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American Journal of Nanomaterials. 2015, 3(2), 40-56
DOI: 10.12691/ajn-3-2-1
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An integrated in Vitro and in Vivo Testing Approach to Assess Pulmonary Toxicity of Engineered Cadmium-Doped Silica Nanoparticles

Uliana De Simone1, Elisa Roda1, Cinzia Signorini2 and Teresa Coccini3,

1Department of Clinical Surgical, Diagnostic and Pediatric Sciences, Faculty of Medicine and Surgery, University of Pavia, Pavia, Italy

2Department of Pathophysiology, Experimental Medicine, and Public Health, University of Siena, Siena, Italy

3Laboratory of Clinical and Experimental Toxicology, Toxicology Division, IRCCS Salvatore Maugeri Foundation, Scientific Institute of Pavia Medical Centre, Pavia, Italy

Pub. Date: November 03, 2015

Cite this paper:
Uliana De Simone, Elisa Roda, Cinzia Signorini and Teresa Coccini. An integrated in Vitro and in Vivo Testing Approach to Assess Pulmonary Toxicity of Engineered Cadmium-Doped Silica Nanoparticles. American Journal of Nanomaterials. 2015; 3(2):40-56. doi: 10.12691/ajn-3-2-1


An in vitro and in vivo testing strategy for assessing the pulmonary effects was used to investigate the safety characteristics of silica nanoparticles doped with cadmium (Cd-SiNPs). In A549 cells, Cd-SiNPs (0.5-100 μg/ml) caused (i) mitochondrial dysfunction and apoptosis at 1 μg/ml, (ii) GSH depletion at 10μg/ml, (iii) membrane alterations at 25 μg/ml, after 1-day, and (iv) cell growth and proliferation inhibition at 0.05 μg/ml after prolonged exposure. Cd-SiNP effects were more pronounced compared to CdCl2. SiNPs affected GSH content only. In vivo results revealed early (1 day) and persistent (until 1 month) rat lung damage after intratracheal instillation of Cd-SiNPs (1mg/rat) in terms of enhanced apoptotic phenomena and altered lung parenchyma morphology. Cd-SiNPs and CdCl2 caused a delayed occurrence of oxidative stress by increasing SOD1, iNOS, and F2-IsoPs. The latter was preceded by marked increase of F2-IsoPs levels in plasma. SiNPs did not cause oxidative stress. Cd-SiNPs showed a higher reactivity than CdCl2 and SiNPs. In vitro and in vivo data on Cd-SiNP toxicity suggest that the lung is a susceptible target tissue. These findings support the concept that multiple assays and an integrated testing strategy should be recommended to characterize toxicological response to NPs.

