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Back to Journal »International Journal of Nanomedicine» Volume 15

The effect of zinc oxide nanoparticles on HUVEC: Cytotoxicity and genotoxicity and functional damage after long-term and repeated exposure in vitro

Authors Poier N, Hochstöger J, Hackenberg S, Scherzad A, Bregenzer M, Schopper D, Kleinsasser N

The 2020 volume will be published on June 22, 2020: 15 pages 4441-4452

DOI https://doi.org/10.2147/IJN.S246797

Single anonymous peer review

Editor approved for publication: 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 1 Department of Otolaryngology, Head and Neck Surgery, Kepler University Hospital, Linz 21, 40 Austria; 2 Department of Medicine, Johannes Kepler University Linz, Linz 4040, Austria; 3 Würzburg University Department of Otolaryngology, Plastic Surgery, Aesthetic and Reconstructive Head and Neck Surgery, Würzburg 97080, Germany Mailing address: Nikolaus Poier Krankenhausstraße 9, Linz 4021, Austria Tel +43 650 240988 HUVEC is the threshold level of cytotoxicity after long-term and repeated exposure as a specific microvascular endothelial system model. In addition, clarified the possible genotoxic effects and functional damage caused by ZnO NPs in HUVEC. Method: Determine the threshold of cytotoxicity by measuring 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Annexin V. To prove DNA damage, single-cell microgel electrophoresis (comet) was performed after exposure to sub-cytotoxic concentrations of ZnO NPs. Proliferation assays, dot blot assays, and capillary formation assays were also performed to analyze functional impairment. Results: NPs are spherical with an average size of 45-55 nm. Long-term exposure and repeated exposure to ZnO NP exceeding 25 μg/mL can cause the vitality of HUVEC to decrease. In addition, after long-term and repeated exposure, comet test indicates DNA damage. 24 hours after long-term exposure, the proliferation assay did not show any difference between the negative control and exposed cells. After 48 hours of exposure, HUVEC showed the ability to proliferate inversely proportional to the concentration. Dot blot analysis provided evidence that ZnO NP caused a reduction in VEGF release, while capillary formation analysis showed that HUVEC’s ability to construct tubes and grids was limited as the first step in angiogenesis. Conclusion: Sub-cytotoxic concentration of ZnO NP can cause DNA damage and dysfunction in HUVEC. Based on these data, ZnO NPs may affect the formation of new blood vessels. Further research based on tissue culture is needed to clarify the effect of ZnO NPs on the human cell system. Keywords: zinc oxide, nanoparticles, cytotoxicity, genetic toxicity, toxicity

In the past few decades, nanotechnology has become increasingly important in industry, biomedicine, and research. 1 In industrialized countries, various types of nanoparticles (NP) based on various chemical structures and particle sizes are used. 2 The European Union proposes to define nanomaterials as particles with one or more external dimensions in the size range of 1 nanometer to 100 nanometers. 3

In the manufacturing process, inhalation is the most relevant route of exposure to nanoparticles. 1 For individuals, the main route of contact is skin application. For example, cosmetics such as sunscreens benefit from the use of small particles instead of large particles to improve lubricity and UV protection. 1 ZnO NPs are the most commonly used NPs in these two fields. 4

An important question that still serves as the basis for conflicting discussions in the literature today is whether ZnO NPs can penetrate human skin and cause systemic damage after skin application. Although there is evidence that ZnO NPs cannot penetrate the intact dermis,5-7 there are other studies showing that ZnO NPs can damage skin keratinocytes,8 and after application on healthy skin, zinc can be detected in blood and urine . 9 Especially if a sunscreen containing ZnO NP is topically applied to pre-damaged skin, such as scratched, sunburned or toxic pre-damaged skin, the potential cumulative systemic effects should be considered. 10 In addition, other adverse effects through phototoxicity may also be important.1

