Stephan Hackenberg

SILVER NANOPARTICLES: EVALUATION OF DNA DAMAGE, TOXICITY AND FUNCTIONAL IMPAIRMENT IN HUMAN MESENCHYMAL STEM CELLS

Silver nanoparticles (Ag-NPs) are the most frequent commercialized nanomaterial currently. Due to a distinct lack of information on hazardous properties of Ag-NPs in human cells, a study was conducted to evaluate Ag-NP induced DNA damage, cell death and functional impairment in human mesenchymal stem cells (hMSCs). Initially, Ag-NPs and their cellular distribution were characterized by transmission electron microscopy (TEM). Ag-NPs were exposed to hMSCs for 1, 3 and 24h. Cytotoxicity was measured by the trypan blue exclusion test and the fluorescein-diacetate test, DNA damage was evaluated by the comet assay and chromosomal aberration test. Cytokine release of IL-6, IL-8 and VEGF was observed using the ELISA technique. Additionally, hMSC migration capability was tested in a transwell system. TEM revealed a Ag-NP distribution to cytoplasm and nucleus. Cytotoxic effects were seen at concentrations of 10 μg/ml for all test exposure periods. Both, comet assay and chromosomal aberration test showed DNA damage after 1, 3, and 24h at 0.1 μg/ml. A significant increase of IL-6, IL-8 and VEGF release indicates hMSC activation. Migration ability was not impaired at subtoxic concentrations. In conclusion, we demonstrated cyto- and genotoxic potential of Ag-NPs in hMSCs at significantly higher concentrations as compared to antimicrobial effective levels.

Cytotoxic, genotoxic and pro-inflammatory effects of zinc oxide nanoparticles in human nasal mucosa cells in vitro

Despite increasing application of zinc oxide nanoparticles (ZnO-NPs) for industrial purposes, data about potential toxic properties is contradictory. The current study focused on the cyto- and genotoxicity of ZnO-NPs in comparison to ZnO powder in primary human nasal mucosa cells cultured in the air-liquid interface. Additionally, IL-8 secretion as a marker for pro-inflammatory effects was measured. Particle morphology and intracellular distribution were evaluated by transmission electron microscopy (TEM). ZnO-NPs were transferred into the cytoplasm in 10% of the cells, whereas an intranuclear distribution could only be observed in 1.5%. While no cyto- or genotoxicity could be seen for ZnO powder in the dimethylthiazolyl-diphenyl-tetrazolium-bromide (MTT) test, the trypan blue exclusion test, and the single-cell microgel electrophoresis (comet) assay, cytotoxic effects were shown at a ZnO-NP concentration of 50 μg/ml (P<0.01). A significant enhancement in DNA damage was observed starting from ZnO-NP concentrations of 10 μg/ml (P<0.05) in comparison to the control. IL-8 secretion into the basolateral culture medium was increased at ZnO-NP concentrations of 5 μg/ml (P<0.05), as shown by ELISA. Our data indicates cyto- and genotoxic properties as well as a pro-inflammatory potential of ZnO-NPs in nasal mucosa cells. Thus, caution should be taken concerning their industrial and dermatological application. Additionally, further investigation on repetitive NP exposure is needed to estimate the impact of repair mechanisms.

Intracellular distribution, geno- and cytotoxic effects of nanosized titanium dioxide particles in the anatase crystal phase on human nasal mucosa cells.

