Comparative Study of Different Methods to Determine the Role of Reactive Oxygen Species Induced by Zinc Oxide Nanoparticles
AbstractAccumulation of reactive oxygen species (ROS) followed by an increase in oxidative stress is associated with cellular responses to nanoparticle induced cell damages. Finding the best method for assessing intracellular ROS production is the key step in the detection of oxidative stress induced injury. This study evaluates and compares four different methods for the measurement of intracellular ROS generation using fluorogenic probe, 2´,7´-dichlorofluorescein diacetate (DCFH-DA). Hydrogen peroxide (H2O2) was utilised as a positive control to assess the reactivity of the probe. Spherically shaped zinc oxide (ZnO) nanoparticles with an average particle size of 85.7 nm were used to determine the diverse roles of ROS in nanotoxicity in Hs888Lu and U937 cell lines. The results showed that different methods exhibit different patterns of ROS measurement. In conclusion this study found that the time point at which the DCFH-DA is added to the reaction, the incubation time and the oxidative species that is responsible for the oxidation of DCFH, have impact on the intracellular ROS measurement.
Ai, H., Bu, Y., and Han, K., 2003. Glycine-Zn+/Zn2+ and their hydrates: On the number of water molecules necessary to stabilize the switterionic glycine-Zn+/Zn2+ over the nonzwitterionic ones. Journal of Chemical Physics, 118 (24), pp.10973-10985 .
Brubacher, J.L., and Bols, N.C., 2001. Chemically de-acetylated 2',7'-dichlorodihydrofluorescein diacetate as a probe of respiratory burst activity in mononuclear phagocytes. Journal of Immunological Methods, 251(1-2), pp.81-91.
Burkitt, M.J., and Wardman, P., 2001. Cytochrome c is a potent catalyst of dichlorofluorescin oxidation: Implications for the role of reactive oxygen species in apoptosis. Biochemical and Biophysical Research, 282(1), pp.329-333.
Curtin, J.F., Donovan, M., and Cotter, T.G., 2002. Regulation and measurement of oxidative stress in apoptosis. Journal of Immunological Methods, 265(1-2), pp.49-72.
Donaldson, K., Stone, V., Borm, P.J., Jimenez, L.A., Gilmour, P.S., Schins, R.P., Knaapen, A.M., Rahman, I., Faux, S.P., Brown, D.M., and MacNee, W., 2003. Oxidative stress and calcium signaling in the adverse effects of environmental particles (PM10). Free Radical Biology and Medicine, 34 (11), pp.1369-1382.
Foucaud, L., Wilson, M.R., Brown, D.M., and Stone, V., 2007. Measurement of reactive species production by nanoparticles prepared in biologically relevant media. Toxicology Letters, 174(1-3), pp.1-9.
Grabinski, C., Hussain, S., Lafdi, K., Braydich-Stolle, L., and Schlager, J., 2007. Effect of particle dimension on biocompatibility of carbon nanomaterials. Carbon, 45(14), pp.2828-2835.
Ischiropoulos, H., Gow, A., Thom, S.R., Kooy, N.W., Royall, J.A. and Crow, J.P., 1999. Detection of reactive nitrogen species using 2,7- dichlorodihydrofluorescein and dihydrorhodamine 123. Methods in Enzymology, 301, pp.367-373.
Kooy, N., Royall, J. and Ischiropoulos, H., 1997. Oxidation of 2',7'-dichlorofluorescein by peroxynitrite. Free Radical Research, 27(3), pp.245-254.
LeBel, C.P., Ischiropoulos, H. and Bondy, S.C., 1992. Evaluation of the probe 2‘,7‘-dichiorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chemical Research in Toxicology, 5(2), pp.227-231.
Li, N., Xia, T. and Nel, A.E., 2008. The role of oxidative stress in ambient particulate matter-induced lung diseases and its implications in the toxicity of engineered nanoparticles. Free Radical Biology and Medicine, 44(9), pp.1689-1699.
Li, Y., Nishimura, T., Teruya, K., Maki, T., Komatsu, T., Hamasaki, T., Kashiwagi, T., Kabayama, S., Shim, S.Y., Katakura, Y., Osada, K., Kawahara, T., Otsubo, K., Morisawa, S., Ishii, Y., Gadek, Z. and Shirahata, S., 2002. Protective mechanism of reduced water against alloxan-induced pancreatic β-cell damage: Scavenging effect against reactive oxygen species. Cytotechnology, 40(1-3), pp.139-149.
Lin, W., Huang, Y., Zhou, X.D. and Ma, Y., 2006. In vitro toxicity of silica nanoparticles in human lung cancer cells. Toxicology and Applied Pharmacology, 217(3), pp.252-259.
Lin, W., Xu, Y., Huang, C.C., Ma, Y., Shannon, K.B., Chen, D.-R. and Huang, Y.-W., 2009. Toxicity of nano- and micro-sized ZnO particles in human lung epithelial cells. Journal of Nanoparticle Research, 11(1), pp.25-39.