Studies have shown that ZnO NPs can cause damage through the blood barrier after inhalation. 11,12 Therefore, they can easily penetrate into the blood 13 and cause adverse systemic effects. Sun et al. demonstrated that human cardiac microvascular endothelial cells treated with ZnO NPs have time, dose, and concentration-dependent cytotoxicity, increased permeability, and inflammatory response. 14 Paszek et al. confirmed these findings in HUVEC, where they observed significant structural deterioration and already dysfunction at subtoxic doses. 15 Suzuki et al. showed that ZnO NPs can accelerate HUVEC atherosclerosis by increasing macrophage adhesion and cholesterol uptake. 16 Yan et al. also demonstrated this in human coronary artery endothelial cells. 17 Other studies have detected pro-inflammatory surface proteins in various endothelial cell systems. 18-20

Different mechanisms seem to contribute to the toxic effects of metal nanoparticles. 21 It is well known that the direct production of ROS will have cytotoxic and genotoxic effects. 22,23 discussed the influence of other factors such as mitochondrial dysfunction, metal ion release and calcium homeostasis on the genotoxicity of ZnO NPs. 24-26

Despite increasing research interest, there is still a lack of data on the biological effects of ZnO NPs on specific microvascular endothelial systems. The functions and cytotoxic effects of ZnO NPs in various endothelial cells and other cell systems have been proven,27 and data on genotoxicity are usually rare and partly contradictory. 1,28-30

Many studies provide evidence that nanomaterials harm cells in different ways. 24,25,31,32 At the same time, they are expected to be used in a large number of medical applications, such as drug delivery, antibacterial coatings, medical imaging and cancer treatment. 33 –36 Therefore, they are regarded as a double-edged sword. 37

As far as we know, there are few data on the cytotoxicity of ZnO NPs in HUVEC, and basically no data on genotoxicity. Therefore, the purpose of this study is to further study the possible cytotoxic and genotoxic effects and functional damage of ZnO NPs in HUVEC as the main cell model of the specific microvascular endothelial system.

30,38 ZnO NPs (ZnO, purity 99.9%, average particle size: 20 nm) were obtained from MK Impex Corp (product number: MKN-ZnO-020, batch number: VA0809; Mississauga, Ontario, Canada). A Zeiss transmission electron microscope EM 900 (Carl Zeiss AG, Oberkochen, Germany) was used to characterize the NPs. In addition, ZetaSizer 3000 HSA (Malvern Instruments Ltd., Malvern, Worcestershire, United Kingdom) was used to evaluate the dynamic light scattering and zeta potential of the dispersion surface. The NP concentration was 50 µg/mL. The medium (pH 7.4) Repeat three times at room temperature. The stock suspension used for cell exposure was prepared as follows: 10 mg of NPs were suspended in 870 µL of sterile distilled water. Ultrasound treatment (Bandelin, Sonopuls HD 60, Berlin, Germany) at a continuous energy level of 4.3 x 105 kJ/m3 for 120 seconds to obtain NP dispersion, 39 30 µL 15 mg/mL bovine serum albumin (BSA; Carl Roth GmbH) +Co.KG, Karlsruhe, Germany) to stabilize the dispersion. Then, 100 µL of 10 x concentrated sterile phosphate buffered saline (PBS) was added to achieve a physiological salt concentration of pH 7.4 in the ZnO NP stock suspension. To determine the working concentration of the cell treatment, the stock suspension was diluted with endothelial cell growth medium containing supplements (ECGM; Provitro GmbH, Berlin, Germany) and 1% penicillin/streptomycin. To analyze the cytotoxic concentration threshold between 1 and 50 µg/mL. For functional impairment and genotoxicity tests, concentrations of 1, 5, 10, and 15 µg/mL were used.

HUVEC (pooled donor, PromoCell GmbH, Heidelberg, Germany) was isolated according to the procedure described in the previous study of our group. 40 Two cell lines were used in the 3rd to 9th generation. ; Provitro GmbH, Berlin, Germany) and 1% penicillin/streptomycin.

