Georg Wultsch

Use of nasal cells in micronucleus assays and other genotoxicity studies.

Genotoxicity experiments with exfoliated nasal mucosa cells are a promising minimally invasive approach for the detection of DNA-damaging compounds in ambient air. Results of single cell gel electrophoresis (SCGE) assays with individual cells and organ cultures from bioptic material show that DNA damage caused by compounds such as nitrosamines, polycyclic aromatic hydrocarbons and pesticides can be detected. Biochemical studies indicate that enzymes involved in the metabolism of environmental mutagens are represented in nasal cells. Several protocols for experiments with nasal cells have been developed and it was shown that formaldehyde, metals, styrene and crystalline silica induce DNA damage in SCGE and/or in micronucleus studies; furthermore, it was also found that polluted urban air causes DNA instability in nasal epithelial cells. Comparisons of these data with results obtained in lymphocytes and buccal cells indicate that nasal cells are in general equally sensitive. Broad variations in the baseline levels, differences of results obtained in various studies as well as the lack of information concerning the impact of confounding factors on the outcome of experiments with these cells indicate the need for further standardisation of the experimental protocols.

Use of the lymphocyte cytokinesis-block micronucleus assay in occupational biomonitoring of genome damage caused by in vivo exposure to chemical genotoxins: Past, present and future

This article concerns the use of the lymphocyte cytokinesis-block micronucleus (CBMN) assay in biomonitoring of occupational and environmental exposures to genotoxic agents. Furthermore, we evaluated the use of this method in different exposure scenarios, in comparison to other DNA damage biomarkers and its regional distribution. So far ca. 400 studies have been published and the number increased substantially in the last years. The most frequently investigated groups are hospital personnel, followed by workers in the chemical industry and agricultural workers. The lymphocyte CBMN assay is more frequently used in occupational studies than MN assays with epithelial cells and other methods, such as chromosomal aberration analyses, Comet assay and DNA-adduct measurements. The use of probes which enable the discrimination between MN containing chromosome fragments and whole chromosomes allows the identification of the molecular mechanisms of MN formation. Most studies were performed in Europe and Asia (ca. 65% and 25%, respectively). Important future developments will be the evaluation of the biological consequences of formation of additional nuclear endpoints (e.g. nucleoplasmic bridges), the improvement of the understanding of the health consequences of their formation, and the use of automated scoring devices. Future applications of the CBMN assay should address new emerging problems, e.g. the potential genotoxic damage induced by the use of nanoparticles and mobile phones. The control of occupational exposures to chemical genotoxins is currently based on chemical measurements which do not reflect interactions of individual factors and the inclusion of the lymphocyte CBMN assay in routine surveillance of workers could contribute substantially to the prevention of adverse health effects.

Results of micronucleus assays with individuals who are occupationally and environmentally exposed to mercury, lead and cadmium

Millions of humans are exposed occupationally and environmentally to lead, mercury and cadmium compounds. Mercury compounds are less abundant but some of them belong to the most toxic chemicals which are known. We evaluated the literature to find out if these metals act in humans as genotoxic carcinogens and if their health effects can be predicted by use of micronucleus (MN) assays with lymphocytes and/or with other genotoxicity tests. Numerous studies showed that lead and mercury induce cancer in humans and also in animals, in vitro experiments with cultured cells indicate that they cause DNA damage via different molecular mechanisms including release of reactive oxygen species and interactions with DNA repair processes. Also in most human studies, positive results were obtained in MN tests with lymphocytes (all 15 occupational studies with lead yielded positive results, with mercury 6 out of 7 investigations were positive). For cadmium, there is clear evidence that it causes cancer in humans; however, induction of chromosomal damage was only seen in high dose experiments with mammalian cells while results of animal and human studies yielded conflicting results (only in 2 of 5MN trials with humans positive findings were reported). Possibly, non-genotoxic mechanisms such as inhibition of apoptosis and interaction with signaling pathways account for the carcinogenic properties of cadmium species. The findings of MN studies with lead and mercury are in excellent agreement with results which were obtained with other endpoints (e.g. chromosomal aberrations and comet formations) and it is evident that this approach can be used for occupational and environmental monitoring of exposed individuals. Important future tasks will be the realization of larger studies with a uniform standardized protocol, the additional evaluation of anomalies other than MN (nuclear buds and bridges) and the combination of such trials with investigations which allow to define the molecular mechanisms relevant for exposed humans.

