Assessment of nicotine-induced DNA damage in a genotoxicological test battery.

The role of the tobacco-alkaloid nicotine in tumour biology is widely discussed in the literature. Due to a strong capacity to induce angiogenesis, a pro-mutagenic potential in non-tumour and cancer cells, and a pro- and anti-apoptotic influence, nicotine seems to promote the growth of established tumours. However, results indicating DNA damage and genetic instability associated with nicotine have been contradictory thus far. A variety of markers and endpoints of genotoxicity are required to characterize the genotoxic potential of nicotine. Induction of DNA single- and double-strand breaks, the formation of micronuclei, and the induction of sister chromatid exchange and chromosome aberrations represent possible genotoxicological endpoints at different cellular levels. Human lymphocytes were exposed to nicotine concentrations between 1µM and 1mM for 24h in vitro. The comet assay, the cytokinesis-block micronucleus test, the chromosome aberration (CA) test, and the sister chromatid exchange (SCE) test were then applied. Viability and apoptosis were measured by flow cytometry in combination with the annexin V-propidium iodide staining test. In this test setting, no enhanced DNA migration was measured by the comet assay. An increase in the micronucleus frequency was detected at a concentration of 100µM nicotine without affecting the frequency of apoptotic cells. A distinct genotoxic effect was determined by the CA test and the SCE test, with a significant increase in CA and SCE at a concentration of 1µM. In the annexin V test, nicotine did not influence the proportion of apoptotic or necrotic cells. The current data indicating the induction of CA by nicotine underscore the necessity of ongoing investigations on the potential of nicotine to initiate mutagenesis and tumour promotion. Taking into account the physiological nicotine plasma levels in smokers or in nicotine-replacement therapy, particularly the long-term use of nicotine should be critically discussed.

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.

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.

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

Industrial application of titanium dioxide nanoparticles (TiO(2) -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 TiO(2) nanotoxicology are inconsistent. The in vitro geno- and cytotoxicity of TiO(2) -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. TiO(2) -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, TiO(2) -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 TiO(2) -NPs. Environ. Mol. Mutagen.

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.

Nicotine and methyl methane sulfonate in mini organ cultures of human parotid gland tissue.

The aim was to demonstrate the applicability of using mini organ cultures (MOC) of the human parotid gland for indicating DNA damage by nicotine. Macroscopically healthy specimens of human parotid glands (1 mm3) were cultured for 7 d. Morphology was examined after HE and immunohistochemical staining of alpha-amylase. MOC were exposed to 2.0 mM nicotine or 100 microM methyl methane sulfonate (MMS) for 1, 2 and 3 h, followed by a regeneration period of 24 h. DNA damage was assessed by the comet assay. Histological findings demonstrated healthy acinar cells up to 8 days of culture and a strong expression pattern of alpha-amylase. Cells in the centre of mini organs showed a granular cytoplasm starting at day 3. 1-3 h nicotine exposure significantly increased DNA damage as determined by DNA in the tail (DT), with no significant differences with increasing exposure time and only a trend towards decreased values of DT after regeneration. MMS demonstrated a time-dependent increase in DNA damage and distinctly reduced DT values after regeneration. MOC may be used to study DNA damage and repair after repetitive exposure to xenobiotics. They provide additional information for in vitro studies of cells growing in an intact tissue structure.

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.

Nicotine induces DNA damage in human salivary glands.

The tobacco alkaloid nicotine is responsible for addiction to tobacco and supposed to contribute to tobacco carcinogensis, too. Recently, genotoxic effects of nicotine have been reported in human cells from blood and upper aerodigestive tract. Because of nicotine accumulation in saliva, the study of possible in vitro genotoxic effects of nicotine have been extended to human salivary gland cells. Specimens of parotid glands of 10 tumor patients were obtained from tumor-free tissue. Single cells were prepared by enzymatic digestion immediately after surgery and exposed for 1h to 0.125-4.0mM of nicotine. Possible genotoxic effects were determined by the Comet assay using the % DNA in tail (DT) as a reliable indicator of DNA damage. Nicotine induced a significant dose-dependent increase of DNA migration in parotid gland single-cells. The mean DT was 1.12-fold (0.125mM) to 2.24-fold (4.0mM) higher compared to control. The lowest concentration eliciting significant DNA damage within 1h, 0.25mM nicotine, is only 10-fold higher than maximal concentrations of nicotine reported in saliva after unrestricted smoking. Although conclusive evidence for a carcinogenic potential of nicotine is still lacking, the safety of long-term nicotine replacement therapy should be carefully monitored.