pulmonary in vivo in vitro nanotoxicology silica cadmium

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[1]  Thomas K. and Sayre P, “Research strategies for safety evaluation of nanomaterials, Part I: evaluating the human health implications of exposure to nanoscale materials,” Toxicology Sciences, 87 (2). 316-321. 2005.
[2]  European Commission, Brussels, 3.10.2012, COM 572 final. Communication from the commission to the European Parliament, the Council and the European Economic and Social Committee. Second Regulatory Review on Nanomaterials, 2012.
[3]  EPA (2008 January), “Draft nanomaterial research strategy (NRS)”, EPA/600/S-08/002, 2008.
[4]  Food and drug Administration U.S.A., “A FDA prospective on nanomedicine current initiatives in the US”, 2010.
[5]  NIOSH (National Institute for Occupational Safety and Health), “Current Intelligence Bulletin: Occupational Exposure to Carbon Nanotubes and Nanofibers,”, 2010.
[6]  Warheit D.B., Borm, P.J.A., Hennes, C. and Lademann J, “Testing strategies to establish the safety of nanomaterials: conclusions of an ECETOC workshop,” Inhalation Toxicology, 19 (8). 631-643. 2007.
[7]  National Institute of Standard and technology (NIST), Htm.
[8]  Card, J.W., Zeldin, D.C., Bonner, J.C. and Nestmann, E.R, “Pulmonary applications and toxicity of engineered nanoparticles,” American Journal of Physiology. Lung Cellular and Molecular Physiology, 295 (3). L400-L411. 2008.
[9]  Donaldson, K., Li, X. and MacNee, W, “Ultrafine (nanometre) particle mediated lung injury,” Journal of Aerosol Science, 29 (5-6). 553-560. 1998.
[10]  Sajid, M., Ilyas, M., Basheer, C., Tariq, M., Daud, M., Baig, N. and Shehzad, F, “Impact of nanoparticles on human and environment: review of toxicity factors, exposures, control strategies, and future prospects,” Environmental Science and Pollution Research International, 22 (6). 4122-4143. 2015.
[11]  Miller, F., Asgharian, B. and Hofmann, W, Dosimetry of nanoparticles in humans: from children to adults, Gardner DE (ed.) Toxicol. Lung, 4th ed. CRC Press, Boca Raton, 2005, 151-194.
[12]  Mühlfeld, C., Gehr, P. and Rothen-Rutishauser, B, “Translocation and cellular entering mechanisms of nanoparticles in the respiratory tract,” Swiss Medical Weekly, 138 (27-28). 387-391. 2008.
[13]  Cho, W.S., Choi, M., Han, B.S., Cho, M., Oh, J., Park, K., Kim, S.J. and Kim, S.H., Jeong, J, “Inflammatory mediators induced by intratracheal instillation of ultrafine amorphous silica particles,” Toxicology Letters, 175 (1-3). 24-33. 2007.
[14]  Kaewamatawong, T., Shimada, A., Okajima, M., Inoue, H., Morita, T., Inoue, K. and Takano, H, “Acute and subacute pulmonary toxicity of low dose of ultrafine colloidal silica particles in mice after intratracheal instillation,” Toxicologic Pathology, 34 (7). 958-965. 2006.
[15]  Eom H.J. and Choi J, “Oxidative stress of silica nanoparticles in human bronchial epithelial cell, Beas-2B,” Toxicology in Vitro, vol. 23, no. 7, pp. 1326-1332, 2009.
[16]  Lin, W., Huang, Y.W., Zhou, X.D. and Ma, Y, “In vitro toxicity of silica nanoparticles in human lung cancer cells,” Toxicology and Applied Pharmacology, 217 (2). 252-259. 2006.
[17]  Panas, A., Marquardt, C., Nalcaci, O., Bockhorn, H., Baumann, W., Paur, H.R., Mülhopt, S., Diabaté, S. and Weiss, C, “Screening of different metal oxide nanoparticles reveals selective toxicity and inflammatory potential of silica nanoparticles in lung epithelial cells and macrophages,” Nanotoxicology, 7 (3). 259-273. 2013.
[18]  Park, E.J. and Park, K, “Oxidative stress and pro-inflammatory responses induced by silica nanoparticles in vivo and in vitro,” Toxicology Letters, 184 (1). 18-25. 2009.
[19]  Borm, P.J., Robbins, D., Haubold S., Kuhlbusch, T., Fissan, H., Donaldson, K., Schins, R., Stone, V., Kreyling, W., Lademann, J., Krutmann, J., Warheit, D. and Oberdorster, E, “The potential risks of nanomaterials: a review carried out for ECETOC,” Particle and Fibre Toxicology, 3.11. 2006.
[20]  Rzigalinski, B.A. and J. Strobl, S, “Cadmium-containing nanoparticles: perspectives on pharmacology and toxicology of quantum dots,” Toxicology and Applied Pharmacology, 238 (3). 280-288. 2009.