To study long-term exposure, the cells were seeded in a 96-well round bottom plate at a density of 104 cells per well and cultured at 37°C and 5% CO2. After 24 hours of incubation, the cells were treated with 20, 25, 30, 35, 40, 45, and 50 µg/mL ZnO NP at 37°C and 5% CO2 for another 24 hours. 1 mM tert-butyl hydroperoxide (t-BHP; Sigma-Aldrich, St. Louis, MO, USA) was used as a positive control, and ECGM without ZnO NPs was used as a negative control. After long-term exposure, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO, USA) colorimetric staining The method was used to evaluate 41 3.5 hours after inoculation with 5% CO2 at 37°C, the MTT solution was replaced by isopropanol. After 20 minutes at room temperature (RT), the color conversion of the blue formazan dye was measured by a microplate absorbance reader (BioTek Instruments, Inc., Winooski, VT, USA) at a wavelength of 570 nm.

For repeated exposure, HUVEC was seeded in a 24-well round bottom plate at a density of 5 x 104 cells per well, and seeded 3 times at each concentration. After 24 hours of incubation, they were exposed to 10, 20, and 50 µg/mL ZnO NP suspension and 10 mM t-BHP for 1 hour, respectively, and then washed with PBS. After the initial exposure, perform the first MTT determination for each concentration (control, 10, 20, 50 µg/mL, t-BHP). The remaining wells were incubated with ECGM for 1 hour without ZnO NPs, and then exposed to ZnO NPs for 1 hour. This process is repeated twice. After the third exposure, a further MTT test was performed, and the remaining cells were incubated in fresh ECGM for a 24-hour regeneration period, after which the final MTT test was performed (Figure 1). Figure 1 The experimental design of long-term exposure to ZnO NPs. 24 hours after inoculation, HUVEC was exposed to ZnO NPs for another 24 hours. Then, MTT assay, comet assay and VEGF dot blot (A) were performed. For the tube formation test, HUVEC was inoculated on Matrigel together with ZnO NP and analyzed after 5 hours (B). The proliferation assay was performed at the time of sowing, before treatment, after long-term treatment, 24 hours after treatment, and 48 hours after treatment (C).

Figure 1 The experimental design of long-term exposure to ZnO NPs. 24 hours after inoculation, HUVEC was exposed to ZnO NPs for another 24 hours. Then, MTT assay, comet assay and VEGF dot blot (A) were performed. For the tube formation test, HUVEC was inoculated on Matrigel together with ZnO NP and analyzed after 5 hours (B). The proliferation assay was performed at the time of sowing, before treatment, after long-term treatment, 24 hours after treatment, and 48 hours after treatment (C).

To assess cell apoptosis, HUVEC was seeded in a 12-well round bottom plate at a density of 105 cells per well. After 24 hours of incubation at 37°C and 5% CO2, the cells were exposed to 20, 25, 30, 35, 40, 45, 50 µg/mL ZnO-NP suspension. Similarly, 1 mM t-BHP was used as a positive control, and ECGM was used as a negative control. The annexin V cell apoptosis assay was performed after 24 hours of long-term exposure. The suspension cells and adherent cells were collected, then centrifuged at 1500 rpm for 5 minutes, and then resuspended in binding buffer (100 µL; 10x Annexin V binding buffer, BD Biosciences, San Jose, CA, USA). The cell suspension was then transferred to a 5 mL culture tube. Add propidium iodide (5 μL; propidium iodide staining solution, BD Biosciences, San Jose, CA, USA) and annexin V (5 μL; APC Annexin V, BD Biosciences, San Jose, CA, USA) Into each tube. After incubating at room temperature for 15 minutes in the dark, the cells were resuspended in 400 μL of binding buffer. The BD FACS Canto flow cytometer (BD Biosciences, San Jose, CA, USA) was used to analyze the samples by BD FACSDiva software (version 5.0.3; BD Biosciences, Oxford, United Kingdom).

