SiO₂ Nanoparticles Induced Cytotoxicity by Oxidative Stress in Human Bronchial Epithelial Cell, Beas-2B

Article information

Environ Health Toxicol. 2011;26.e2011013

Publication date (electronic) : 2011 September 08

doi : https://doi.org/10.5620/eht.2011.26.e2011013

School of Environmental Engineering, College of Urban Science, University of Seoul, Seoul, Korea.

Correspondence: Jinhee Choi, PhD. 90 Jeonnong-dong, Dongdaemun-gu, Seoul 130-743, Korea. Tel: +82-2-2210-5622, Fax: +82-2-2244-2245, jinhchoi@uos.ac.kr

Received 2011 May 11; Accepted 2011 July 28.

Abstract

Objectives

In this study, we investigated the potential harmful effect of the exposure to silicon dioxide (SiO₂) nanoparticles through in vitro toxicity assay using human bronchial epithelial cell, Beas-2B with a focus on the involvement of oxidative stress as the toxic mechanism.

Methods

SiO₂-induced oxidative stress was assessed by examining formation of reactive oxygen species (ROS), the induction of superoxide dismutase (SOD) and heme oxygenase-1 (HO-1), as well as cytotoxicity effect was evaluation by cell viability. Subsequently, to understand the molecular mechanism of nanoparticle-induced oxidative stress, the involvement of oxidative stress-responding transcription factors, such as, nuclear factor-kappaB (NF-κB) and nuclear factor-E2-related factor-2 (Nrf-2), and mitogen-activated protein (MAP) kinase signal transduction pathway was also investigated.

Results

5-d i phenyltera zolium bromide (MTT) assay results show that decrease 20% in cell viability and the number of cells in the subG1 phase increased. The increase in ROS formation was observed in SiO₂ nanoparticle treated cells. The expression of SOD protein was not changed, whereas that of HO-1 was increased by SiO₂ nanoparticle exposure. transcription factors Nrf-2 and the expression of phosphorylated form of extracellular signal-regulating kinase (ERK) was strongly induced by SiO₂ nanoparticle exposure

Conclusions

SiO₂ nanoparticles exert their toxicity through oxidative stress as they cause the significant increase ROS level. SiO₂ nanoparticles induce induction of HO-1 via Nrf-2-ERK MAP kinase pathway. Our tested oxidative stress parameters are rather limited in terms of allowing the full understanding of oxidative stress and cellular response by SiO₂ nanoparticle exposure.

Keywords: Extracellular signal-regulating kinase; Heme oxygenase-1; Nuclear factor-E2-related factor-2; Oxidative stress; Silicon dioxide nanoparticles

INTRODUCTION

In recent years, as nanotechnology has rapidly developed, nanomaterials have been widely used in the fields of biomedicine, pharmaceutical, and other industry. Of the various manufactured nanomaterials, silicon dioxide (SiO₂) nanoparticles have the potential for widespread applications. SiO₂ nanoparticles are being used in these fields such as chemical mechanical polishing and as additives to drugs, cosmetics, printer toners, varnishes, and food [1,2]. In recent years, the use of SiO₂ nanoparticles has been extended to biomedical and biotechnological fields, such as biosensors for simultaneous assay of glucose, lactate, L-glutamate, and hypoxanthine levels in rat striatum [3], biomarkers for leukemia [4,5], DNA delivery [6,7], targeted drug delivery [8] and controlled drug release for genes and proteins [9,10]

Considering their wide range of applications, the potential adverse effect of SiO₂ nanoparticles on human health and on the environment is of great interest. In this study, we investigated the potential harmful effect of exposure to SiO₂ nanoparticles by conducting an in vitro toxicity assay with a focus on the involvement of oxidative stress. Numerous previous studies on nanoparticle toxicity, with various cell types and various nanoparticle types, reported that oxidative stress is one of the most important toxicity mechanisms related to exposure to nanoparticles [11-15]. Indeed, previous studies reported oxidative stress as the toxic mechanism of SiO₂ [13,16-18]

