Analysis of zinc oxide nanoparticle-associated mechanisms of toxicity using the example of a human squamous cell carcinoma cell line and primary human mesenchymal stem cells

Einleitung: Zinkoxid (ZnO) ist eines der am häufigsten für industrielle Produktionen und Konsumgüter eingesetzten Nanomaterialien. Zytotoxizität von ZnO-Nanopartikeln (NP) war bereits Gegenstand einiger Studien, jedoch sind die molekularen Schädigungsmechanismen nicht gänzlich geklärt. Über genotoxische Eigenschaften, die bereits bei sub-zytotoxischen Dosen auftreten können, ist im Allgemeinen weniger bekannt. Ziel dieser Studie war die Erstellung eines umfassenden Toxizitätsprofils von ZnO-NP durch Einsatz multipler Testsysteme. Methoden: Neben der Plattenepithelkarzinom-Zelllinie FaDu wurden humane mesenchymale Knochenmarkstammzellen (BMSC) für unterschiedliche Zeiträume und Konzentrationen mit ZnO-NP behandelt. Zytotoxizität, Apoptoseinduktion und Zellzyklusalteration wurden durch MTT-Test, PCR und Durchflusszytometrie analysiert. Mit dem fpg-modifizierten Comet Assay wurden der Einfluss von oxidativem Stress auf das Gesamtausmaß der DNA-Schädigung untersucht. Ergebnisse: ZnO-NP führten im MTT-Test ab 8 µg/ml zu einer Reduktion der Vitalität in FaDu-Zellen. Durchflusszytometrisch wurden eine dosis- und zeitabhängige Zunahme von Apoptose sowie Veränderungen des Zellzyklus nachgewiesen. Im Comet Assay konnte nach Inkubation mit 5 µg/ml ZnO-NP eine signifikante DNA-Fragmentierung in BMSC nachgewiesen werden. Bei allen getesteten Konzentrationen wurde oxidativer Stress als wichtiger Einflussfaktor der Schädigung nachgewiesen. Diskussion: Vorliegende Studie liefert Hinweise dafür, dass ZnO-NP toxisch sind. Gegenwärtig ist eine definitive Aussage über das schädigende Potenzial von NP nicht zu treffen, da der Vergleich verschiedener Studien kaum möglich ist. Gerade die Verwendung von ZnO-NP als Bestandteil von Kosmetikprodukten, die repetitiv in geringen Mengen von Verbrauchern appliziert werden, sollte jedoch kritisch betrachtet werden.Introduction: Zinc oxide (ZnO) is among the most commonly used nanomaterials for consumer products. Cytotoxicity of ZnO-nanoparticles (ZnO-NP) has already been addressed in several studies; however, there is still a lack of knowledge concerning the molecular mechanisms of toxicity. As to genotoxicity, which can already occur at sub-toxic doses, data is generally rare. It was therefore the aim of this study to establish a broad profile of toxicity for ZnO-NP by applying multiple test systems. Methods: Besides the head and neck squamous cell carcinoma-derived cell line FaDu, primary human bone marrow-derived mesenchymal stem cells (BMSC) were treated with ZnO-NP with variable doses and for different time periods. Cytotoxicity, induction of apoptosis and cell cycle alteration were assessed by MTT-Test, PCR and flow cytometry. Fpg-modified Comet Assay was used to determine the influence of oxidative stress with regard to the total DNA-damage. Results: 8 µg/ml ZnO-NP reduced cell viability in FaDu cells in the MTT-Test. Dose- and time-dependent increase of apoptosis and alterations of the cell cycle were verified by flow cytometry. After incubation with 5 µg/ml ZnO-NP a significant DNA-fragmentation was measured in BMSC with the Comet Assay. In all tested concentrations oxidative stress could be identified as an important factor of cell damage. Discussion: This study provides evidence that ZnO-NP are toxic. At the current point a definite statement concerning the damaging potential of ZnO-NP is not to be made because the comparison between different studies is not possible. Yet especially the repetitive low-dose application of ZnO-NP as component of cosmetic products should be investigated toxicologically

The radiosensitizing effect of zinc oxide nanoparticles in sub-cytotoxic dosing is associated with oxidative stress in vitro