The alkaline version of single cell microgel electrophoresis (comet) assay is used to show DNA strand breaks and alkali instability and incomplete excision repair sites in single cells. 42

Based on previous cytotoxicological analysis by MTT assay and Annexin V assay, ZnO NPs were tested in HUVEC at sub-cytotoxic doses of 1, 5, and 10 µg/mL. In addition, pure medium was used as a negative control, and 200 µM directly alkylated methyl methanesulfonate (MMS, Sigma-Aldrich) was used as a positive control. Apply the test concentration for 24 hours. Three subsequent treatments were performed in the repeated exposure setting for 1 hour, with intermittent washing steps and another hour of regeneration period (Figure 2). Figure 2 The experimental design of cells repeatedly exposed to ZnO NP. HUVEC was exposed for 1 hour, followed by 3 times, with a washing step and a 1-hour regeneration period, followed by a 24-hour regeneration period. The MTT measurement was performed after 1 hour of treatment, 3 hours of treatment, and 24 hours of regeneration period. The comet test was performed after 3 hours of treatment.

Figure 2 The experimental design of cells repeatedly exposed to ZnO NP. HUVEC was exposed for 1 hour, followed by 3 times, with a washing step and a 1-hour regeneration period, followed by a 24-hour regeneration period. The MTT measurement was performed after 1 hour of treatment, 3 hours of treatment, and 24 hours of regeneration period. The comet test was performed after 3 hours of treatment.

The comet test was performed and analyzed as described above. 43,44 For each test concentration, use a fluorescence microscope to semi-automatically analyze 50 randomly selected single cells on each of the two slides (100 cells in total for each test concentration) (Leica Microsystems, Wetzlar, Germany) ) Use green excitation filter, two-color beam splitter (580 nm long pass) and emission filter (590 nm long pass), and the magnification is 400 times. Use the software COMET 5.5 imaging system (Kinetic Imaging, Liverpool, UK). The percentage of DNA in the tail (TD), tail length (TL), and olive tail moment (OTM) are taken as the product of the median migration distance and the percentage of DNA in the tail as the result parameters. 45 In our research, OTM is used for statistical analysis.

In order to determine the effect of ZnO NPs on the proliferation ability of HUVEC, the cell number and viability were measured before and after 1 day of culture. Therefore, before treatment with ZnO NPs, NPs were treated with various test concentrations of ZnO for 24 hours, and 24 hours after treatment. Hours and 48 hours. All measurements were performed as follows: each well was inoculated with 3x104 HUVEC and cultured in a 12-well plate at 37°C and 5% CO2. ECGM included supplements and 1% penicillin/streptomycin. After trypsinization and stopping the enzymatic reaction as described above, the number of cells was measured by a Casy Modell TT cell counter (Innovatis AG, Reutlingen, Germany). Each test is performed 3 times and the arithmetic average is calculated to achieve higher statistical validity.

To examine the effect of ZnO NPs on HUVEC's ability to paracrine production and secretion of cytokines (such as vascular endothelial growth factor (VEGF)), dot blot analysis (Raybiotec Inc., Norcross, GA, USA) was performed. The inspection of this semi-quantitative analysis is carried out in accordance with the manufacturer's agreement. Therefore, 5x104 HUVEC per well was cultured in a 12-well plate for 24 hours. To avoid test fraud, since fetal bovine serum usually contains VEGF, ECGM without supplements (ie, no FCS) is used. In addition, before incubating with the treatment solution for another 24 hours, perform a washing step with PBS. Next, collect the supernatant and centrifuge at 1500 rpm for 15 minutes at room temperature. This centrifugation step is performed twice. After that, the supernatant was frozen and analyzed later. Use detection buffer and exposure to X-ray film to show the presence of labeled cytokines by chemiluminescence. The size and intensity of the dots represent the concentration level. Use NIH ImageJ (https://imagej.nih.gov/ij/) to automatically analyze the integral density of points.