In this study, to understand the potential harmful effect of nanoparticles on human health, the oxidative stress-related toxicity was investigated by exposure to SiO₂ nanoparticles. SiO₂-induced oxidative stress was assessed by examining formation of reactive oxygen species (ROS), the induction of superoxide dismutase (SOD) and heme oxygenase-1 (HO-1), as well as cytotoxicity effect was evaluation by cell viability. Subsequently, to understand the molecular mechanism of nanoparticle-induced oxidative stress, the involvement of oxidative stress-responding transcription factors, such as, nuclear factor-kappaB (NF-κB) and nuclear factor-E2-related factor-2 (Nrf-2), and mitogen-activated protein (MAP) kinase signal transduction pathway was also investigated.

MATERIALS AND METHODS

I. Cell Culture and Nanoparticle Treatment

Human bronchial epithelial cells, Beas-2B, were maintained in DMEM / F12 (GIBCO BRL Life Technologies, Rockville, MD, USA), supplemented with 10% (v/v) fetal bovine serum and 1% antibiotics, at 37℃ in a humidified atmosphere of air and 5% CO₂. SiO₂ nanoparticles were purchased from Sigma (St. Louis, MO, USA). Test solution of SiO₂ nanoparticles were prepared in the culture media and dispersed for 20 minutes using a sonicator (Branson Inc., Danbury, CT, USA) to prevent aggregation. During the testing periods, the suspension of nanoparticles was stable and uniform in the culture media. For surface area measurement, Branauer, Emmett & Teller (BET) method was used with BELSORP-mini II, a volumetric adsorption apparatus (BEL Japan, Inc., Osaka, Japan). To investigate the size and shape of SiO₂ nanoparticles, 20 uL of particle suspension form test media was dried on a 400 mesh carbon-coated copper grid and imaged with a JEM 1010 transmission electron microscopy (TEM; JEOL, Tokyo, Japan) at 40-100 kV. The size distribution of nanoparticles was evaluated using a Photal dynamic light scattering (DLS) spectrometer, DLS-7000 (Otsuka Electronics Co., Inc., Osaka, Japan). The concentration we used in this study was 1 mg/L to prevent aggregation and/or precipitation of particles. The cells were treated with various sizes of nanoparticles for 24 hours for toxicological studies.

II. Cell Viability Assay

Beas-2B cells were plated on 96-well plates and the plates were incubated with the nanoparticles for 24 hours. Cell viability was measured using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-di phenyltera zolium bromide (MTT) reagent [19].

III. Flow Cytometry

Flow cytometry was performed on the treated and control cells for analysis of the cell cycle and apoptosis. Propidium iodide (PI) stained cells were analyzed using a flow cytometer (BD Science, San Jose, CA, USA) [20]. The effect on apoptosis was determined by the increase in the proportion of subG1 hypo-diploid cells.

IV. Formation of ROS

To detect ROS generation in nanoparticles-treated cells, a fluorometric assay using intracellular oxidation of 2,7-dichlorofluoroscein diacetate (DCFH-DA, Sigma) was performed [21,22]. Cells grown to confluence were treated with SiO₂ nanoparticles for 24 hours, washed with PBS, and then incubated with DCFH-DA (40 uM) for 30 minutes. Following DCFH-DA incubation, the fluorescence of dichlorofluoroscein (DCF), which is the oxidized product of DCFH-DA, was visualized using a fluorescent microscope (Nikon, Tokyo, Japan) with excitation and emission wavelengths of 485 and 530 nm, respectively.

