TY - JOUR T1 - Cyto- and genotoxicity of copper (II) oxide (CuO) nanoparticles in HeLa cells AU - Abudayyak [m. Fırat Kenanoğlu], Mahmoud AU - Abo Ras, Fedaa AU - Özhan, Gül PY - 2023 DA - August DO - 10.26650/IstanbulJPharm.2023.1255310 JF - İstanbul Journal of Pharmacy JO - iujp PB - Istanbul University WT - DergiPark SN - 2587-2087 SP - 126 EP - 132 VL - 53 IS - 2 LA - en AB - Background and Aims: Cancer is a widespread disease responsible for the death of millions every year. Different approaches and drugs are in use to treat cancer, however, there is a need for new drugs with low cost, high activity, and low side effect risks. Nanotechnology and nanomaterials are important to develop those drugs. Copper-based nanoparticles (NPs) are shown to have biological activity as the antibacterial, and cytotoxic potential. Copper (II) oxide (CuO) NPs are widely used among Cu-based NPs. Different studies evaluated its anticancer and cytotoxic activity; however, the results are still controversial.Methods: It was planned to characterize the NPs using Transmission Electron Microscopy (TEM) in cell culture medium and distilled water and then to evaluate their cytotoxicity in human cervical cancer cells (HeLa) using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay and neutral red uptake (NRU) assays. As one of the cytotoxicity mechanisms, the DNA damage induction potential was evaluated by Comet assay.Results: The CuO NPs have an average diameter of about 35 nm in distilled water and 39 nm in cell culture medium. The IC50 levels of NPs were 10.7 µg/mL and 6.73 µg/mL by MTT and NRU assays, respectively. The results reveal the NPs dosedependently increased in the DNA damage. The tail moment was 1.3-fold at 3.125 µg/mL, 2.5-fold at 6.25 µg/mL, and 3.8-fold at 12.5 µg/mL.Conclusion: CuO NPs have high cytotoxic activity in HeLa cancerous cells. The induction of DNA damage could be an important step in the induction of cell death. Further in vivo and in vitro studies in need to improve the safety/low toxicity and understand the molecular mechanism of CuO-induced activity. KW - Copper (II) oxide KW - nanoparticles KW - HeLa KW - genotoxicity KW - cytotoxicity CR - Abudayyak, M., Guzel, E.E., Özhan, G. (2016a). Copper (II) oxide nanoparticles induce high toxicity in human neuronal cell. Global Journal of Medical Research: B Pharma, Drug Discovery, Toxicology & Medicine; XVI (III):6-15. google scholar CR - Abudayyak, M., Altincekic Gurkaynak, T., & Özhan, G. (2016c). In vitro toxicological assessment of cobalt ferrite nanoparticles in several mammalian cell types. Biological Trace Element Research, 175(2), 458-465. doi:10.1007/s12011-016-0803-3 google scholar CR - Abudayyak, M., Guzel, E. E., & Özhan, G. (2016b). Copper (II) oxide nanoparticles induced nephrotoxicity in vitro conditions. Applied In Vitro Toxicology, 2(3), 157-164. doi:10.1089/aivt.2016.0008 google scholar CR - Abudayyak, M., Guzel, E., & Özhan, G. (2017). Nickel oxide nanopar-ticles are highly toxic to SH-SY5Y neuronal cells. Neurochemistry International, 108, 7-14. doi:10.1016/j.neuint.2017.01.017 google scholar CR - Abudayyak, M., Guzel, E., & Özhan, G. (2020). Cupric oxide nanoparticles induce