Nanomaterials are defined as substances with at least one dimension smaller than 100nm in size and are used for a multitude of purposes. Titanium dioxide nanoparticles (TiO(2)-NPs) are an important material used as an additive in pharmaceutical and cosmetic products. Due to their high surface-to-mass index, TiO(2) nanoparticles show different physical and chemical characteristics compared to the bulk substance. The knowledge about geno- or cytotoxic effects of TiO(2)-NPs is incomplete since existing studies show contrary results. Human nasal mucosa cells were obtained from 10 donors and exposed to TiO(2)-NPs in increasing concentrations of 10, 25, 50 und 100mug/ml. Transmission electron microscopy (TEM) was applied to document particle morphology and size distribution, the degree of particle aggregation and the distribution of particles in inter- and intracellular spaces. Furthermore, DNA fragmentation and cytotoxicity caused by TiO(2)-NPs were evaluated. DNA strand breakage was detected by single-cell microgel electrophoresis (comet) assay. Cytotoxic effects were analyzed by trypan blue exclusion test and fluorescein diacetate (FDA) assay. TiO(2) particles used in this study were mainly nanosized but also showed a strong tendency to aggregate in spite of sonication of the suspension. Particles entered the cytoplasm in 11% and the cell nucleus in 4%. The trypan blue exclusion test and the FDA assay did not show any loss of cell viability. In the comet assay, there was no evidence of increased DNA damage for TiO(2)-NPs. In this pilot project, no cyto- or genotoxic effects could be shown for TiO(2)-NPs on human nasal epithelial cells. Further investigations will focus on a variety of metal oxide nanoparticles to describe the biocompatibility in the human organism.

Repetitive exposure to zinc oxide nanoparticles induces dna damage in human nasal mucosa mini organ cultures.

Data on the toxicological properties of zinc oxide nanoparticles (ZnO‐NPs) is incomplete. ZnO‐NPs may enter humans via inhalation or ingestion. The aim of the current study was to evaluate ZnO‐NP‐induced genotoxicity in three‐dimensional (3D) mini organ cultures (MOCs) of human nasal mucosa following repeated exposure to ZnO‐NP and regeneration. Nasal MOCs of 10 patients and ZnO‐NPs were cultivated for one week and then characterized by electron microscopy. Nasal MOCs were partially covered by ciliated epithelium after one week of cultivation. ZnO‐NPs were distributed to the cytoplasm and the nucleus. MOCs were exposed once, twice, or three times to 0.1 or 5 μg/ml of ZnO‐NPs for 1 hr per exposure and were then evaluated for cytotoxicity and genotoxicity. MOCs were cultivated for 24 hr after the triple ZnO‐NP exposure to allow for regeneration. ZnO‐NP exposure did not result in significant cytotoxicity or apoptosis, as determined by trypan blue exclusion and caspase‐3 activity, respectively. A significant increase in DNA damage was detected following repetitive exposure compared to single exposure to ZnO‐NPs at 5 μg/ml, but not 0.1 μg/ml ZnO‐NPs. At both concentrations of ZnO‐NP, DNA fragmentation increased after 24 hr of regeneration. In contrast, DNA damage which was induced by the positive control, methyl methanesulfonate, was significantly reduced after 24‐hr regeneration. Thus, our results suggest that repetitive exposure to low concentrations of ZnO‐NPs results in persistent or ongoing DNA damage. Environ. Mol. Mutagen. 2011.

Nanosized titanium dioxide particles do not induce DNA damage in human peripheral blood lymphocytes

Industrial application of titanium dioxide nanoparticles (TiO2‐NPs) as an additive in pharmaceutical and cosmetic products is increasing. However, the knowledge about the toxicity of this material is still incomplete and data concerning health and environmental safety and results of recent studies on TiO2 nanotoxicology are inconsistent. The in vitro geno‐ and cytotoxicity of TiO2‐NPs in the anatase crystal phase was evaluated in human peripheral blood lymphocytes from 10 male donors. Initially, transmission electron microscopy (TEM) was performed to describe particle morphology and size, the degree of particle aggregation, and the intracellular distribution. Cells were exposed to nanoparticles in increasing concentrations of 20, 50, 100, and 200 μg/ml for 24 hr. Cytotoxic effects were analyzed by trypan blue exclusion test and the single‐cell microgel electrophoresis (comet) assay was applied to detect DNA double‐strand breakage. TiO2‐NPs were sphere shaped with a diameter of 15–30 nm. Despite dispersive pretreatment, a strong tendency to form aggregates was observed. Particles were detected in the cytoplasm of lymphocytes, but also a transfer into the nucleus was seen. The trypan blue exclusion test did not show any decrease in lymphocyte viability, and there was no evidence of genotoxicity in the comet assay for any of the tested concentrations. In conclusion, TiO2‐NPs reached the cytoplasm as well as the nucleus and did not induce cyto‐ or genotoxic effects in human peripheral blood lymphocytes. Complement investigations on different human cell systems will be performed to estimate the biocompatibility of TiO2‐NPs. Environ. Mol. Mutagen., 2011.

