Pascal Ickrath

Time-Dependent Toxic and Genotoxic Effects of Zinc Oxide Nanoparticles after Long-Term and Repetitive Exposure to Human Mesenchymal Stem Cells

Zinc oxide nanoparticles (ZnO-NP) are widely spread in consumer products. Data about the toxicological characteristics of ZnO-NP is still under controversial discussion. The human skin is the most important organ concerning ZnO-NP exposure. Intact skin was demonstrated to be a sufficient barrier against NPs; however, defect skin may allow NP contact to proliferating cells. Within these cells, stem cells are the most important toxicological target for NPs. The aim of this study was to evaluate the genotoxic and cytotoxic effects of ZnO-NP at low-dose concentrations after long-term and repetitive exposure to human mesenchymal stem cells (hMSC). Cytotoxic effects of ZnO-NP were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Furthermore, genotoxicity was evaluated by the comet assay. For long-term observation over 6 weeks, transmission electron microscopy (TEM) was applied. The results of the study indicated cytotoxic effects of ZnO-NP beginning at high concentrations of 50 μg/mL and genotoxic effects in hMSC exposed to 1 and 10 μg/mL ZnO-NP. Repetitive exposure enhanced cyto- but not genotoxicity. Intracellular NP accumulation was observed up to 6 weeks. The results suggest cytotoxic and genotoxic potential of ZnO-NP. Even low doses of ZnO-NP may induce toxic effects as a result of repetitive exposure and long-term cellular accumulation. This data should be considered before using ZnO-NP on damaged skin.

The Proinflammatory Potential of Nitrogen Dioxide and Its Influence on the House Dust Mite Allergen Der p 1

Asthma and allergies are both major global health problems with an increasing prevalence, and environmental data implicate an influence of air pollutants on their development. The present study focuses on the influence of nitrogen dioxide (NO2) and the major allergen of the house dust mite Der p 1 on human nasal epithelial cells of nonallergic patients in vitro. Nasal epithelial mucosa samples of 11 donors were harvested during nasal air passage surgery and cultured as an air-liquid interface. Exposure to 0.1, 1 and 10 ppm NO2 or synthetic air as a control was performed for 1 h. Subsequently, the cells were exposed to Der p 1 for 24 h. The release of interleukin (IL)-6 and IL-8 was measured by ELISA, and the production of IL-6 mRNA and IL-8 mRNA was measured by RT-PCR. NO2 exposure resulted in a concentration-dependent release of IL-6, but not IL-8 release. The coexposure of 0.1 ppm NO2 and Der p 1, or 1 ppm NO2 and Der p 1 significantly increased both IL-6 and IL-8 release. Exposure to NO2, Der p 1, or their combination, did not significantly influence the production of IL-6 or IL-8 mRNA. In conclusion, NO2 increases the release of inflammatory cytokines in human nasal epithelial cells, especially in coexposure with Der p 1, as a mechanism of allergotoxicology.

Genotoxic effects of zinc oxide nanoparticles in nasal mucosa cells are antagonized by titanium dioxide nanoparticles

Titanium dioxide nanoparticles (TiO2-NPs) and zinc oxide nanoparticles (ZnO-NPs) are often used in sunscreens and other consumer products due to their photoprotective properties. However, concern exists regarding them possibly causing cyto- and genotoxic effects. The aim of this study was to assess cyto- and genotoxicity of these nanomaterials after single or combined exposure. For this purpose, a battery of cell culture test systems for human nasal mucosa (monolayer, air-liquid interface and mini organ culture) were exposed to 0.1-20μg/ml of TiO2- and ZnO-NPs alone and in combination. Cytotoxicity was measured by the MTT assay, and DNA damage and repair capacity were investigated using the comet assay. TiO2-NPs did not exhibit any cyto- or genotoxic potential within the tested concentrations. However, results of the study indicated cyto- and genotoxicity resulting from ZnO-NPs. The genotoxicity could be antagonized by TiO2-NPs. Furthermore, the DNA repair capacity after ZnO-NP-induced DNA damage was enhanced by TiO2-NPs. The adsorption of dissolved zinc ions onto TiO2-NPs is discussed as the major antagonistic mechanism. The combination of both metal oxide nanoparticles interferes with the genotoxicity of ZnO-NPs and should be discussed as a reasonable and safe alternative to the sole use of ZnO-NPs in consumer products.

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.

Impact and Modulations of Peripheral and Edaphic B Cell Subpopulations in Chronic Rhinosinusitis With Nasal Polyposis

Objectives The pathophysiological mechanisms of chronic rhinosinusitis with nasal polyposis (CRSwNP) still are discussed controversially. Regulatory B cells (Breg) are responsible for the suppression of T cell activity: deficiencies for Breg have been demonstrated to contribute to autoimmune disorders, e.g., systemic lupus erythematosus. In order to evaluate the influence of B cell subpopulations, especially Breg, on the etiology of this disease, the aim of this study was to characterize subpopulations of peripheral and edaphic B cells in CRSwNP. Methods Polypoid tissue and blood samples were collected from 10 patients undergoing paranasal sinus surgery and lymphocytes were analyzed by multicolor flow cytometry. Results There was a significantly lower frequency of B cells in nasal polyps compared to peripheral blood mononuclear cells (PBMC) in patients with CRSwNP. Mature resting B cells were the main population within B cells in PBMC, and memory B cells in nasal polyps. Remarkably, Breg and mature B cells significantly decreased in nasal polyps compared to PBMC. Memory B cells significantly increased and represented the main subpopulation in nasal polyps in patients with CRSwNP. Conclusion In this study a detailed contemporary characterization of B cell subpopulations in patients with CRSwNP is presented. The influence of edaphic B cells could play a key role in the maintenance of this chronic infectious disease.