Radioresistance is an important cause of head and neck cancer therapy failure. Zinc oxide nanoparticles (ZnO-NP) mediate tumor-selective toxic effects. The aim of this study was to evaluate the potential for radiosensitization of ZnO-NP. The dose-dependent cytotoxicity of ZnO-NP$_{20 nm}$ and ZnO-NP$_{100 nm}$ was investigated in FaDu and primary fibroblasts (FB) by an MTT assay. The clonogenic survival assay was used to evaluate the effects of ZnO-NP alone and in combination with irradiation on FB and FaDu. A formamidopyrimidine-DNA glycosylase (FPG)-modified single-cell microgel electrophoresis (comet) assay was applied to detect oxidative DNA damage in FB as a function of ZnO-NP and irradiation exposure. A significantly increased cytotoxicity after FaDu exposure to ZnO-NP$_{20 nm}$ or ZnO-NP$_{100 nm}$ was observed in a concentration of 10 µg/mL or 1 µg/mL respectively in 30 µg/mL of ZnO-NP$_{20 nm}$ or 20 µg/mL of ZnO-NP$_{100 nm}$ in FB. The addition of 1, 5, or 10 µg/mL ZnO-NP$_{20 nm}$ or ZnO-NP$_{100 nm}$ significantly reduced the clonogenic survival of FaDu after irradiation. The sub-cytotoxic dosage of ZnO-NP$_{100 nm}$ increased the oxidative DNA damage compared to the irradiated control. This effect was not significant for ZnO-NP$_{20 nm}$. ZnO-NP showed radiosensitizing properties in the sub-cytotoxic dosage. At least for the ZnO-NP$_{100 nm}$, an increased level of oxidative stress is a possible mechanism of the radiosensitizing effect

Investigation of the immune modulatory potential of zinc oxide nanoparticles in human lymphocytes

Zinc oxide nanoparticles (ZnO-NP) are commonly used for a variety of applications in everyday life. In addition, due to its versatility, nanotechnology supports promising approaches in the medical sector. NP can act as drug-carriers in the context of targeted chemo- or immunotherapy, and might also exhibit autonomous immune-modulatory characteristics. Knowledge of potential immunosuppressive or stimulating effects of NP is indispensable for the safety of consumers as well as patients. In this study, primary human peripheral blood lymphocytes of 9 donors were treated with different sub-cytotoxic concentrations of ZnO-NP for the duration of 1, 2, or 3 days. Flow cytometry was performed to investigate changes in the activation profile and the proportion of T cell subpopulations. ZnO-NP applied in this study did not induce any significant alterations in the examined markers, indicating their lack of impairment in terms of immune modulation. However, physicochemical characteristics exert a major influence on NP-associated bioactivity. To allow a precise simulation of the complex molecular processes of immune modulation, a physiological model including the different components of an immune response is needed

Cultivation of head and neck squamous cell carcinoma cells with wound fluid leads to cisplatin resistance via epithelial-mesenchymal transition induction

Locoregional recurrence is a major reason for therapy failure after surgical resection of head and neck squamous cell carcinoma (HNSCC). The physiological process of postoperative wound healing could potentially support the proliferation of remaining tumor cells. The aim of this study was to evaluate the influence of wound fluid (WF) on the cell cycle distribution and a potential induction of epithelial-mesenchymal transition (EMT). To verify this hypothesis, we incubated FaDu and HLaC78 cells with postoperative WF from patients after neck dissection. Cell viability in dependence of WF concentration and cisplatin was measured by flow cytometry. Cell cycle analysis was performed by flow cytometry and EMT-marker expression by rtPCR. WF showed high concentrations of interleukin (IL)-6, IL-8, IL-10, CCL2, MCP-1, EGF, angiogenin, and leptin. The cultivation of tumor cells with WF resulted in a significant increase in cell proliferation without affecting the cell cycle. In addition, there was a significant enhancement of the mesenchymal markers Snail 2 and vimentin, while the expression of the epithelial marker E-cadherin was significantly decreased. After cisplatin treatment, tumor cells incubated with WF showed a significantly higher resistance compared with the control group. The effect of cisplatin-resistance was dependent on the WF concentration. In summary, proinflammatory cytokines are predominantly found in WF. Furthermore, the results suggest that EMT can be induced by WF, which could be a possible mechanism for cisplatin resistance