Aspects of nitrogen dioxide toxicity in environmental urban concentrations in human nasal epithelium.

Cytotoxicity and genotoxicity of nitrogen dioxide (NO(2)) as part of urban exhaust pollution are widely discussed as potential hazards to human health. This study focuses on toxic effects of NO(2) in realistic environmental concentrations with respect to the current limit values in a human target tissue of volatile xenobiotics, the epithelium of the upper aerodigestive tract. Nasal epithelial cells of 10 patients were cultured as an air-liquid interface and exposed to 0.01 ppm NO(2), 0.1 ppm NO(2), 1 ppm NO(2), 10 ppm NO(2) and synthetic air for half an hour. After exposure, genotoxicity was evaluated by the alkaline single-cell microgel electrophoresis (Comet) assay and by induction of micronuclei in the micronucleus test. Depression of proliferation and cytotoxic effects were determined using the micronucleus assay and trypan blue exclusion assay, respectively. The experiments revealed genotoxic effects by DNA fragmentation starting at 0.01 ppm NO(2) in the Comet assay, but no micronucleus inductions, no changes in proliferation, no signs of necrosis or apoptosis in the micronucleus assay, nor did the trypan blue exclusion assay show any changes in viability. The present data reveal a possible genotoxicity of NO(2) in urban concentrations in a screening test. However, permanent DNA damage as indicated by the induction of micronuclei was not observed. Further research should elucidate the effects of prolonged exposure.

Analysis of nicotine-induced DNA damage in cells of the human respiratory tract.

Epithelium of the upper and lower airways is a common origin of tobacco-related cancer. The main tobacco alkaloid nicotine may be associated with tumor progression. The potential of nicotine in inducing DNA mutations as a step towards cancer initiation is still controversially discussed. Different subtypes of nicotinic acetylcholine receptors (nAChR) are expressed in human nasal mucosa and a human bronchial cell line representing respiratory mucosa as a possible target for receptor-mediated pathways. In the present study, both cell systems were investigated with respect to DNA damage induced by nicotine and its mechanisms. Specimens of human nasal mucosa were harvested during surgery of the nasal air passage. After enzymatic digestion over night, single cells were exposed to an increasing nicotine concentration between 0.001 mM and 4.0mM. In a second step co-incubation was performed using the antioxidant N-acetylcysteine (NAC) and the nAChR antagonist mecamylamine. DNA damage was assessed using the alkali version of the comet assay. Dose finding experiments for mecamylamine to evaluate the maximal inhibitory effect were performed in the human bronchial cell line BEAS-2B with an increasing mecamylamine concentration and a constant nicotine concentration. The influence of nicotine in the apoptotic pathway was evaluated in BEAS-2B cells with the TUNEL assay combined with flow cytometry. After 1h of nicotine exposure with 0.001, 0.01, 0.1, 1.0 and 4.0mM, significant DNA damage was determined at 1.0mM. Further co-incubation experiments with mecamylamine and NAC were performed using 1.0mM of nicotine. The strongest inhibitory effect was measured at 1.0mM mecamylamine and this concentration was used for co-incubation. Both, the antioxidant NAC at a concentration of 1.0mM, based on the literature, as well as the receptor antagonist were capable of complete inhibition of the nicotine-induced DNA migration in the comet assay. A nicotine-induced increase or decrease in apoptosis as assessed by the TUNEL assay in BEAS-2B could not be detected. These results support the hypothesis that oxidative stress is responsible for nicotine-induced DNA damage. Similar results exist for other antioxidants in different cell systems. The decrease in DNA damage after co-incubation with a nAChR antagonist indicates a receptor-dependent pathway of induction for oxidative stress. Further investigations concerning pathways of receptor-mediated DNA damage via nAChR, the role of reactive oxygen species and apoptosis in this cell system will elucidate underlying mechanisms.