Micronucleus assay with urine derived cells (UDC): a review of its application in human studies investigating genotoxin exposure and bladder cancer risk.

The first micronucleus (MN) study with urine derived cells (UDC) appeared 30 years ago. So far, 56 investigations have been published with this method and it was shown that it can be used for the detection of chromosomal damage caused by environmental and lifestyle factors as well as by occupational exposures and certain diseases This approach may be also useful as a diagnostic tool for the detection and prognosis of bladder cancer. The test system has been improved in the last years, i.e., it was shown that, apart from MN also other nuclear anomalies can be evaluated in UDC which are found in other types of epithelial cells as well (e.g., in oral and nasal cells) and are indicative for acute toxicity (pyknosis, karyorrhexis, karyolysis, condensed chromatin) and genomic instability (nuclear buds, binucleates). Furthermore, an improved protocol with Carnoy I fixation and Papanicolaou stain was developed which enables the discrimination between cells which originate from the cervix and those from the urothelium. The evaluation of the currently available results indicates that exposures and health conditions which are associated with increased cancer rates in the bladder (and possibly also in other organs) lead to positive results in MN-UDC assays and a limited number of studies indicate that this method may be equally sensitive as other more frequently used human biomonitoring assays. The major shortcoming of the UDC-MN method is the lack of standardization; the evaluation of the current data shows that a variety of staining and fixation methods are used and that the numbers of evaluated cells vary over a broad range. These inconsistencies may account for the large inter-laboratory variations of the background frequencies. In order to improve the reliability of the method, further standardization and validation is required. Therefore an international program should be initiated in which a similar strategy could be used as in previous validation/standardization projects concerning MN-cytome assays with lymphocytes and buccal cells.

Formation of micronuclei and other nuclear anomalies in exfoliated nasal and oral cells: Results of a human study with workers in a power plant processing poultry litter

Handling of chicken litter leads to exposure to toxic gases, endotoxins and airborne microorganisms. Aim of this study was to investigate if this results in acute cytotoxicity and to damage of the genetic material which is involved in the etiology of various diseases including cancer. Nuclear anomalies which reflect genotoxic and cytotoxic effects were monitored in exfoliated buccal and nasal cells which were collected from workers (n = 25) of a power plant which processes chicken manure and from controls (n = 21). Furthermore, biochemical parameters of the redox status (malondialdehyde, oxLDL and TEAC) and C-reactive protein (CRP) in plasma and the concentrations of toxic gases and endotoxins in the air were determined. No increase of anomalies which reflect chromosomal damage (micronuclei, binucleates, nuclear buds) but significantly higher rates of nuclear aberrations which are indicative for cytotoxicity (karyolysis, karyorrhexis, condensed chromatin) were found in the workers. These effects were in nasal cells more pronounced as in buccal cells. MDA, oxLDL and CRC levels were in both study groups similar. Chemical analyses show that the workers are exposed to high concentrations of NO and endotoxins, while the levels of NO2, NH3 and H2S were below the MAK levels. Taken together, the results show that anomalies that are due to cytotoxicity are increased in the workers and suggest that the exposure may lead to inflammations in the respiratory tract. However, the lack of induction of anomalies that reflect chromosomal damage indicate that no health effects will take place which are due to instability of the genetic material.