cellular toxicity in liver and intestine cell lines. Advanced Pharmaceutical Bulletin, 10(2), 213-220. doi:10.34172/apb.2020.025 google scholar CR - Ahamed, M., Siddiqui, M. A., Akhtar, M. J., Ahmad, I., Pant, A. B., & Alhadlaq, H. A. (2010). Genotoxic potential of copper oxide nanoparticles in human lung epithelial cells. Biochemical and Bio-physical Research Communications, 396(2), 578-583. doi:10.1016/j. bbrc.2010.04.156 google scholar CR - Aitken, R. J., Chaudhry, M. Q., Boxall, A. B., & Hull, M. (2006). Manu-facture and use of nanomaterials: Current status in the UK and global trends. Occupational Medicine, 56(5), 300-306. doi:10.1093/ occmed/kql051 google scholar CR - Akhtar, M. J., Ahamed, M., Fareed, M., Alrokayan, S. A., & Kumar, S. (2012). Protective effect of sulphoraphane against oxidative stress mediated toxicity induced by Cuo nanoparticles in mouse em-bryonic fibroblasts Balb 3t3. The Journal of Toxicological Sciences, 37(1), 139-148. doi:10.2131/jts.37.139 google scholar CR - Akhtar, M. J., Kumar, S., Alhadlaq, H. A., Alrokayan, S. A., Abu-Salah, K. M., & Ahamed, M. (2016). Dose-dependent genotoxicity of cop-per oxide nanoparticles stimulated by reactive oxygen species in human lung epithelial cells. Toxicology and Industrial Health, 32(5), 809-821. doi:10.1177/0748233713511512 google scholar CR - Alishah, H., Pourseyedi, S., Ebrahimipour, S.Y., Mahani, S.E., & Rafiei, N. (2017). Green synthesis of starch-mediated CuO nanoparticles: preparation, characterization, antimicrobial activities and in vitro MTT assay against MCF-7 cell line. Rendiconti Lincei. Scienze Fisiche e Naturali 28, 65-71. https://doi.org/10.1007/s12210-016-0574-y google scholar CR - • Andleeb, A., Andleeb, A., Asghar, S., Zaman, G., Tariq, M., Mehm-ood, A., . . . Abbasi, B. H. (2021). A systematic review of biosynthe-sized metallic nanoparticles as a promising Anti-Cancer-Strategy. Cancers, 13(11), 2818. doi:10.3390/cancers13112818 google scholar CR - Chang, Y., Zhang, M., Xia, L., Zhang, J., & Xing, G. (2012). The toxic effects and mechanisms of Cuo and zno nanoparticles. Materials, 5(12), 2850-2871. doi:10.3390/ma5122850 google scholar CR - Chen, J., Zhu, J., Cho, H., Cui, K., Li, F., Zhou, X., . . . Huang, X. (2008). Differential cytotoxicity of metal oxide nanopar-ticles. Journal of Experimental Nanoscience, 3(4), 321-328. doi:10.1080/17458080802235765 google scholar CR - Chen, Z., Meng, H., Xing, G., Chen, C., Zhao, Y., Jia, G., . . . Wan, L. (2006). Acute toxicological effects of copper nanoparticles in vivo. Toxicology Letters, 163(2), 109-120. doi:10.1016/j.tox-let.2005.10.003 google scholar CR - Cioffi, N., Ditaranto, N., Torsi, L., Picca, R. A., Sabbatini, L., Valen-tini, A., . . . Zambonin, P. G. (2005). Analytical characterization of bioactive fluoropolymer ultra-thin coatings modified by copper nanoparticles. Analytical and Bioanalytical Chemistry, 381(3), 607616. doi:10.1007/s00216-004-2761-4 google scholar CR - Collins, A. R. (2004). The comet assay for DNA damage and repair: Principles, applications, and limitations. Molecular Biotechnology, 26(3), 249-261. doi:10.1385/mb:26:3:249 google scholar CR - Dadure K.M., Mahapatra D., Haldar A., Potbhare