 13, 2014
12
International Journal of Toxicology
24. Olive PL, Banath JP. The comet assay: a method to measure DNA damage in individual cells. Nat Protoc. 2006;1(1):23-29. 25. Hansen MB, Nielsen SE, Berg K. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J Immunol Methods. 1989;119(2):203-210. 26. Royall JA, Ischiropoulos H. Evaluation of 2,7-dichlorofluorescein and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells. Arch Biochem Biophys. 1993; 302(2):348-352. 27. Lee JY, Park W, Yi DK. Immunostimulatory effects of gold nanorod and silica-coated gold nanorod on RAW 264.7 mouse macrophages. Toxicol Lett. 2012;209(1):51-57. 28. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem. 1976;72:248-254. 29. Wills ED. Lipid peroxide formation in microsomes. Relation of hydroxylation to lipid peroxide formation. Biochem J. 1969; 113(2):333-341. 30. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959;82(1):70-77. 31. Marklund S, Marklund G. Involvement of superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem. 1974;47(3):469-474. 32. Aebi H. Catalase. In: Packer L, ed. Methods in Enzymology. Vol 105. Orlando, FL: Academic pres; 1984:121-126. 33. Carlberg I, Mannervik B. Glutathione reductase. Methods Enzymol. 1985;113:484-490. 34. Floche L, Gunzler WA. Assay of glutathione peroxidase. In: Packer L, ed. Methods Enzymol. Vol 105. New York: Academic Press; 1984:114-121. 35. Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferase. The first enzymatic step in mercapturic acid formation. J Biol Chem. 1974;249(22):7130-7139. 36. Organisation for Economic Co-operation and Development. Guideline for Testing of Chemicals: In Vitro Mammalian Cell Micronucleus Test (MNvit) (487). Paris: Organization for Economic Co-operation and Development; 2007. 37. Tice RR, Agurell E, Anderson D, et al. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen. 2000;35(3):206-221. 38. Zhou X, Wang B, Chen Y, Mao Z, Gao C. Uptake of cerium oxide nanoparticles and their influences on functions of A549 cells. J Nanosci Nanotechnol. 2013;13(1):204-215.
39. Rosenkranz P, Fernandez-Cruz ML, Conde E, et al. Effects of cerium oxide nanoparticles to fish and mammalian cell lines: an assessment of cytotoxicity and methodology. Toxicol In Vitro. 2012;26(6):888-896. 40. Kim IS, Baek M, Choi SJ. Comparative cytotoxicity of Al2O3, CeO2, TiO2 and ZnO nanoparticles to human lung cells. J Nanosci Nanotechnol. 2010;10(5):3453-3458. 41. Afonso V, Champy R, Mitrovic D, Collin P, Lomri A. Reactive oxygen species and superoxide dismutases: role in joint diseases. Joint Bone Spine. 2007;74(4):324-329. 42. Zhang H, He X, Zhang Z, et al. Nano-CeO2 exhibits adverse effects at environmental relevant concentrations. Environ Sci Technol. 2011;45(8):3725-3730. 43. Cheng G, Guo W, Han L, et al. Cerium oxide nanoparticles induce cytotoxicity in human hepatoma SMMC-7721 cells via oxidative stress and the activation of MAPK signaling pathways. Toxicol In Vitro. 2013;27(3):1082-1088. 44. Sies H. Glutathione and its role in cellular functions. Free Radic Biol Med. 1999;27(9-10):916-921. 45. Perreault F, Melegari SP, da Costa CH, Rossetto ALOF, Popovic R, Matias WG. Genotoxic effects of copper oxide nanoparticles in Neuro 2A cell cultures. Sci Total Environ. 2012;441:117-124. 46. Hussain SM, Hess KL, Gearhart JM, Geiss KT, Schlager JJ. In vitro toxicity of nanoparticles in BRL 3A rat liver cells. Toxicol In Vitro. 2005;19(7):975-983. 47. Kimura A, Miyata A, Honma M. A combination of in vitro comet assay and micronucleus test using human lymphoblastoid TK6 cells. Mutagenesis. 2013;28(5):583-590. 48. Auffan M, Rose J, Orsiere T, et al. CeO2 nanoparticles induce DNA damage towards human dermal fibroblasts in vitro . Nanotoxicology. 2009;3(2):161-171. 49. Marzi LD, Monaco A, Lapuente JD, et al. Cytotoxicity and genotoxicity of ceria nanoparticles on different cell lines in vitro . Int J Mol Sci. 2013;14(2):3065-3077. 50. Lanone S, Rogerieux F, Geys J, et al. Comparative toxicity of 24 manufactured nanoparticles in human alveolar epithelial and macrophage cell lines. Part Fibre Toxicol. 2009;6:14. 51. Gurr JR, Wang AS, Chen CH, Jan KY. Ultrafine titanium dioxide particles in the absence of photoactivation can induce oxidative damage to human bronchial epithelial cells. Toxicology. 2005; 213(1-2):66-73. 52. Schins RPF, Knaapen AdM. Genotoxicity of poorly soluble particles. Inhal Toxicol. 2007;19(s 1):189-198.
Downloaded from ijt.sagepub.com at Scientific library of Moscow State University on February 13, 2014
