Ag- but Not ZnO-Nanoparticles Disturb the Airway Epithelial Barrier at Subtoxic Concentrations.

Inhalation is considered to be the most relevant source of human exposure to nanoparticles (NPs); however, only a few investigations have addressed the influence of exposing the respiratory mucosal barrier to subcytotoxic doses. In the nasal respiratory epithelium, cells of the mucosa represent one of the first contact points of the human organism with airborne NPs. Disruption of the epithelial barrier by harmful materials can lead to inflammation in addition to potential intrinsic toxicity of the particles. The aim of this study was to investigate whether subtoxic concentrations of zinc oxide (ZnO)- and silver (Ag)-NPs have an influence on upper airway barrier integrity. Nasal epithelial cells from 17 donors were cultured at the air-liquid interface and exposed to ZnO- and Ag-NPs. Barrier function, quantified by transepithelial electrical resistance (TEER), decreased after treatment with 10 µg/mL Ag-NPs, but FITC-dextran permeability remained stable and no change in mRNA levels of tight junction proteins and E-cadherin was detected by real-time quantitative PCR (RT-qPCR). The results indicate that subtoxic concentrations of Ag-NPs may already induce damage of the upper airway epithelial barrier in vitro. The lack of similar disruption by ZnO-NPs of similar size suggests a specific effect by Ag-NPs.

Influence of Wound Fluid on the Transdifferentiation of Human Mesenchymal Bone Marrow Stem Cells into Cancer-Associated Fibroblasts.

Cancer-associated fibroblasts (CAF) in the tumor microenvironment have a decisive influence on tumor growth and metastatic behavior. The cellular origins as well as the stimuli leading to CAF formation are heterogenous, impeding a precise characterization. Aim of this study was to analyze the influence of cytokines secreted in the process of wound healing, tumor cell-associated paracrine-secreted factors, and direct cell-cell contact on the expression of the CAF-associated markers fibroblast activation protein (FAP), α-smooth muscle actin (α-SMA), thrombospondin-1 (THBS1), and tenascin-c (TNC) by RT-PCR in mesenchymal stem cells (MSC). Cells developed different morphological characteristics after incubation with wound fluid (WF). Moreover, expression of FAP and α-SMA in MSC was significantly reduced after WF compared to tumor-conditioned medium and in co-culture with tumor cells; THBS1 and TNC were not significantly altered after any of the different incubation methods. There were no alterations of expression patterns of FAP and α-SMA in the immunohistochemical analysis. Differ-ences in the cytokine composition of the media were found in the dot blot. The heterogeneity of the results emphasizes the complexity of the interactions of tumor cells and cells of the microenvironment, particularly through the addition of human-derived WF.

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.

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.

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-NP20 nm and ZnO-NP100 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-NP20 nm or ZnO-NP100 nm was observed in a concentration of 10 µg/mL or 1 µg/mL respectively in 30 µg/mL of ZnO-NP20 nm or 20 µg/mL of ZnO-NP100 nm in FB. The addition of 1, 5, or 10 µg/mL ZnO-NP20 nm or ZnO-NP100 nm significantly reduced the clonogenic survival of FaDu after irradiation. The sub-cytotoxic dosage of ZnO-NP100 nm increased the oxidative DNA damage compared to the irradiated control. This effect was not significant for ZnO-NP20 nm. ZnO-NP showed radiosensitizing properties in the sub-cytotoxic dosage. At least for the ZnO-NP100 nm, an increased level of oxidative stress is a possible mechanism of the radiosensitizing effect.

Toxicological characterization of ZnO nanoparticles in malignant and non-malignant cells.

The increasing usage of zinc oxide nanoparticles (ZnO-NPs) in industrial applications as well as in consumer products raises concern regarding their potential adverse effects to a greater extend. Numerous studies have demonstrated toxic properties of NPs, however there is still a lack of knowledge concerning the underlying mechanisms. This study was designed to systematically investigate cytotoxicity, apoptosis, cell cycle alterations, and genotoxicity induced by ZnO-NP. Moreover, it was an aim of the investigations to specify the diverse effects of nanoparticle exposure in malignant in comparison with non-malignant cells. Therefore, human head and neck squamous cell carcinoma-derived FaDu cells were incubated with 4-20 µg/ml of ZnO-NPs for 1-48 hr and tested for cell viability, cell cycle alterations, apoptosis and caspase-3 gene expression as a sensitive marker of molecular apoptotic processes with regard to time- and dose-dependent effects. Human mesenchymal bone marrow stem cells were used as non-malignant representatives to examine oxidative stress-related genotoxicity. Results showed a significant reduction in cell viability as well as dose- and time-dependent increase of apoptotic cells following nanoparticle treatment. Likewise, caspase-3 gene expression enhanced already before first apoptotic cells were detectable. It could be observed that doses that were cytotoxic in tumor cells did not reduce viability in stem cells. However, the same concentrations already induced significant DNA damage. The findings of the study suggest to keep a more critical eye on the use of nanoparticles as anti-cancer agents. Yet, additional in vivo studies are needed to assess safety concerns for consumers and patients. Environ. Mol. Mutagen. 59:247-259, 2018. © 2017 Wiley Periodicals, Inc.