Loetchutinat, C., Kothan, S., Dechsup, S., Meesungnoen, J., Jay-Gerin, J.-P. and Mankhetkorn, S., 2005. Spectrofluorometric determination of intracellular levels of reactive oxygen species in drug-sensitive and drug-resistant cancer cells using the 2',7'-dichlorofluorescein diacetate assay. Radiation Physics and Chemistry, 72(2-3), pp.323-331.
Myhre, O., Andersen, J.M., Aarnes, H. and Fonnum, F., 2003. Evaluation of the probes 2',7'-dichlorofluorescin diacetate, luminol, and lucigenin as indicators of reactive species formation. Biochemical Pharmacology, 65(10), pp.1575-1582.
Najim, N., Rusdi, R., Zain, M.M., Hamzah, A.S., Shaameri, Z. and Kamarulzaman, N., 2014. Effects of the absorption behaviour of ZnO nanoparticles on the optical measurements of cytotoxicity studies: In normal and cancer cell lines. Journal of Nanomaterials, 2014, pp.1-8.
Oberdorster, E., 2004. Manufactured nanomaterials (fullerenes, C60) induce oxidative stress in the brain of juvenile largemouth bass. Environmental Health Perspectives, 112(10), pp.1058-1062.
Ohashi, T., Mizutani, A., Murakami, A., Kojo, S., Ishii, T. and Taketani, S., 2002. Rapid oxidation of dichlorodihydrofluorescin with heme and hemoproteins: formation of the fluorescein is independent of the generation of reactive oxygen species. FEBS Letters, 511(1-3), pp.21-27.
Ostrovsky, S., Kazimirsky, G., Gedanken, A. and Brodie, C., 2009. Selective cytotoxic effect of ZnO nanoparticles on glioma cells. Nano Research, 2(11), pp.882-890.
Possel, H., Noack, H., Augustin, W., Keilhoff, G. and Wolf, G., 1997. 2',7'-Dihydrodichlorofluorescein diacetate as a fluorescent marker for peroxynitrite formation. FEBS Letters, 416(2), pp.175-178.
Reeves, J.F., Davies, S.J., Dodd, N.J.F. and Jha, A.J., 2008. Hydroxyl radicals (•OH) are associated with titanium dioxide (TiO2) nanoparticle induced cytotoxicity and oxidative DNA damage in fish cells. Mutation Research, 640(1-2), pp.113-122.
Reinisch, N., Wiedermann, C.J. and Ricevuti, G., 2000. Inhibition of human peripheral blood neutrophil respiratory burst by alcohol-based venipuncture site disinfection. Clinical and Diagnostic Laboratory Immunology, 7(6), pp.980-982.
Rota, C., Chignell, C.F. and Mason, R.P., 1999. Evidence for free Radical Formation during the oxidation of 2'-7'-dichlorofluorescin to the fluorescent dye 2'-7'-dichlorofluorescein by horseradish peroxidase: possible implications for oxidative stress measurements. Free Radical Biology and Medicine, 27(7), pp.873-881.
Royall, J. and Ischiropoulos, H., 1993. Evaluation of 2',7'-dichlorofluorescein and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells Archives of Biochemistry and Biophysics, 302(2), pp.348-355.
Rusdi, R., Rahman, A., Mohamed, N., Kamarudin, N. and Kamarulzaman, N., 2011. Preparation and band gap energies of ZnO nanotubes, nanorods and spherical nanostructures. Powder Technology, 210(1), pp.18-22.
Sharma, V., Shukla, R.K., Saxena, N., Parmar, D., Das, M. and Dhawan, A., 2009. DNA damaging potential of zinc oxide nanoparticles in human epidermal cells. Toxicology Letters, 185(3), pp.211-218.
Song, W., Zhang, J., Guo, J., Zhang, J., Ding, F., Li, L. and Sun, Z., 2010. Role of the dissolved zinc ion and reactive oxygen species in cytotoxicity of ZnO nanoparticles. Toxicology Letters, 199(3), pp.389-397.
Ubezio, P. and Civoli, F., 1994. Flow cytometric detection of hydrogen peroxide production induced by doxorubicin in cancer cells. Free Radical Biology and Medicine, 16(4), pp.509-516.
Wang, H. and Joseph, J.A., 1999. Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radical Biology and Medicine, 27(5-6), pp.612-616.
Winterbourn, C.C. and Sutton, H.C., 1984. Hydroxyl radical production from hydrogen peroxide and enzymatically generated paraquat radicals: Catalytic requirements and oxygen dependence. Archives of Biochemistry and Biophysics, 235(1), pp.116-126.
Xia, T., Kovochich, M., Liong, M., Madler, L., Gilbert, B., Shi, H., Yeh, J.I., Zink, J.I. and Nel, A.E., 2008. Comparison of the mechanism of toxicity of zinc oxide and cerium oxide nanoparticles based on dissolution and oxidative stress properties. ACS nano, 2(10), pp.2121-2134.
Xia, T., Kovochich, M. and Nel, A., 2006. The role of reactive oxygen species and oxidative stress in mediating particulate matter injury. Occupational and Environmental Medicine, 5(4), pp.817-836.
Copyright (c) 2016 Nigar A. Najim
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.
Authors who publish with this journal agree to the following terms:
- Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License [CC BY-NC-SA 4.0] that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).