The capillary formation test was conducted to show the possible effect of ZnO NPs on the proliferation of vascular endothelial cells and the ability to form microtubules as a model for neovascularization and tissue repair. 46 Therefore, 10 µL Matrigel (Sigma-Aldrich, St. Louis, Missouri, USA) was transferred to a special plate (μ-Slide Angiogenesis Plate, ibidi GmbH, Martinsried, Germany) and stored in the culture under standard conditions In the box. Thereafter, transfer 50 µL of the test cell suspension (containing 104 cells) to each well. The plates were incubated as described above and analyzed after 5 and 24 hours. In order to capture the entire hole, four pictures of each hole were acquired by a phase-contrast inverted microscope (Leica Microsystems, Wetzlar, Germany). In order to achieve the highest possible statistical validity and objectivity, all test concentrations are performed in triplicate, and each test concentration produces 12 pictures. One of the four pictures with the best capillary formation was carefully selected and used for automatic image analysis. Finally, three analysis pictures were generated for each test concentration. According to the recommendations of DeCicco-Skinner et al., NIH ImageJ uses the Angiogenesis Analyzer plug-in 47 to automatically analyze tube formation. 46

The average value was used to analyze the dose-dependent effects within the treatment group by Friedman's test. The Friedman test is the best available method for detecting differences in three or more related samples, and therefore, it is the preferred method for elucidating dose dependence. For the assessment of viability, the negative control is defined as 100%. Use SPSS Statistics 25.0 for windows (IBM Corp., Armonk, NY, USA) for statistical analysis. P value<0.05 was considered statistically significant.

To characterize the nanoparticles, transmission electron microscopy (TEM) and dynamic light scattering were performed. The average diameter of the nanoparticles is 45-55 nm, and they are spherical. The average diameter of the aggregates is 121 nm, the zeta potential is -11 mV, and the polydispersity index is 0.14. These findings did not match the specifications provided by commercial suppliers.

In order to determine the cytotoxicity threshold of ZnO NPs to HUVEC, MTT and Annexin V assays were used. Compared with the untreated control in the MTT assay, viability is expressed as a percentage (Figure 3). Neither test showed any effect on viability at concentrations between 5 and 20 µg/mL. In the MTT assay, a concentration of 35 to 50 µg/mL results in a reduction of viable cells. The difference was significant in Friedman's test (p<0.01). In the annexin V assay, an increase in apoptosis and necrotic cells can also be observed at concentrations higher than 30 µg/mL. Cells treated with 25 µg/mL showed a tendency to decrease the survival rate of MTT, which was similar to that found in the Annexin V test: 2.0% of apoptotic cells after exposure to 20 µg/mL, and after exposure to 25 µg/mL It is 5.7%, using 27.6% 30 µg/mL (Figure 4). Figure 3 The viability of HUVEC after 24 hours of exposure to ZnO NP. The untreated control is defined as 100%. 10 mM tBHP was used as a positive control (pos). A significant reduction in HUVEC vitality was observed in the Friedman test. Figure 4 continued. Figure 4 Annexin V measurement. The results of HUVEC treated with ZnO NP concentrations of 20, 25, 30, 35, 40, 45, and 50 µg/mL are shown, and negative and positive controls are shown. Q1, percentage of damaged cells; Q2, percentage of necrotic cells; Q3, percentage of live cells; Q4, percentage of apoptotic cells.

Figure 3 The viability of HUVEC after 24 hours of exposure to ZnO NP. The untreated control is defined as 100%. 10 mM tBHP was used as a positive control (pos). A significant reduction in HUVEC vitality was observed in the Friedman test.