Antitumor activity of photo-stimulated zinc oxide nanoparticles combined with paclitaxel or cisplatin in HNSCC cell lines

Zinc oxide nanoparticles (ZnO-NPs) exhibit photocatalytic properties and are used in sunscreen cosmetics and for the degradation of environmental pollutants. Furthermore, ZnO-NPs have proven to induce tumor-selective cell death in human squamous cell carcinoma (HNSCC) in vitro. The aim of the current study was to evaluate cytotoxic effects of UVA-1-activated ZnO-NPs in combination with paclitaxel and cisplatin in HNSCC. Three HNSCC cell lines (HLaC-78, Cal-27 and PJ-41) were incubated with ZnO-NPs at concentrations of 0.02 and 0.2 μg/ml in combination with paclitaxel (0.5-5 nM) or cisplatin (0.03-3 μM) for 24 h. Afterwards, cells were irradiated with UVA-1 for 15 min. Viability was measured by MTT assay, fluorescein-diacetate (FDA) test and annexin/propidiumiodide flow cytometry. A significant decrease in viable cells could be observed in all three HNSCC cell lines treated by photocatalytic therapy with 0.2 μg/cm(2) ZnO-NPs alone. A combination with paclitaxel or cisplatin at low concentrations resulted in a further increase in cytotoxicity in vitro revealing a synergistic effect. Flow cytometry revealed a combination of apoptosis and necrosis. These results indicate that photocatalytic therapy of HNSCC with ZnO-NPs could enhance the cytotoxic action of chemotherapeutic agents synergistically, indicating a promising potential for ZnO-NPs in antitumor applications.

BMSC enhance the survival of paclitaxel treated squamous cell carcinoma cells in vitro

The 5-year survival rate of patients suffering from head and neck squamous cell carcinoma (HNSCC) is unsatisfying despite the advances in carcinoma treatment. Recent studies suggest that stem cells can be used as a gene therapy carrier for cancer treatment. Stem cells produce different cytokines such as growth factors in a paracrine manner and cancer cells may show drug resistance in the presence of such growth factors. Reports in the literature concerning treatment of cancer using bone marrow derived stem cells (BMSC) are controversial, which led us to investigate the effects of paclitaxel on human HNSCC cell lines (FaDu and HLaC 78) cultivated simultaneously with BMSC in a transwell system (co-culture). Co-culture and HNSCC cell lines were treated with 10nM of paclitaxel for 24h. Morphology, viability and apoptosis were measured by microscopy, the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and the Annexin V-propidium iodide test. The survival of HNSCC cell lines treated with paclitaxel in co-culture increased significantly compared to control cells. Apoptosis of HNSCC cell lines in co-culture was attenuated significantly. In conclusion, BMSC increase HNSCC resistance to treatment with paclitaxel in vitro. Tumor-stroma interactions are critical components of tumor biology including tumor invasion and metastatic potential. Therefore particular attention must be paid to the complex tumor-stroma interactions to fully understand how tumor cells become chemoresistant.

Effects of salinomycin on human bone marrow-derived mesenchymal stem cells in vitro

Various hypotheses on the origin of cancer stem cells (CSCs) exist, including that CSCs develop from transformed human bone marrow mesenchymal stem cells (hBMSC). Since the polyether antibiotic salinomycin selectively kills CSCs, the present study aims to elucidate the effects of salinomycin on normal hBMSC. The immunophenotype of hBMSC after salinomycin exposure was observed by flow cytometry. The multi-differentiation capacity of hBMSC was evaluated by Oil Red O and van Kossa staining. Cytotoxic effects of salinomycin were monitored by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) assay. Furthermore, spheroid formation and migration capacity were assessed. There were no differences in the immunophenotype and multi-differentiation capacity of hBMSC induced by salinomycin treatment. Cytotoxic effects were observed at concentrations of 30 μM and above. Neither the migration capability nor the ability to form spheroids was affected. Essential functional properties of hBMSC were unaffected by salinomycin. However, dose-dependent cytotoxicity effects could be observed. Overall, low dose salinomycin showed no negative effects on hBMSC. Since mesenchymal stem cells from various sources respond differently, further in vitro studies are needed to clarify the effect of salinomycin on tissue-specific stem cells.