Effects of Zinc Oxide Nanoparticles in HUVEC: Cyto- and Genotoxicity and Functional Impairment After Long-Term and Repetitive Exposure in vitro
Authors Poier N, Hochstöger J, Hackenberg S, Scherzad A, Bregenzer M, Schopper D, Kleinsasser N
Received 21 January 2020
Accepted for publication 17 April 2020
Published 22 June 2020 Volume 2020:15 Pages 4441—4452
Checked for plagiarism Yes
Review by Single anonymous peer review
Peer reviewer comments 4
Editor who approved publication: Prof. Dr. Thomas Webster
Nikolaus Poier,1,2 Johannes Hochstöger,1,2 Stephan Hackenberg,3 Agmal Scherzad,3 Maximilian Bregenzer,3 Dominik Schopper,1,2 Norbert Kleinsasser1,3
1Department of Otorhinolaryngology, Head and Neck Surgery, Kepler University Hospital, Linz 4021, Austria; 2Medical Faculty, Johannes Kepler University Linz, Linz 4040, Austria; 3Department of Otorhinolaryngology, Plastic, Aesthetic and Reconstructive Head and Neck Surgery, University of Wuerzburg, Würzburg 97080, Germany
Correspondence: Nikolaus Poier Krankenhausstraße 9, Linz 4021, Austria
Tel +43 650 2409889
Purpose: The present study focuses on threshold levels for cytotoxicity after long-term and repetitive exposure for HUVEC as a model for the specific microvascular endothelial system. Furthermore, possible genotoxic effects and functional impairment caused by ZnO NPs in HUVEC are elucidated.
Methods: Thresholds for cytotoxic effects are determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Annexin V assay. To demonstrate DNA damage, single-cell microgel electrophoresis (comet) assay is performed after exposure to sub-cytotoxic concentrations of ZnO NPs. The proliferation assay, dot blot assay and capillary tube formation assay are also carried out to analyze functional impairment.
Results: NPs showed to be spherical in shape with an average size of 45– 55 nm. Long-term exposure as well as repetitive exposure with ZnO NPs exceeding 25 μg/mL lead to decreased viability in HUVEC. In addition, DNA damage was indicated by the comet assay after long-term and repetitive exposure. Twenty-four hours after long-term exposure, the proliferation assay does not show any difference between negative control and exposed cells. Forty-eight hours after exposure, HUVEC show an inverse concentration-related ability to proliferate. The dot blot assay provides evidence that ZnO NPs lead to a decreased release of VEGF, while capillary tube formation assay shows restriction in the ability of HUVEC to build tubes and meshes as a first step in angiogenesis.
Conclusion: Sub-cytotoxic concentrations of ZnO NPs lead to DNA damage and functional impairment in HUVEC. Based on these data, ZnO NPs may affect neo-angiogenesis. Further investigation based on tissue cultures is required to elucidate the impact of ZnO NPs on human cell systems.
Keywords: zinc oxide, nanoparticles, cytotoxicity, genotoxicity, toxicity
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