Figure 4 Annexin V measurement. The results of HUVEC treated with ZnO NP concentrations of 20, 25, 30, 35, 40, 45, and 50 µg/mL are shown, and negative and positive controls are shown. Q1, percentage of damaged cells; Q2, percentage of necrotic cells; Q3, percentage of live cells; Q4, percentage of apoptotic cells.

In addition, we used the MTT assay to study the effects of repeated exposure to ZnO NPs. Friedman's test showed that there was a significant dose-dependent difference between 1 (p=0.032) and 3 (p=0.029) after a continuous 1-hour treatment period and after a 24-hour regeneration period (p=0.042; Figure 5). Figure 5 The viability of HUVEC after exposure to ZnO NPs (µg/mL). The untreated control is defined as 100%, and tBHP 10 mM is used as the positive control (pos). The figure shows the results after 1 and 3 consecutive 1 hour treatment periods and 24 hours regeneration period (reg).

Figure 5 The viability of HUVEC after exposure to ZnO NPs (µg/mL). The untreated control is defined as 100%, and tBHP 10 mM is used as the positive control (pos). The figure shows the results after 1 and 3 consecutive 1 hour treatment periods and 24 hours regeneration period (reg).

Compared with the control group, in HUVEC exposed to sub-cytotoxic concentrations of ZnO NPs, DNA damage was significantly increased as determined by comet assay. Compared with untreated cells, long-term exposure (p<0.001) and repeated exposure (p<0.001) and ZnO NP resulted in an increase in OTM. In both test environments, MMS-treated cells as a positive control also showed enhanced DNA migration (Figure 6). Figure 6 DNA fragments expressed in the olive tail moment (OTM) in HUVEC after exposure to ZnO NPs (µg/mL). ECGM without ZnO NPs was used as a negative control (c) and MMS 200 µM was used as a positive control (pos). The graph shows the results of long-term (24 hours) and repeated (rep) exposure to ZnO NP (µg/mL) for 24 hours.

Figure 6 DNA fragments expressed in the olive tail moment (OTM) in HUVEC after exposure to ZnO NPs (µg/mL). ECGM without ZnO NPs was used as a negative control (c) and MMS 200 µM was used as a positive control (pos). The graph shows the results of long-term (24 hours) and repeated (rep) exposure to ZnO NP (µg/mL) for 24 hours.

The proliferation assay showed that there was no significant difference between the cells treated with subcytotoxic ZnO NPs concentration and the negative control within 24 hours after long-term treatment. At 48 hours after treatment, the cells showed an inverse concentration-dependent proliferation ability. Although the negative control showed the greatest decrease in cell number, increasing the ZnO NP concentration seemed to lead to an increase in the ability to further proliferate (Figure 7). Figure 7 Cell number of HUVEC. Measure the number of cells at the time of inoculation, before treatment (BT), immediately after treatment (AT), 24 hours after treatment (24 hours AT), and 48 hours after treatment (48 hours AT), using 1, 5, 10, and 15 µg /mL ZnO nanoparticles. ECGM serves as a negative control (control). There was no difference in AT until 24 hours. At 48 hours, the reverse dose-dependent proliferation rate can be observed in AT. The higher the ZnO NP concentration, the better the proliferation of HUVEC. We interpret this as a sign that cells have reached the pore limit earlier when exposed to lower concentrations of ZnO NP.

Figure 7 Cell number of HUVEC. Measure the number of cells at the time of inoculation, before treatment (BT), immediately after treatment (AT), 24 hours after treatment (24 hours AT), and 48 hours after treatment (48 hours AT), using 1, 5, 10, and 15 µg /mL ZnO nanoparticles. ECGM serves as a negative control (control). There was no difference in AT until 24 hours. At 48 hours, the reverse dose-dependent proliferation rate can be observed in AT. The higher the ZnO NP concentration, the better the proliferation of HUVEC. We interpret this as a sign that cells have reached the pore limit earlier when exposed to lower concentrations of ZnO NP.

