Abstract
In this paper, we investigated the toxicological behavior of metal nanoparticles (gold, silver) and metal oxide nanoparticles (copper oxide, zinc oxide, titanium dioxide) in vitro in human colorectal adenocarcinoma cells (HT29). We analyzed the cellular uptake by ICP-MS and TEM, the influence on cell viability by MTT assay and trypan blue exclusion test, their effect on DNA damage and/or generation of oxidized bases by alkaline comet assay, and their potential to induce apoptosis by flow cytometry after 24-h nanoparticle treatment with concentrations between 2 and 10 µg/ml. We determined the amount of metal taken up by a single HT29 cell, ranging from 0.02 pg/cell up to 1.39 pg/cell. Cell viability assays showed a significantly decrease for metal oxide nanoparticles using trypan blue exclusion test and for all nanoparticles, except titanium dioxide, using MTT assay. Genotoxic effects after nanoparticle treatment were not observed for the tested concentrations. Apoptosis induction was significantly increased for silver nanoparticles (tested for two sizes) as well as for titanium dioxide and zinc oxide nanoparticles. Our results indicate potential health risks of oral NP uptake by food ingredients or food contamination, making further mechanistic investigations on cellular uptake and toxicity necessary.
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References
Aueviriyavit S, Phummiratch D, Maniratanachote R (2014) Mechanistic study on the biological effects of silver and gold nanoparticles in Caco-2 cells–induction of the Nrf2/HO-1 pathway by high concentrations of silver nanoparticles. Toxicol Lett 224(1):73–83. doi:10.1016/j.toxlet.2013.09.020
Azqueta A, Arbillaga L, Lopez de Cerain A, Collins A (2013) Enhancing the sensitivity of the comet assay as a genotoxicity test, by combining it with bacterial repair enzyme FPG. Mutagenesis 28(3):271–277. doi:10.1093/mutage/get002
Böhmert L, Niemann B, Thunemann AF, Lampen A (2012) Cytotoxicity of peptide-coated silver nanoparticles on the human intestinal cell line Caco-2. Arch Toxicol 86(7):1107–1115. doi:10.1007/s00204-012-0840-4
Borowicki A, Stein K, Scharlau D et al (2010) Fermented wheat aleurone inhibits growth and induces apoptosis in human HT29 colon adenocarcinoma cells. Br J Nutr 103(3):360–369. doi:10.1017/S0007114509991899
Botelho MC, Costa C, Silva S et al (2014) Effects of titanium dioxide nanoparticles in human gastric epithelial cells in vitro. Biomed Pharmacother 68(1):59–64. doi:10.1016/j.biopha.2013.08.006
Bouwmeester H, Dekkers S, Noordam MY et al (2009) Review of health safety aspects of nanotechnologies in food production. Regul Toxicol Pharmacol 53(1):52–62. doi:10.1016/j.yrtph.2008.10.008
Bumbudsanpharoke N, Ko S (2015) Nano-food packaging: an overview of market, migration research, and safety regulations. J Food Sci 80(5):R910–R923. doi:10.1111/1750-3841.12861
Demir E, Creus A, Marcos R (2014) Genotoxicity and DNA repair processes of zinc oxide nanoparticles. J Toxicol Environ Health A 77(21):1292–1303. doi:10.1080/15287394.2014.935540
Di Bucchianico S, Cappellini F, Le Bihanic F, Zhang Y, Dreij K, Karlsson HL (2016) Genotoxicity of TiO2 nanoparticles assessed by mini-gel comet assay and micronucleus scoring with flow cytometry. Mutagenesis. doi:10.1093/mutage/gew030
DIN EN ISO (17294-2:2005-02)
Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35(4):495–516. doi:10.1080/01926230701320337
Fadeel B, Garcia-Bennett AE (2010) Better safe than sorry: understanding the toxicological properties of inorganic nanoparticles manufactured for biomedical applications. Adv Drug Deliv Rev 62(3):362–374. doi:10.1016/j.addr.2009.11.008
Flower NA, Brabu B, Revathy M et al (2012) Characterization of synthesized silver nanoparticles and assessment of its genotoxicity potentials using the alkaline comet assay. Mutat Res 742(1–2):61–65. doi:10.1016/j.mrgentox.2011.12.003
