Effects of Zinc Oxide Nanoparticles in HUVEC: Cyto- and Genotoxicity and Functional Impairment After Long-Term and Repetitive Exposure in vitro

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.