Dot blot analysis showed that HUVECs treated with sub-cytotoxic concentrations of ZnO NPs released less VEGF than the negative control (Figure 8). Figure 8 Dot blot. The dot blot showed that HUVEC treated with ZnO NPs released less VEGF. The pictures of the negative control (A) and 5 µg/mL (B) show the VEGF dots highlighted by the red oval. Figure (C) shows the integrated density of VEGF points released by HUVEC after exposure to ECGM (c; negative control) and ZnO NPs (μg/mL) analyzed using ImageJ.

Figure 8 Dot blot. The dot blot showed that HUVEC treated with ZnO NPs released less VEGF. The pictures of the negative control (A) and 5 µg/mL (B) show the VEGF dots highlighted by the red oval. Figure (C) shows the integrated density of VEGF points released by HUVEC after exposure to ECGM (c; negative control) and ZnO NPs (μg/mL) analyzed using ImageJ.

The capillary formation test showed that the number of meshes and nodes and the length of the tube were reduced. The number of grids showed a significant dose-dependent reduction (p = 0.033), while the number of nodes and tube length did not (p = 0.053 for both) (Figures 9 and 10). Figure 9 Capillary formation test. This measurement showed that the function of HUVEC decreased after exposure to ZnO NPs. The picture shows the tube formation after treatment with ECGM ((A); negative control) and 15 µg/mL ZnO NPs (B). ImageJ uses the Angiogenesis Analyzer plug-in to automatically analyze tube formation. Figure 10 The capillary formation test shows that the number of meshes (A), the number of nodes (B) and the length of the tube ((C), tube length (mm)) are reduced. Although the number of grids is significantly reduced (p = 0.033), the number of nodes and tube length are not significantly reduced (p = 0.053 for both).

Figure 9 Capillary formation test. This measurement showed that the function of HUVEC decreased after exposure to ZnO NPs. The picture shows the tube formation after treatment with ECGM ((A); negative control) and 15 µg/mL ZnO NPs (B). ImageJ uses the Angiogenesis Analyzer plug-in to automatically analyze tube formation.

Figure 10 The capillary formation test shows that the number of meshes (A), the number of nodes (B) and the length of the tube ((C), tube length (mm)) are reduced. Although the number of grids is significantly reduced (p = 0.033), the number of nodes and tube length are not significantly reduced (p = 0.053 for both).

ZnO NPs are an important part of medical solutions, cosmetics and dietary supplements. 49 There is no doubt that ZnO NPs come into contact with human cells in various ways; however, it is not yet clear how they affect undamaged cells and tissues. In this regard, general nanomaterials must consider the balance between human health risks and economic benefits. 37 Risk stratification is becoming more and more indispensable.

The main purpose of this study is to determine the cytotoxicity threshold of ZnO NPs in HUVEC after long-term exposure. According to the results of another study, 15 we found that concentrations higher than 25 µg/mL produce cytotoxic effects and increase cell apoptosis and necrosis. Paszek et al. demonstrated the time and dose-dependent changes in HUVEC cell membrane and cytoskeleton after a single exposure of 4 hours and 24 hours. Long-term and repeated exposure have shown dysfunction in VEGF expression and tube formation.

Many studies have proven that ZnO NPs can induce DNA damage in various cell systems,8,29,31,49, and determining the underlying mechanism of these effects is the goal of many recent investigations. The small size, large surface area, and physicochemical properties of ZnO NPs, as well as the fact that they can be transferred into cells through various mechanisms, are key factors in ZnO nanocytotoxicology and nanogenotoxicology. 49,50 Our results indicate that product information does not necessarily match the characteristics of NP observed in the laboratory. Therefore, we recommend a detailed analysis and description of NP to provide comparable data. Zeta potential, electromotive force in colloidal dispersion 51 and polydispersity index, relative molecular mass distribution measurement 52 are widely used to describe NP in suspension.