[21]  Mahmoudi, M., Serpooshan V. and Laurent, S, “Engineered nanoparticles for biomolecular imaging,” Nanoscale, 3 (8). 3007-3026. 2011.
[22]  Simovic, S., Ghouchi-Eskandar, N., Sinn, A.M., Losic, D. and Prestidge, C.A, “Silica materials in drug delivery applications,” Current Drug Discovery Technologies, vol. 8, no. 3, pp. 269-27., 2011.
[23]  Vivero-Escoto, J.L., Slowing, I.I., Trewyn, B.G. and Lin, V.S, “Mesoporous silica nanoparticles for intracellular controlled drug delivery,” Small, 6 (18). 1952-1967. 2010.
[24]  Napierska. D., Thomassen. L.C., Lison, D., Martens, J.A. and Hoet, P.H, “The nanosilica hazard: another variable entity” Particle and Fibre Toxicology, 7 (1). 39. 2010.
[25]  Oberdörster, G., Cherian, M.G. and Baggs, R.B, “Correlation between cadmium induced pulmonary carcinogenicity, metallothionein expression, and inflammatory processes: a species comparison,” Environmental Health Perspectives, 102 (3). 257-263. 1994.
[26]  IARC (International Agency for Research on Cancer), “Arsenic, metals, fibres, and dusts. A review of human carcinogens,” IARC Monographs, vol. 100 C, pp. 1-526, 2009.
[27]  National Toxicology Program U.S.A, “Report on Carcinogens,” Twelfth Edition, U.S. Department of Health and Human Services Public Health Service National Toxicology Program, 2011.
[28]  Shapiro, D.L., Nardone, L.L., Rooney S.A., Motoyama, E.K. and Munozm, J.L, “Phospholipid biosynthesis and secretion by a cell line (A549) which resembles type II aleveolar epithelial cells,” Biochimica et Biophysica Acta, 530 (2). 197-207. 1978.
[29]  Stringer, B., Imrich, A. and Kobzik, L, “Lung epithelial cell (A549) interaction with un opsonized environmental particulates: quantitation of particle specific binding and IL-8 production,” Experimental Lung Research., 22 (5). 495-508. 1996.
[30]  Stearns, R.C., Paulauskis, J.D. and Godleski, J.J, “Endocytosis of ultrafine particles by A549 cells,” American Journal of Respiratory Cell and Molecular Biology, 24 (2). 108-115. 2001.
[31]  Coccini, T., Barni, S., Vaccarone, R., Mustarelli, P., Manzo, L. and Roda, E, “Pulmonary toxicity of instilled cadmium-doped silica nanoparticles during acute and subacute stages in rats,” Histology and Histopathology, 28 (2). 195-209. 2013.
[32]  De Simone, U., Manzo, L., Profumo, A. and Coccini, T, “In vitro toxicity evaluation of engineered cadmium-coated silica nanoparticles on human pulmonary cells” Journal of Toxicology, 2013; 2013: 931785.
[33]  De Simone, U., Manzo, L., Ferrari, C., Bakeine, J., Locatelli, C. and Coccini, T, “Short and long-term exposure of CNS cell lines to BPA-f a radiosensitizer for Boron Neutron Capture Therapy: safety dose evaluation by a battery of cytotoxicity tests,” Neurotoxicology, 35. 84-90. 2013.
[34]  Damiano, V.V., Cherian, P.V., Frankel, F.R., Steeger, J.R., Sohn, M., Oppenheim, D. and Weinbaum, G, “Intraluminal fibrosis induced unilaterally by lobar instillation of CdCl2 into the rat lung,” The American Journal of Pathology, 137 (4). 883-894. 1990.
[35]  Driscoll, K.E., Maurer, J.K., Poynter, J., Higgins, J., Asquith, T. and Miller, N.S, “Stimulation of rat alveolar macrophage fibronectin release in a cadmium chloride model of lung injury and fibrosis,” Toxicology and Applied Pharmacology, 116 (1). 30-37. 1992.
[36]  Weibel, E.R, Stereological methods, Vol. 1: Practical methods for biological morphometry. Academic Press Inc. London 1979.
[37]  Signorini, C., Comporti, M. and Giorgi, G, “Ion trap tandem mass spectrometric determination of F2-isoprostanes,” Journal of Mass Spectrometry, 38 (10). 1067-1074. 2003.
[38]  Signorini, C., Ciccoli, L., Leoncini, S., Carloni, S., Perrone, S., Comporti, M., Balduini, W. and Buonocore, G, “Free iron, total F2-isoprostanes and total F4-neuroprostanes in a model of neonatal hypoxicischemic encephalopathy: neuroprotective effect of melatonin,” Journal of Pineal Research, 46 (2).148-154. 2009.
[39]  De Felice, C., Ciccoli, L., Leoncini, S., Signorini, C., Rossi, M., Vannuccini, L., Guazzi, G., Latini, G., Comporti, M., Valacchi, G. and Hayek, J, “Systemic oxidative stress in classic Rett syndrome,” Free Radical Biology & Medicine, 47 (4). 440-448. 2009.