V. Western Blotting

For western blotting analysis, aliquots of the cell lysates were subjected to electrophoresis on a 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel, and the gel transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membranes were incubated with a primary antibody for 1 hours, subjected to further incubation with a secondary antibody and then exposed to X-ray film. Protein bands were detected using an enhanced chemiluminescence western blotting detection kit (Amersham, Little Chalfont, Buckinghamshire, UK). The antibodies for the detection of p38, phosphorylated p38, ERK-2, phosphorylated ERK and Nrf-2 were purchased from Santa Cruz (Santa Cruz, CA, USA), antibodies for JNK, phosphorylated JNK were from Cell Signaling (Beverly, MA, USA), antibody for HO-1 was from Stressgen (Victorya, BC, Canada) and antibody for Cu/Zn SOD was from Biodesign (Saco, ME, USA).

VI. Data Analysis

Statistical differences between the control and treated cells were examined with the aid of a parametric t test using SPSS version 12.0 KO (SPSS Inc., Chicago, Il, USA). An alpha level of 0.05 was used to determine significance in all statistical analyses.

RESULTS

I. Characterization of SiO₂ Nanoparticles

The result from characterization of SiO₂ nanoparticles was summarized in Figure 1. The BET surface areas of was 201.01 m³/g, respectively. The TEM images indicated that silica nanoparticles had an even distribution with 20-40 nm individual particle sizes. The size distribution in the test medium was investigated using a DLS method, as the size of the nanoparticles distributed in the test medium were about 25 and 40 nm.

Figure 1

Characterization of different sized SiO₂ nanoparticles using TEM and DLS methods. Particles shape were analysed by TEM (A) and the size distribution in the test media were evaluated by DLS (B).

SiO₂: silicon dioxide, TEM: transmission electron microsopy, DLS: dynamic light scattering.

II. Cytotoxicity of SiO₂ Nanoparticles

To investigate of SiO₂ nanoparticle induced cytotoxicity effects on Beas-2B cells, cell viability and apoptosis were examined. MTT assay results show that decrease in cell viability was observed only about 20% compared with that of the control by SiO₂ exposure (Figure 2). Flow cytometry analysis was conducted using PI staining to quantify the number of cells with a subdiploid DNA content (Figure 3). In Beas2B cells exposed to SiO₂ nanoparticles, the number of cells in the subG1 phase increased. The degree of increase induced by SiO₂ nanoparticles was about 3-fold compared with that of the control.

Figure 2

The effect of 1 mg/L SiO₂ nanoparticles on the cell viability investigated using MTT assay. Results were presented as relative units compared to control.

SiO₂: silicon dioxide, MTT: 5-d i phenyltera zolium bromide.

Figure 3

Flow cytometry analysis to quantify the number of cells with a subdiploid DNA content in 1 mg/L SiO₂ nanoparticles exposed Beas-2B cells. H₂O₂ (100 uM) was used as positive control.

SiO₂: silicon dioxide.

III. SiO₂ Nanoparticles Induced Oxidative Stress

The formation of ROS was visualized in Beas2-B cells exposed to SiO₂ nanoparticles by inspection under fluorescent microscope (Figure 4). DCFH-DA staining revealed increased concentration of ROS in SiO₂ nanoparticles treated cells. As the increase in ROS formation was observed in SiO₂ nanoparticle treated cells (Figure 4), a marker for cellular defense mechanism against oxidative stress (i.e. induction of antioxidant enzymes) was investigated in Beas2-B cells exposed to SiO₂ nanoparticles (Figure 5). The expression of SOD protein was not changed, whereas that of HO-1 was increased by SiO₂ nanoparticle exposure.

Figure 4

ROS induced by nanoparticles in Beas-2B cells. The cells were incubated with 1 mg/L SiO₂ nanoparticles for 24 hours and 40 uM DCFH-DA at 37℃ for 30 min and observed fluorescence microscope. H₂O₂ (100 uM) was used as positive control.

ROS: reactive oxygen species, DCFH-DA: 2,7-dichlorofluoroscein diacetate, SiO₂: silicon dioxide.