A.K., Chaudhary R.G. (2022). Utilization of mother nature’s gift for the biofabrication of copper/ copper oxide nanoparticles for therapeutic applica-tions. Jordan Journal of Physics, 15(1), 89-99. doi:10.47011/15.1.12 Gnanavel, V., Palanichamy, V., & Roopan, S. M. (2017). Biosynthesis and characterization of copper oxide nanoparticles and its an-ticancer activity on Human Colon Cancer Cell Lines (HCT-116). Journal of Photochemistry and Photobiology B: Biology, 171, 133138. doi:10.1016/j.jphotobiol.2017.05.001 google scholar CR - Farshori, N.N., Siddiqui, M.A., Al-Oqail, M.M., Al-Sheddi, E.S., Al-Massarani, S.M., Ahamed, M, ... Al-Khedhairy, A.A. (2022). Copper Oxide Nanoparticles Exhibit Cell Death Through Oxidative Stress Responses in Human Airway Epithelial Cells: a Mechanistic Study. Biological Trace Element Research 200, 5042-5051 (2022). https:// doi.org/10.1007/s12011-022- google scholar CR - Gosens, I., Cassee, F. R., Zanella, M., Manodori, L., Brunelli, A., Costa, A. L., . . . Stone, V. (2016). Organ burden and pulmonary toxicity of nano-sized copper (II) oxide particles after short-term inhalation exposure. Nanotoxicology, 10(8), 1084-1095. doi:10.3109/174353 90.2016.1172678 google scholar CR - Ingle, A. P., Duran, N., & Rai, M. (2014). Bioactivity, mechanism of action, and cytotoxicity of copper-based nanoparticles: A Re-view. Applied Microbiology and Biotechnology, 98(3), 1001-1009. doi:10.1007/s00253-013-5422-8 google scholar CR - Kadammattil, A. V., Sajankila, S. P., Prabhu, S., Rao, B. N., & Rao, B. S. (2018). Systemic toxicity and teratogenicity of copper oxide nanoparticles and copper sulfate. Journal of Nanoscience and Nanotechnology, 18(4), 2394-2404. doi:10.1166/jnn.2018.14542 google scholar CR - Karlsson, H. L., Cronholm, P., Gustafsson, J., & Möller, L. (2008). Cop-per oxide nanoparticles are highly toxic: A comparison between metal oxide nanoparticles and carbon nanotubes. Chemical Re-search in Toxicology, 21(9), 1726-1732. doi:10.1021/tx800064j google scholar CR - Khalid, S., Afzal, N., Khan, J. A., Hussain, Z., Qureshi, A. S., Anwar, H., & Jamil, Y. (2018). Antioxidant resveratrol protects against copper ox-ide nanoparticle toxicity in vivo. Naunyn-Schmiedeberg’s Archives of Pharmacology, 391(10), 1053-1062. doi:10.1007/s00210-018-1526-0 google scholar CR - Lei, R., Wu, C., Yang, B., Ma, H., Shi, C., Wang, Q., . . . Liao, M. (2008). Integrated metabolomic analysis of the nano-sized copper parti-cle-induced hepatotoxicity and nephrotoxicity in rats: A rapid in vivo screening method for nanotoxicity. Toxicology and Applied Pharmacology, 232(2), 292-301. doi:10.1016/j.taap.2008.06.026 google scholar CR - Liu, Y., Gao, Y., Zhang, L., Wang, T., Wang, J., Jiao, F., . . . Chen, C. (2009). Potential health impact on mice after nasal instillation of nano-sized copper particles and their translocation in mice. Journal of Nanoscience and Nanotechnology, 9(11), 6335-6343. doi:10.1166/jnn.2009.1320 google scholar CR - Maksoudian, C., Saffarzadeh, N., Hesemans, E., Dekoning, N., Butt-iens, K., & Soenen, S. J. (2020). Role of inorganic nanoparticle deg-radation in cancer therapy. Nanoscale Advances, 2(9), 3734-3763. doi:10.1039/d0na00286k google scholar