Different mechanisms may cause the toxic effects of ZnO NPs. Bai et al. revealed that mitochondrial dysfunction leads to increased ROS production and continuous DNA damage and cell death. 53 Another study showed that the up-regulation of lipoxygenase in neuroblastoma cells stimulates the production of ROS. 54 It has been suggested that the continuous generation of ROS after dissolving ZnO NPs into Zn2+ ions and incorporation may be the cause of the genotoxic effect. 50,55 This seems more likely because zinc is a component of many intracellular enzymes and transcription factors in the human organism. 27,32,56 Compared with normal cells, it is controversial whether ZnO NPs induce cytotoxicity in cancer cells at a lower concentration than normal cells. 21,57,58 This is an important question that needs to be answered because they are discussed as promising drugs for drug delivery in cancer treatment. 59

In our study, we observed dose-dependent DNA damage after treatment with sub-cytotoxic concentrations of ZnO NPs in HUVEC, which may affect the formation of new blood vessels. Considering the application of sunscreen, this may be a problem for damaged skin. On the other hand, it may be beneficial to fast-growing tissues such as tumors.

Since Vermylen et al. pointed out in their comments that particulate air pollutants can enter the circulatory system through ingestion and inhalation and cause coronary heart disease, there is increasing interest in the underlying mechanism of how NPs affect the vascular system. 60 Despite in-depth research, especially on ZnO NPs, data on the effect of NP on HUVEC as the main cell model of the specific microvascular endothelial system is still quite lacking. In our current study, we have demonstrated the functional impairment of HUVEC in different observations as a function of various sub-cytotoxic ZnO NP concentrations. The proliferation rate of HUVEC cells was monitored during the first 24 hours after exposure, and no difference was found between cells exposed to ZnO NPs and untreated cells. After another 24 hours, the proliferation capacity of the negative control showed significant contraction, and the proliferation of cells exposed to NP was dependent on the NP concentration. This may be the result of cells reaching the pore limit earlier when exposed to lower NP concentrations. VEGF secretion is also measured as a representative protein that induces blood vessel formation. This study shows that at the sub-cytotoxic concentration of HUVEC, the production of VEGF is severely restricted in a dose-dependent manner. Finally, when analyzing the ability of HUVEC to form typical tubes, nodes, and grids as its main function, it was demonstrated that the ability to decrease related to the increase in the concentration of ZnO NPs. Regarding HUVEC, these findings indicate a lack of new blood vessel formation under the influence of ZnO NPs. Again, this may have a negative impact on pre-damaged tissues, and at the same time, it may help slow tumor progression.

Generally speaking, ZnO NP exposure to workers as well as consumers and patients can be considered repetitive. However, the existing literature clearly focuses on a single exposure setting. Our research team previously demonstrated that repeated exposure can cause sustained or sustained DNA damage in various cell lines. 30,31 In this study, there was no difference between long-term or repeated exposure to sub-cytotoxic concentrations of ZnO NPs because both exposure methods induced cell death and increased DNA fragmentation in a similar manner.

Nevertheless, the available information on the genotoxic effects and dysfunction caused by ZnO NPs is still limited. In particular, the underlying molecular mechanism needs further research. The generation of dissolved Zn2+ ions and ROS seems to be the related mechanism of cytotoxicity and genotoxicity caused by ZnO NPs. 61 Therefore, we recommend further research on the basis of tissue culture. Analyzing the behavior of ZnO NPs in tissue structure may help us understand how they affect human cells. In particular, the degree of solubility of ionized Zn2+ should be clarified.

In this study, the cytotoxicity threshold of ZnO NPs in HUVEC can be defined. In addition, genotoxic effects have been demonstrated after prolonged and repeated exposure. In several ways, the dysfunction of the microvascular endothelial model caused by ZnO NPs was confirmed. This study shows that ZnO NPs have anti-angiogenic effects.

Supported by the Johannes Kepler Open Access Publishing Fund.

The authors report no conflicts of interest in this work.

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