Figure 5

Expression of HO-1 and Cu/Zn SOD in Beas-2B cells exposed to 1 mg/L SiO₂ nanoparticles Densitometric values of expression of HO-1 and Cu/Zn SOD were normalized using that of Actin and were presented as relative units compared to control. The control value was set to 1; data represent the mean ± standard error of the mean; n=3.

SOD: superoxide dismutase, SiO2: silicon dioxide,

HO-1: heme oxygenase-1.

^*p < 0.05 (B).

IV. Oxidative Signaling Pathway

SiO₂ increased formation of ROS and the induction of antioxidant enzyme, HO-1 (Figure 4,5). In order to understand the molecular mechanism of the observed oxidative stress and related physiological alteration by SiO₂ nanoparticles exposure, NF-κB and Nrf-2 were examined in the cytosolic and nuclear fraction of cells treated with SiO₂ nanoparticles. Neither nuclear localization of NF-κB nor the degradation of cytosolic I-κB was observed, whereas nuclear localization of Nrf-2 increased in cells treated with SiO₂ nanoparticles (Figure 6). In our study, SiO₂ nanoparticles induced the translocation of Nrf-2 into the nucleus as evidenced by the results of the western blot analysis (Figure 6).

Figure 6

Expression of Nrf-2 in cytosolic and nuclear fraction of Beas-2B cells exposed to 1 mg/L SiO₂ nanoparticles. Densitometric values of expression of Nrf-2 were normalized using that of Actin and were presented as relative units compared to control.

Nrf: nuclear factor-E2-related factor-2, SiO₂: silicon dioxide.

To further clarify the possible upstream oxidative signaling pathway involved in Nrf-2 activation leading to HO-1 induction, we examined the activation of MAP kinases, known as major signaling kinases, involved in cell survival against oxidative stress through the Nrf-2 signaling pathway (Figure 7). The expression of unphosphorlyated forms of MAP kinases was constant regardless of exposure of SiO₂ nanoparticles, whereas, the expression of phosphorylated form of ERK was strongly induced by SiO₂ nanoparticles. The phosphorylation of ERK was significant by SiO₂ nanoparticles (about 5-fold compared with that of the control). The expression level of phosphorylated p38 or JNK was not changed by SiO₂ nanoparticles.

Figure 7

Expression of intact and phosphorylated ERK, p38 and JNK in Beas-2B cells exposed to 1 mg/L SiO₂ nanoparticles. Densitometric values of expression of ERK, p38 and JNK were normalized using that of Actin and were presented as relative units compared to control. The control value was set to 1; data represent the mean ± standard error of the mean; n=3.

ERK: extracellular signal-regulating kinase, JNK: c-Jun N-terminal kinase, SiO₂: silicon dioxide.

^*p < 0.05, ^†p < 0.01.

DISCUSSION

There are many studies have reported that nanoparticle toxicity deal with oxidative stress [11-14,23-27]. However, the mechanism by which oxidative stress is involved in nanotoxicity has been poorly addressed. We measured the ROS level (Figure 4), as well as the induction of antioxidant enzyme (Figure 5), provided strong evidence for the involvement of oxidative stress in SiO₂ nanoparticles-induced cytotoxicity. To understand the molecular mechanism of the observed SiO₂ nanoparticle-induced cytotoxicity by oxidative stress, activation of transcription factors (i.e NF-κB, Nrf-2) and signal transduction pathway (i.e. MAP kinase pathway) investigated.

In this study, to provide cellular consequence of the oxidative stress response observed (Figure 4-7), and cytotoxicity test were conducted (Figure 2,3). Exposed to SiO₂ nanoparticles seemed to affect the cell viability and apoptosis by induction of ROS. ROS production was investigated as an initial step of oxidative stress, which revealed an increase in ROS formation in the cells exposed to SiO₂ compared to control.