[40]  Signorini, C., Perrone, S., Sgherri, C., Ciccoli, L., Buonocore, G., Leoncini, S., Rossi, V., Vecchio, D. and Comporti, M, “Plasma esterified F2-isoprostanes and oxidative stress in newborns: role of nonprotein-bound iron,” Pediatric Research, 63 (3). 287-291. 2008.
[41]  Soffler, C., Campbell, V.L. and Hassel, D.M, “Measurement of urinary F2- isoprostanes as markers of in vivo lipid peroxidation: a comparison of enzyme immunoassays with gas chromatography-mass spectrometry in domestic animal species,” Journal of Veterinary Diagnostic Investigation, 22 (22). 200-209. 2010.
[42]  Maurer-Jones, M.A. and Haynes, C.L, “Toward correlation in in vivo and in vitro nanotoxicology studies,” The Journal of Law, Medicine & Ethics, 40 (4). 795-801. 2013.
[43]  Driscoll, K.E., Costa, D.L., Hatch, G., Henderson, R., Oberdorster, G., Salem, H. and Schlesinger, R.B, “Intratracheal instillation as an exposure technique for the evaluation of respiratory tract toxicity: uses and limitations,” Toxicological Sciences, 55 (1). 24-25. 2000.
[44]  Oberdörster, G., Baumert, H.P. and Hochrainer, D, “The clearance of cadmium aerosols after inhalation exposure,” American Industrial Hygiene Association Journal, 40 (6). 443-450. 1979.
[45]  Oberdörster, G, Pulmonary deposition, clearance and effects of inhaled soluble and insoluble cadmium compounds, Nordberg GF, Herber RFM, Alessio L, eds. Cadmium in the human environment: Toxicity and carcinogenicity. Lyon: International Agency for Research on Cancer, 1992, 189-204.
[46]  Oberdörster, G., Cherian, M.G. and Baggs, R.B, “Importance of species differences in experimental pulmonary carcinogenicity of inhaled cadmium for extrapolation to humans,” Toxicology Letters, 72 (1-3). 339-343. 1994.
[47]  ATSDR (Agency for Toxic Substances and Disease Registry) “Toxicological profile for Cadmium (Draft for Public Comment)”, Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service, 2008.
[48]  Coccini, T., Roda, E., Barni, S., Signorini, C. and Manzo, L, “Long-lasting oxidative pulmonary insult in rat after intratracheal instillation of silica nanoparticles doped with cadmium,” Toxicology, 302 (2-3). 203-211. 2012.
[49]  Ochi, T., Takahashi, K. and Ohsawa, M, “Indirect evidence for the induction of a prooxidant state by cadmium chloride in cultured mammalian cells and a possible mechanism for the induction,” Mutation Research, vol. 180 (2). 257-266. 1987.
[50]  Chin, T.A., and Templeton, D.M, “Protective elevations of glutathione and metallothionein in cadmium-exposed mesangial cells,” Toxicology, 77 (1-2). 145-156. 1993.
[51]  Gaubin, Y., Vaissade, F., Croute, F., Beau, B., Soleilhavoup, J. and Murat, J, “Implication of free radicals and glutathione in the mechanism of cadmium-induced expression of stress proteins in the A549 human lung cell-line,” Biochimica et Biophysica Acta, 1495 (1). 4-13, 2000.
[52]  Sauvageau, J.A. and Jumarie, C, “Different mechanisms for metal-induced adaptation to cadmium in the human lung cell lines A549 and H441,” Cell Biology and Toxicology, 29 (3). 159-173. 2013.
[53]  Li, K.G., Chen, J.T., Bai, S.S., Wen, X., Song, S.Y., Yu, Q., Li, J. and Wang, Y.Q, “Intracellular oxidative stress and cadmium ions release induce cytotoxicity of unmodified cadmium sulfide quantum dots,” Toxicology In Vitro, 23 (6). 1007-1013. 2009.
[54]  Aoyagi, T., Hayakawa, K., Miyaji, K., Ishikawa, H. and Hata, M, “Cadmium nephrotoxicity and evacuation from the body in a rat modeled subchronic intoxication,” International Journal of Urology, 10 (6). 332-338. 2003.
[55]  Park, E.J., Yi, J., Kim, Y., Choi, K. and Park, K, “Silver nanoparticles induce cytotoxicity by a Trojan-horse type mechanism,” Toxicology In Vitro, 24 (3). 872-878. 2010.
[56]  Limbach, L.K., Wich, P., Manser, P., Grass, R.N., Bruinink, A. and Stark, W.J, “Exposure of engineered nanoparticles to human lung epithelial cells: influence of chemical composition and catalytic activity on oxidative stress,” Environmental Science & Technology, 41 (11). 4158-4163. 2007.