Fraga S, Faria H, Soares ME et al (2013) Influence of the surface coating on the cytotoxicity, genotoxicity and uptake of gold nanoparticles in human HepG2 cells. J Appl Toxicol 33(10):1111–1119. doi:10.1002/jat.2865
Frohlich E, Roblegg E (2012) Models for oral uptake of nanoparticles in consumer products. Toxicology 291(1–3):10–17. doi:10.1016/j.tox.2011.11.004
Frohlich E, Roblegg E (2016) Oral uptake of nanoparticles: human relevance and the role of in vitro systems. Arch Toxicol 90(10):2297–2314. doi:10.1007/s00204-016-1765-0
Glei M, Latunde-Dada GO, Klinder A et al (2002) Iron-overload induces oxidative DNA damage in the human colon carcinoma cell line HT29 clone 19A. Mutat Res 519(1–2):151–161
Glei M, Kirmse A, Habermann N, Persin C, Pool-Zobel BL (2006) Bread enriched with green coffee extract has chemoprotective and antigenotoxic activities in human cells. Nutr Cancer 56(2):182–192. doi:10.1207/s15327914nc5602_9
Glei M, Schneider T, Schlormann W (2016) Comet assay: an essential tool in toxicological research. Arch Toxicol. doi:10.1007/s00204-016-1767-y
Gojova A, Guo B, Kota RS, Rutledge JC, Kennedy IM, Barakat AI (2007) Induction of inflammation in vascular endothelial cells by metal oxide nanoparticles: effect of particle composition. Environ Health Perspect 115(3):403–409. doi:10.1289/ehp.8497
Govender R, Phulukdaree A, Gengan RM, Anand K, Chuturgoon AA (2013) Silver nanoparticles of Albizia adianthifolia: the induction of apoptosis in human lung carcinoma cell line. J Nanobiotechnology 11:5. doi:10.1186/1477-3155-11-5
Hsin YH, Chen CF, Huang S, Shih TS, Lai PS, Chueh PJ (2008) The apoptotic effect of nanosilver is mediated by a ROS- and JNK-dependent mechanism involving the mitochondrial pathway in NIH3T3 cells. Toxicol Lett 179(3):130–139. doi:10.1016/j.toxlet.2008.04.015
Kahru A, Dubourguier HC (2010) From ecotoxicology to nanoecotoxicology. Toxicology 269(2–3):105–119. doi:10.1016/j.tox.2009.08.016
Kettiger H, Schipanski A, Wick P, Huwyler J (2013) Engineered nanomaterial uptake and tissue distribution: from cell to organism. Int J Nanomedicine 8:3255–3269. doi:10.2147/IJN.S49770
Knoll N, Weise A, Claussen U et al (2006) 2-Dodecylcyclobutanone, a radiolytic product of palmitic acid, is genotoxic in primary human colon cells and in cells from preneoplastic lesions. Mutat Res 594(1–2):10–19. doi:10.1016/j.mrfmmm.2005.07.015
Krug HF (2014) Nanosafety research–are we on the right track? Angew Chem Int Ed Engl 53(46):12304–12319. doi:10.1002/anie.201403367
Kumari A, Yadav SK (2014) Nanotechnology in agri-food sector. Crit Rev Food Sci Nutr 54(8):975–984. doi:10.1080/10408398.2011.621095
Landsiedel R, Fabian E, Ma-Hock L et al (2012) Toxico-/biokinetics of nanomaterials. Arch Toxicol 86(7):1021–1060. doi:10.1007/s00204-012-0858-7
Liu W, Wu Y, Wang C et al (2010) Impact of silver nanoparticles on human cells: effect of particle size. Nanotoxicology 4(3):319–330. doi:10.3109/17435390.2010.483745
Ma Y, Huang J, Song S, Chen H, Zhang Z (2016) Cancer-targeted nanotheranostics: recent advances and perspectives. Small. doi:10.1002/smll.201600635
Mahmoud A, Ezgi O, Merve A, Ozhan G (2016) In vitro toxicological assessment of magnesium oxide nanoparticle exposure in several mammalian cell types. Int J Toxicol 35(4):429–437. doi:10.1177/1091581816648624
McQuaid HN, Muir MF, Taggart LE et al (2016) Imaging and radiation effects of gold nanoparticles in tumour cells. Sci Rep 6:19442. doi:10.1038/srep19442
Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65(1–2):55–63
Munjal U, Scharlau D, Glei M (2012) Gut fermentation products of inulin-type fructans modulate the expression of xenobiotic-metabolising enzymes in human colonic tumour cells. Anticancer Res 32(12):5379–5386