Neither nuclear localization of NF-κB nor the degradation of cytosolic I-κB was observed, whereas nuclear localization of Nrf-2 increased in cells treated with SiO₂ nanoparticles (Figure 6). In our study, SiO₂ nanoparticles induced the translocation of Nrf-2 into the nucleus as evidenced by the results of the western blot analysis (Figure 6). The translocation of Nrf-2 into the nucleus following nanoparticles treatment was associated with the increase in HO-1 protein, which suggests that nanoparticles activate Nrf-2 in association with the upregulation of HO-1 in Beas2-B cells. However, in this study, SiO₂ nanoparticle-induced NF-κB activation was not observed, which was unexpected, as NF-κB is the major stress response transcription factor that has been reported to respond to a wide variety of environment stressors. Therefore, although SiO₂-induced NF-κB activation was not observed in this study, the relative importance of NF-κB vs Nrf-2 signaling in terms of contribution to antioxidant response, such as HO-1 upregulation, may merit further investigation with various oxidative stress-inducing nanoparticles. To confirm SiO₂ nanoparticles-induced for activation of transcription factor, more direct evidence, using electrophoretic mobility shift assay (EMSA) may be needed.

The upstream signaling mechanism responsible for regulating oxidative stress is poorly defined. Most studies have focused that oxidative stress may evoke the induction of antioxidant-related transcription factors, such as Nrf-2 or NF-kB via basal signal transduction systems, such as MAP kinase cascade. The MAP kinase cascades are multifunctional signaling pathways that are evolutionally well conserved in all eukaryotic cells. Three MAP kinase cascades that converge on ERKs, JNKs, and p38 MAP kinases have already been characterized [28,29]. Two of those three MAP kinase cascades converge on JNKs and p38 MAP kinases are preferentially activated by cytotoxic stresses, such as X-ray/UV irradiation, heat/osmotic shock, and oxidative/nitrosative stress [30-33]. To further clarify the possible upstream oxidative signaling pathway involved in Nrf-2 activation leading to HO-1 induction, we examined the activation of MAP kinases, known as major signaling kinases, involved in cell survival against oxidative stress through the Nrf-2 signaling pathway (Figure 7). The expression of unphosphorlyated forms of MAP kinases was constant regardless of exposure of SiO₂ nanoparticles, whereas, the expression of phosphorylated form of ERK was strongly induced by SiO₂ nanoparticles.

Activation of ERK MAP kinase pathway by SiO₂nanoparticles suggests that the induction of HO-1 may be mediated through Nrf-2-ERK MAP kinase signaling pathway. ERK has been known to respond to internal stimuli, such as growth factors. However, our study revealed that SiO₂ nanoparticles strongly induce the phosphorylation of ERK. Increase expression of phosphorylated ERK was also observed in our previous study with CeO₂ nanoparticles exposure [34]. These results suggest that the ERK signaling pathway also responds to environmental stressors. The activation of the ERK signaling pathway by external stimuli has already been reported [30-33].

CONCLUSIONS

SiO₂ nanoparticles exert their toxicity through oxidative stress as they cause the significant increase in cellular H₂O₂ concentrations. SiO₂ nanoparticles induce induction of HO-1 via Nrf-2-ERK MAP kinase pathway. Our tested oxidative stress parameters are rather limited in terms of allowing the full understanding of oxidative stress and cellular response by SiO₂ nanoparticle exposure. Further studies on the mechanism by which SiO₂ nanoparticles induce the Nrf2-ERK MAP kinase pathway are warranted to better understand the nanoparticle-induced cytotoxicity by oxidative stress, as are studies with dose-response and time-course studies.

ACKNOWLEDGEMENTS

This work was supported by the Midcareer Researcher Program through National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0027722)

Notes

The authors have no conflict of interest to declare on this study.

This article is available from: http://e-eht.org/

References

1. Lu J, Liong M, Zink JI, Tamanoi F. Mesoporous silica nanoparticles as a delivery system for hydrophobic anticancer drugs. Small 2007;3(8):1341–1346. 17566138.