[57]  Jin, Y., Kannan, S., Wu, M. and Zhao, J.X, “Toxicity of luminescent silica nanoparticles to living cells,” Chemical Research in Toxicology, 20 (8). 1126-1133. 2007.
[58]  Shi, Y., Yadav, S., Wang, F. and Wang, H, “Endotoxin promotes adverse effects of amorphous silica nanoparticles on lung epithelial cells in vitro,” Journal of Toxicology and Environmental Health. Part A, 73 (11). 748-756. 2010.
[59]  Akhtar, M.J., Ahamed, M., Kumar, S., Siddiqui, H., Patil, G., Ashquin, M. and Ahmad, I, “Nanotoxicity of pure silica mediated through oxidant generation rather than glutathione depletion in human lung epithelial cells,” Toxicology, 276 (2). 95-102. 2010.
[60]  Choi, S.J., Oh, J.M. and Choy, J.H, “Toxicological effects of inorganic nanoparticles on human lung cancer A549 cells,” Journal of Inorganic Biochemistry, 103 (3). 463-471. 2009.
[61]  Henderson, R.F., Driscoll, K.E., Harkema, J.R., Lindenschmidt, R.C., Chang, I.Y., Maples, K.R. and Barr, E.B, “A comparison of the inflammatory response of the lung to inhaled versus instilled particles in F-344 rats,” Fundamental and Applied Toxicology, 24 (2). 183-197. 1995.
[62]  Jacobsen, N.R., Moller, P., Jensen, K.A., Vogel, U., Ladefoged, O., Loft, S. and Wallin, H, “Lung inflammation and genotoxicity following pulmonary exposure to nanoparticles in ApoE−/− mice,” Particle and Fibre Toxicology, 6 (2). doi: 10.1186/1743-8977-6-2. 2009.
[63]  Morimoto, Y., Hirohashi, M., Ogami, A., Oyabu, T., Myojo, T., Todoroki, M., Yamamoto, M., Hashiba, M., Mizuguchi, Y., Lee, B.W,, Kuroda, E., Shimada, M., Wang, W.N., Yamamoto ,K., Fujita, K., Endoh, S., Uchida, K., Kobayashi, N., Mizuno, K., Inada, M., Tao, H., Nakazato, T., Nakanishi, J. and Tanaka, I, “Pulmonary toxicity of well-dispersed multi-wall carbon nanotubes following inhalation and intratracheal instillation,” Nanotoxicology, 6 (6). 587-599. 2012.
[64]  Ogami, A., Morimoto, Y., Myojo, T. and Oyabu, T, “Biological effect of fullerene (C60) to lung by inhalation or instillation,” Journal of UOEH, 34 (1). 65-75. 2012.
[65]  Jackson, P., Hougaard, K.S., Boisen, A.M., Jacobsen, N.R., Jensen, K.A., Møller, P., Brunborg, G., Gutzkow, K.B., Andersen, O., Loft, S., Vogel, U. and Wallin, H, “Pulmonary exposure to carbon black by inhalation or instillation in pregnant mice: effects on liver DNA strand breaks in dams and offspring,” Nanotoxicology, 6 (5). 486-500. 2012.
[66]  Baisch, B.L., Corson, N.M, Wade-Mercer, P., Gelein, R., Kennell, A.J., Oberdörster, G. and Elder, A1, “Equivalent titanium dioxide nanoparticle deposition by intratracheal instillation and whole body inhalation: the effect of dose rate on acute respiratory tract inflammation,” Particle and Fibre Toxicology, 2014.
[67]  Roberts, J.R., Antonini, J.M., Porter, D.W., Chapman, R.S., Scabilloni, J.F., Young, S.H., Schwegler-Berry, D., Castranova, V. and Mercer, R.R, “Lung toxicity and biodistribution of Cd/Se-ZnS quantum dots with different surface functional groups after pulmonary exposure in rats,” Particle and Fibre Toxicology. 2013.
[68]  Bolognin, M., Kirschvink, N., Leemans, J., De Buscher, V., Snaps, F., Gustin, P., Peeters, D. and Clercx, C, “Characterization of the acute and reversible airway inflammation induced by cadmium chloride inhalation in healthy dogs and evaluation of the effects of salbutamol and prednisolone,” Veterinary Journal, 179 (3). 443-50. 2009.
[69]  Blum, J.L., Rosenblum, L.K., Grunig, G., Beasley, M.B., Xiong, J.Q. and Zelikoff, J.T, “Short-term inhalation of cadmium oxide nanoparticles alters pulmonary dynamics associated with lung injury, inflammation, and repair in a mouse model,” Inhalation Toxicology, 26 (1). 48-58. 2014.
[70]  Sayes, C.M., Reed, K.L. and Warheit, D.B, “Assessing toxicity of fine and nanoparticles: comparing in vitro measurements to in vivo pulmonary toxicity profiles,” Toxicological Sciences, 97 (1). 163-80. 2007.
[71]  Han, Y.G., Xu, J., Li, Z.G., Ren, G.G. and Yang, Z, “In vitro toxicity of multi-walled carbon nanotubes in C6 rat glioma cells,” NeuroToxicology, 33 (5). 1128-1134. 2012.