Long‑term changes in the properties of skin-derived fibroblasts following irradiation of the head and neck

The tumor stroma performs an important role in carcinogenesis. It predominantly consists of fibroblasts and the connective tissue produced by them, and undergoes a multitude of interactions with the surrounding cancer cells. Since irradiation is part of the majority of therapeutic strategies for head and neck squamous cell carcinoma, more information regarding the effects of a previous irradiation on the tumor stroma is desirable. In the present study, fibroblasts were cultivated from human non-irradiated and pre-irradiated skin of the neck for 48 h. Subsequently, analyses of cell viability, apoptosis, necrosis and motility were conducted via MTT assay, Annexin V/propidium iodide staining, electronic cell counting for 4 consecutive days, and scratch assay. Pre-irradiated fibroblasts exhibited a significantly slower growth rate as well as increased rates of apoptosis and necrosis. They also exhibited significantly decreased motility compared with non-irradiated fibroblasts. These results indicated the long-term effects of irradiation on fibroblasts, which may affect cancer recurrence in the irradiated region via the tumor stroma. More information, such as that regarding the secretory capacities of pre-irradiated fibroblasts, is required to evaluate the possible therapeutic implications of these findings.

Zinc oxide nanoparticles antagonize the effect of Cetuximab on head and neck squamous cell carcinoma in vitro

ABSTRACT Zinc oxide nanoparticles (ZnO-NPs) are being used in many cosmetic products and have been shown to induce tumor-selective cell death in human head and neck squamous cell carcinoma (HNSCC) in vitro. Cetuximab is a monoclonal antibody directed against the epidermal growth factor receptor (EGFR), whose effectiveness for HNSCC, alone or in combination with cytostatic drugs, has been demonstrated intensively in the last decades. Nanoparticles are known to interact with protein structures and thus may influence their functionality. The aim of the current study was to evaluate the effect of ZnO-NPs on the antitumor properties of Cetuximab in HNSCC in vitro. Two HNSCC cell lines (FaDu and HLaC-78) were treated with 0.1, 1 or 10 μM Cetuximab as well as 0, 0.1 or 1 μg/ml ZnO-NP. Qualitative assessment of ZnO-NP was conducted via transmission electron microscopy (TEM) and immunofluorescence staining. Evaluation was done via the MTT-assay after 24, 48 and 72 hours of incubation with Cetuximab and ZnO-NPs. ZnO-NPs were shown to antagonize the anti-tumor effects of Cetuximab in a time-dependent as well as dose-dependent way. These findings suggest an inhibitory interaction of ZnO-NPs with Cetuximab, which warrants further investigation.

Characterization of T-cell subpopulations in patients with chronic rhinosinusitis with nasal polyposis.

Background There is an ongoing discussion concerning the potential origins of chronic rhinosinusitis with nasal polyposis (CRSwNP). Objective The aim of this study was to quantify subpopulations of T cells in peripheral blood and nasal polyps in CRSwNP to examine their influence on the etiology of this disease. Methods Tissue and blood samples were collected from 11 patients who underwent nasal sinus surgery, and these samples were analyzed by multicolor flow cytometry. Results There was a significantly lower frequency of CD4+ T-helper (Th) cells and a significantly higher frequency of CD8+ T cells among lymphocytes isolated from nasal polyps compared with peripheral blood mononuclear cells (PBMC). In both T-cell subpopulations, a shift mainly from naive T cells among peripheral blood lymphocytes toward an effector memory and terminally differentiated subtype predominance in nasal polyps was observed. Among CD4+ T cells, the frequencies of cluster of differentiation (CD) 45RA- Forkhead-Box-Protein P3high (FoxP3high) cytotoxic T-lymphocyte-associated Protein 4high (CTLA-4high) activated regulatory T (Treg) cells, and CD45RA- Forkhead-Box-Protein P3low (FoxP3low) memory T cells were significantly increased in nasal polyps compared with PBMC. Conclusion In this study, we presented a detailed characterization of CD4+ and CD8+ T-cell subpopulations in patients with CRSwNP. CD8+ T cells were more prominent in nasal polyps than in CD4+ T cells. Both nasal CD8+ T cells and CD4+ T cells predominantly had an effector memory phenotype. Among CD4+ T cells, activated Treg cells were increased in nasal polyps compared with PBMC. The data point toward a local regulation of T-cell composition within the microenvironment of nasal polyps, which might be further exploited in the future to develop novel immunotherapeutic strategies.