2. Zhao J, Wu P, Brancewicz C, Li Y. A liposome-containing slurry for tungsten chemical mechanical polishing. J Electrochem Soc 2007;154(3):H225–H230.

3. Zhang FF, Wan Q, Li CX, Wang XL, Zhu ZQ, Xian YZ, et al. Simultaneous assay of glucose, lactate, L-glutamate and hypoxanthine levels in a rat striatum using enzyme electrodes based on neutral red-doped silica nanoparticles. Anal Bioanal Chem 2004;380(4):637–642. 15517210.

4. Santra S, Zhang P, Wang K, Tapec R, Tan W. Conjugation of biomolecules with luminophore-doped silica nanoparticles for photostable biomarkers. Anal Chem 2001;73(20):4988–4993. 11681477.

5. Hirsch LR, Stafford RJ, Bankson JA, Sershen SR, Rivera B, Price RE, et al. Nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance. Proc Natl Acad Sci U S A 2003;100(23):13549–13554. 14597719.

6. Bharali DJ, Klejbor I, Stachowiak EK, Dutta P, Roy I, Kaur N, et al. Organically modified silica nanoparticles: a nonviral vector for in vivo gene delivery and expression in the brain. Proc Natl Acad Sci U S A 2005;102(32):11539–11544. 16051701.

7. Gemeinhart RA, Luo D, Saltzman WM. Cellular fate of a modular DNA delivery system mediated by silica nanoparticles. Biotechnol Prog 2005;21(2):532–537. 15801794.

8. Zhao W, Gu J, Zhang L, Chen H, Shi J. Fabrication of uniform magnetic nanocomposite spheres with a magnetic core/mesoporous silica shell structure. J Am Chem Soc 2005;127(25):8916–8917. 15969545.

9. Roy I, Ohulchanskyy TY, Bharali DJ, Pudavar HE, Mistretta RA, Kaur N, et al. Optical tracking of organically modified silica nanoparticles as DNA carriers: a nonviral, nanomedicine approach for gene delivery. Proc Natl Acad Sci U S A 2005;102(2):279–284. 15630089.

10. Slowing II, Vivero-Escoto JL, Wu CW, Lin VS. Mesoporous silica nanoparticles as controlled release drug delivery and gene transfection carriers. Adv Drug Deliv Rev 2008;60(11):1278–1288. 18514969.

11. Shvedova AA, Castranova V, Kisin ER, Schwegler-Berry D, Murray AR, Gandelsman VZ, et al. Exposure to carbon nanotube material: assessment of nanotube cytotoxicity using human keratinocyte cells. J Toxicol Environ Health A 2003;66(20):1909–1926. 14514433.

12. Green M, Howman E. Semiconductor quantum dots and free radical induced DNA nicking. Chem Commun (Camb) 2005;(1):121–123. 15614393.

13. Lin W, Huang YW, Zhou XD, Ma Y. In vitro toxicity of silica nanoparticles in human lung cancer cells. Toxicol Appl Pharmacol 2006;217(3):252–259. 17112558.

14. Monteiller C, Tran L, MacNee W, Faux S, Jones A, Miller B, et al. The pro-inflammatory effects of low-toxicity low-solubility particles, nanoparticles and fine particles, on epithelial cells in vitro: the role of surface area. Occup Environ Med 2007;64(9):609–615. 17409182.

15. Eom HJ, Choi J. p38 MAPK activation, DNA damage, cell cycle arrest and apoptosis as mechanisms of toxicity of silver nanoparticles in Jurkat T cells. Environ Sci Technol 2010;44(21):8337–8342. 20932003.

16. Kaewamatawong T, Shimada A, Okajima M, Inoue H, Morita T, Inoue K, et al. Acute and subacute pulmonary toxicity of low dose of ultrafine colloidal silica particles in mice after intratracheal instillation. Toxicol Pathol 2006;34(7):958–965. 17178696.