CR - Masters, J. R. (2002). Hela cells 50 years on: The good, the bad and the ugly. Nature Reviews Cancer, 2(4), 315-319. doi:10.1038/nrc775 google scholar CR - Meng, H., Chen, Z., Xing, G., Yuan, H., Chen, C., Zhao, F., . . . Zhao, Y. (2007). Ultrahigh reactivity provokes nanotoxicity: Explanation of oral toxicity of nano-copper particles. Toxicology Letters, 175(1-3), 102-110. doi:10.1016/j.toxlet.2007.09.015 google scholar CR - Nagajyothi, P., Muthuraman, P., Sreekanth, T., Kim, D. H., & Shim, J. (2017). Green synthesis: In-vitro anticancer activity of copper oxide nanoparticles against human cervical carcinoma cells. Arabian Journal of Chemistry, 10(2), 215-225. doi:10.1016/j.arab-jc.2016.01.011 google scholar CR - Mahmoud, N.M.R., Mohamed, H.I., Ahmed, S.B., & Akhtar, S. (2020). Efficient biosynthesis of CuO nanoparticles with poten-tial cytotoxic activity. Chemical Papers 74, 2825-2835 https://doi. org/10.1007/s11696-020-01120-6 google scholar CR - Oza, G., Calzadilla-Avila, A. I., Reyes-Calderon, A., Anna, K. K., Ramfrez-Bon, R., Tapia-Ramirez, J., & Sharma, A. (2020). Ph-depen-dent biosynthesis ofcopper oxide nanoparticles using Galphimia glauca for their cytocompatibility evaluation. Applied Nanosci-ence, 10(2), 541-550. doi:10.1007/s13204-019-01159-2 google scholar CR - Perreault, F., Melegari, S. P., Da Costa, C. H., De Oliveira Franco Rossetto, A. L., Popovic, R., & Matias, W. G. (2012). Genotoxic ef-fects of copper oxide nanoparticles in Neuro 2a Cell Cultures. Science of The Total Environment, 441, 117-124. doi:10.1016/j.sci-totenv.2012.09.065 google scholar CR - Piret, J., Jacques, D., Audinot, J., Mejia, J., Boilan, E., Noel, F., . . . Tous-saint, O. (2012). Copper (II) oxide nanoparticles penetrate into HEPG2 cells, exert cytotoxicity via oxidative stress and induce pro-inflammatory response. Nanoscale, 4(22), 7168. doi:10.1039/ c2nr31785k google scholar CR - Rani, N., & Saini, K. (2022). Biogenic metal and metal oxides nanoparticles as anticancer agent: A Review. IOP Confer-ence Series: Materials Science and Engineering, 1225(1), 012043. doi:10.1088/1757-899x/1225/1/012043 google scholar CR - Rehana, D., Mahendiran, D., Kumar, R. S., & Rahiman, A. K. (2017). In vitro antioxidant and antidiabetic activities of zinc oxide nanoparticles synthesized using different plant extracts. Bio-process and Biosystems Engineering, 40(6), 943-957. doi:10.1007/ s00449-017-1758-2 google scholar CR - Repetto, G., Del Peso, A., & Zurita, J. L. (2008). Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nature Proto-cols, 3(7), 1125-1131. doi:10.1038/nprot.2008.75 google scholar CR - Sankar, R., Maheswari, R., Karthik, S., Shivashangari, K. S., & Ravi-kumar, V. (2014). Anticancer activity of Ficus religiosa engineered copper oxide nanoparticles. Materials Science and Engineering: C, 44, 234-239. doi:10.1016/j.msec.2014.08.030 google scholar CR - Schrand, A. M., Rahman, M. F., Hussain, S. M., Schlager, J. J., Smith, D. A., & Syed, A. F. (2010). Metal-based nanoparticles and their toxicity assessment. WIREs Nanomedicine and Nanobiotechnology, 2(5), 544-568. doi:10.1002/wnan.103 google scholar CR - Sekhon, B. (2014). Nanotechnology in agri-food production: An overview. Nanotechnology, Science and Applications, 31. doi:10.2147/nsa.s39406 google scholar CR - Siddiqui, M. A., Alhadlaq, H. A., Ahmad, J., Al-Khedhairy, A. A., Musarrat, J., & Ahamed, M. (2013). Copper oxide nanoparticles induced mitochondria mediated apoptosis in human Hepatocar-cinoma cells. PLoS ONE, 8(8), doi:10.1371/journal.pone.0069534 google scholar CR - Speit, G., & Hartmann, A. (1999). The comet assay (single-cell gel test): A sensitive genotoxicity test for the detection of DNA damage and Repair. DNA Repair Protocols, 203-212. doi:10.1385/1-59259-675-4:203 google scholar CR - Sun, J., Wang, S., Zhao, D., Hun, F. H., Weng, L., & Liu, H. (2011). Cyto-toxicity, permeability, and inflammation of metal oxide nanopar-ticles in human cardiac microvascular endothelial cells. Cell Biolo-gy and Toxicology, 27(5), 333-342. doi:10.1007/s10565-011-9191-9 google scholar CR - Sung, H., Ferlay, J., Siegel, R. L., Laversanne, M., Soerjomataram, I., Jemal, A., & Bray, F. (2020). Global cancer statistics 2020: Globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians, 71(3), 209-249. doi:10.3322/caac.21660 google scholar CR - Thit, A., Selck, H., & Bjerregaard, H. F. (2013). Toxicity of cuo nanoparticles and Cu ions to tight epithelial cells from xenopus laevis (A6): Effects on proliferation, cell cycle progression and cell death. Toxicology in Vitro, 27(5), 1596-1601. doi:10.1016/j. tiv.2012.12.013 google scholar CR - Thit, A., Selck, H., & Bjerregaard, H. F. (2015). Toxic mechanisms of copper oxide nanoparticles in epithelial kidney cells. Toxicology in Vitro, 29(5), 1053-1059. doi:10.1016/j.tiv.2015.03.020 google scholar CR - Tuli, H. S., Kashyap, D., Bedi, S. K., Kumar, P., Kumar, G., & Sandhu, S. S. (2015). Molecular aspects of Metal Oxide Nanoparticle (MO-NPS) mediated pharmacological effects. Life Sciences, 143, 71-79. doi:10.1016/j.lfs.2015.10.021 google scholar CR - Van Meerloo, J., Kaspers, G. J., & Cloos, J. (2011). Cell sensitiv-ity assays: The MTT assay. Methods in Molecular Biology, 237-245. doi:10.1007/978-1-61779-080-5_20 google scholar CR - Verma, R., & Hansch, C. (2006). Chemical toxic-ity on Hela cells. Current Medicinal Chemistry, 13(4), 423-448. doi:10.2174/092986706775527910 google scholar CR - Wang, Y., Aker, W.G., Hwang, H.M., Yedjou, C.G., Yu, H., Tchoun-wou, P.B. (2011). A study of the mechanism of in vitro cytotox-icity of metal oxide nanoparticles using catfish primary hepato-cytes and human HepG2 cells. Science of the Total Environment. 409(22):4753-62. doi: 10.1016/j. scitotenv.2011.07.039 google scholar CR - Wang, Z., Li, N., Zhao, J., White, J. C., Qu, P., & Xing, B. (2012). Cuo nanoparticle interaction with human epithelial cells: Cellular up-take, location, export, and genotoxicity. Chemical Research in Toxi-cology, 25(7), 1512-1521. doi:10.1021/tx3002093 google scholar CR - Xu, J., Li, Z., Xu, P., Xiao, L., & Yang, Z. (2012). Nanosized copper oxide induces apoptosis through oxidative stress in podocytes. Archives of Toxicology, 87(6), 1067-1073. doi:10.1007/s00204-012-0925-0 google scholar UR - https://doi.org/10.26650/IstanbulJPharm.2023.1255310 L1 - https://dergipark.org.tr/en/download/article-file/2969701 ER -