Park E, Glei M, Knobel Y, Pool-Zobel BL (2007) Blood mononucleocytes are sensitive to the DNA damaging effects of iron overload–in vitro and ex vivo results with human and rat cells. Mutat Res 619(1–2):59–67. doi:10.1016/j.mrfmmm.2007.01.012
Piperigkou Z, Karamanou K, Engin AB et al (2016) Emerging aspects of nanotoxicology in health and disease: from agriculture and food sector to cancer therapeutics. Food Chem Toxicol 91:42–57. doi:10.1016/j.fct.2016.03.003
Sahu SC, Zheng J, Graham L et al (2014) Comparative cytotoxicity of nanosilver in human liver HepG2 and colon Caco2 cells in culture. J Appl Toxicol 34(11):1155–1166. doi:10.1002/jat.2994
Schneider T, Jahr N, Jatschka J, Csaki A, Stranik O, Fritzsche W (2013) Localized surface plasmon resonance (LSPR) study of DNA hybridization at single nanoparticle transducers. J Nanopart Res 15(4):1–10. doi:10.1007/s11051-013-1531-7
Sharma V, Anderson D, Dhawan A (2012) Zinc oxide nanoparticles induce oxidative DNA damage and ROS-triggered mitochondria mediated apoptosis in human liver cells (HepG2). Apoptosis 17(8):852–870. doi:10.1007/s10495-012-0705-6
Siddiqui MA, Alhadlaq HA, Ahmad J, Al-Khedhairy AA, Musarrat J, Ahamed M (2013) Copper oxide nanoparticles induced mitochondria mediated apoptosis in human hepatocarcinoma cells. PLoS ONE 8(8):e69534. doi:10.1371/journal.pone.0069534
Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175(1):184–191
Sun J, Wang S, Zhao D, Hun FH, Weng L, Liu H (2011) Cytotoxicity, permeability, and inflammation of metal oxide nanoparticles in human cardiac microvascular endothelial cells: cytotoxicity, permeability, and inflammation of metal oxide nanoparticles. Cell Biol Toxicol 27(5):333–342. doi:10.1007/s10565-011-9191-9
Tsai SW, Liaw JW, Kao YC et al (2013) Internalized gold nanoparticles do not affect the osteogenesis and apoptosis of MG63 osteoblast-like cells: a quantitative, in vitro study. PLoS ONE 8(10):e76545. doi:10.1371/journal.pone.0076545
Vales G, Rubio L, Marcos R (2015) Long-term exposures to low doses of titanium dioxide nanoparticles induce cell transformation, but not genotoxic damage in BEAS-2B cells. Nanotoxicology 9(5):568–578. doi:10.3109/17435390.2014.957252
Venable JH, Coggeshall R (1965) A simplified lead citrate stain for use in electron microscopy. J Cell Biol 25(2(p1)):407–408. doi:10.1083/jcb.25.2.407
Vidlarova L, Romero GB, Hanus J, Stepanek F, Muller RH (2016) Nanocrystals for dermal penetration enhancement—effect of concentration and underlying mechanisms using curcumin as model. Eur J Pharm Biopharm 104:216–225. doi:10.1016/j.ejpb.2016.05.004
Wang H, Wang F, Tao X, Cheng H (2012) Ammonia-containing dimethyl sulfoxide: an improved solvent for the dissolution of formazan crystals in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Anal Biochem 421(1):324–326. doi:10.1016/j.ab.2011.10.043
Woodruff RS, Li Y, Yan J et al (2012) Genotoxicity evaluation of titanium dioxide nanoparticles using the Ames test and Comet assay. J Appl Toxicol 32(11):934–943. doi:10.1002/jat.2781
Zhang S, Gao H, Bao G (2015) Physical principles of nanoparticle cellular endocytosis. ACS Nano 9(9):8655–8671. doi:10.1021/acsnano.5b03184
Acknowledgements
The project was funded by Thüringer Aufbaubank (Project number: 2012PE0141). The authors thank Dr. Andrea Csáki (IPHT Jena) for preparation of metal NPs and Dr. Ulrich Schäfer for linguistic support. We express our gratitude to the Food GmbH Jena for all ICP-MS measurements.
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Schneider, T., Westermann, M. & Glei, M. In vitro uptake and toxicity studies of metal nanoparticles and metal oxide nanoparticles in human HT29 cells. Arch Toxicol 91, 3517–3527 (2017). https://doi.org/10.1007/s00204-017-1976-z
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DOI: https://doi.org/10.1007/s00204-017-1976-z