17. Park EJ, Park K. Oxidative stress and pro-inflammatory responses induced by silica nanoparticles in vivo and in vitro. Toxicol Lett 2009;184(1):18–25. 19022359.

18. Yang X, Liu J, He H, Zhou L, Gong C, Wang X, et al. SiO₂ nanoparticles induce cytotoxicity and protein expression alteration in HaCaT cells. Part Fibre Toxicol 2010;7:1. 20180970.

19. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65(1-2):55–63. 6606682.

20. Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 1991;139(2):271–279. 1710634.

21. Fotakis G, Cemeli E, Anderson D, Timbrell JA. Cadmium chloride-induced DNA and lysosomal damage in a hepatoma cell line. Toxicol In Vitro 2005;19(4):481–489. 15826806.

22. Elbekai RH, El-Kadi AO. The role of oxidative stress in the modulation of aryl hydrocarbon receptor-regulated genes by As3+, Cd2+, and Cr6+. Free Radic Biol Med 2005;39(11):1499–1511. 16274885.

23. Xia T, Korge P, Weiss JN, Li N, Venkatesen MI, Sioutas C, et al. Quinones and aromatic chemical compounds in particulate matter induce mitochondrial dysfunction: implications for ultrafine particle toxicity. Environ Health Perspect 2004;112(14):1347–1358. 15471724.

24. 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. 16125895.

25. Sayes CM, Gobin AM, Ausman KD, Mendez J, West JL, Colvin VL. Nano-C60 cytotoxicity is due to lipid peroxidation. Biomaterials 2005;26(36):7587–7595. 16005959.

26. Foster KA, Galeffi F, Gerich FJ, Turner DA, Müller M. Optical and pharmacological tools to investigate the role of mitochondria during oxidative stress and neurodegeneration. Prog Neurobiol 2006;79(3):136–171. 16920246.

27. Limbach LK, Wick P, Manser P, Grass RN, Bruinink A, Stark WJ. Exposure of engineered nanoparticles to human lung epithelial cells: influence of chemical composition and catalytic activity on oxidative stress. Environ Sci Technol 2007;41(11):4158–4163. 17612205.

28. Kyriakis JM, Avruch J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 2001;81(2):807–869. 11274345.

29. Takeda K, Matsuzawa A, Nishitoh H, Ichijo H. Roles of MAPKKK ASK1 in stress-induced cell death. Cell Struct Funct 2003;28(1):23–29. 12655147.

30. Roberts ES, Richards JH, Jaskot R, Dreher KL. Oxidative stress mediates air pollution particle-induced acute lung injury and molecular pathology. Inhal Toxicol 2003;15(13):1327–1346. 14569496.

31. Liu JP, Schlosser R, Ma WY, Dong Z, Feng H, Lui L, et al. Human alphaA- and alphaB-crystallins prevent UVA-induced apoptosis through regulation of PKCalpha, RAF/MEK/ERK and AKT signaling pathways. Exp Eye Res 2004;79(6):393–403.

32. Kim J, Sharma RP. Cadmium-induced apoptosis in murine macrophages is antagonized by antioxidants and caspase inhibitors. J Toxicol Environ Health A 2006;69(12):1181–1201. 16728380.

33. Iles KE, Dickinson DA, Wigley AF, Welty NE, Blank V, Forman HJ. HNE increases HO-1 through activation of the ERK pathway in pulmonary epithelial cells. Free Radic Biol Med 2005;39(3):355–364. 15993334.

34. Eom HJ, Choi J. Oxidative stress of CeO₂ nanoparticles via p38-Nrf-2 signaling pathway in human bronchial epithelial cell, Beas-2B. Toxicol Lett 2009;187(2):77–83. 19429248.

Article information Continued

(open-access, http://creativecommons.org/licenses/by-nc/3.0) :

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.