Genotoxic effects of occupational exposure measured in lymphocytes of waste-incinerator workers

Workers of solid-waste incinerators are exposed to a variety of pollutants such as dioxins, polycyclic aromatic hydrocarbons and heavy metals. It has been shown that the emissions and the fly ash produced by incineration have mutagenic properties. To our knowledge, no studies have been conducted in which genotoxic effects were investigated in exposed workers. Therefore, we monitored DNA damage by means of the single-cell gel electrophoresis (SCGE) and micronucleus (MN) assays in lymphocytes of individuals (n=23) who were temporarily (1-11 months) conducting maintenance works of an incinerator and in unexposed controls (n=19). Additionally, we measured the urinary concentrations of selected metals (Cr, Mn, Ni, As) with atomic absorption resonance. We found no differences in the levels of DNA migration and in the MN frequencies between different exposure groups and controls. Likewise, we also failed to find differences in the metal concentrations. Taken together, our results indicate that incinerator workers at the site investigated here have no increased health risks due to DNA damage.

The sensitivity of biomarkers for genotoxicity and acute cytotoxicity in nasal and buccal cells of welders.

Welders are inhalatively exposed to fumes which contain genotoxic carcinogens and it was found in epidemiological studies that they have increased cancer rates which may be causally related to DNA damage. In order to assess their health risks and to find out which chemicals cause the adverse effects, bioassays can be performed which enable the detection of genetic damage. The aim of the present study was a comparative investigation with exfoliated buccal and nasal cells in regard to induction of chromosomal alterations and acute cytotoxicity in welders and unexposed controls (n=22 per group). To elucidate the factors which account for genotoxic and cytotoxic effects, additional biochemical parameters were monitored reflecting the redox status as well as concentrations of different metals and 1-hydroxypyrene (1-OHP) in body fluids. We found in the nasal cells significant induction of alterations which are indicative for DNA damage, i.e. of micronuclei (MNi) and nuclear buds, while elevated rates of nuclear anomalies reflecting cytotoxic effects (condensed chromatin, karyorrhexis, karyolylsis) were detected in cells from both organs. The levels of certain metals (Cr, Cu, Mn, Mo, Ni), but not markers of oxidative damage were significantly higher in the body fluids of the welders. Multivariate Poisson regression analyses indicate that exposure to Mo (15% MNi increase by one standard deviation increase of Mo in serum), Ni (9% increase) and Mn (14% increase) are positively associated with the induction of MNi in nasal cells while Ni was associated with cytotoxic effects in both types of cells (12 and 16% increase). Taken together, our findings indicate that epithelial cells from the respiratory tract are suitable for the detection of DNA-damaging and cytotoxic effects in welders and can be used to assess health risks associated with genomic instability.

Impact of exposure to wood dust on genotoxicity and cytotoxicity in exfoliated buccal and nasal cells

Wood dust was classified by the IARC as a human carcinogen which causes sinonasal tumours. However, the exposure in different industries varies strongly and the risks of workers depend on the specific situation which can be assessed by the use of biomonitoring methods. The aim of this study was to investigate the workers who are exposed to low dust levels (below the permitted concentrations) with cytogenetic and biochemical methods. Micronuclei (MNi) which are indicative for genomic damage, nuclear buds which reflect gene amplification, binucleated cells which are caused by mitotic disturbances and acute cytotoxicity parameters (pyknosis, karyorrhexis, condensed chromatin, karyolysis) were monitored in buccal and nasal cells of workers of a veneer factory (n = 51) who are exposed to volatile wood-derived compounds, in carpenters of a furniture factory which use no synthetic chemicals (n=38) and in a control group (n = 65). Additionally, markers were measured in blood plasma which reflect inflammations (C-reactive protein, CRP) and the redox status, namely malondialdehyde (MDA) and oxidised low density proteins (oxLDL). No induction of micronucleated cells was observed in both epithelia in the two exposure groups while all other nuclear anomalies except pyknosis were increased; also one health-related biochemical marker (MDA) was significantly elevated in the workers. Taken together, the results of our study show that exposure to low levels of wood dust does not cause formation of MNi indicating that the cancer risks of the workers are not increased as a consequence of genetic damage while positive results were obtained in earlier studies with workers who are exposed to high dust levels. However, our findings indicate that wood dust causes cytotoxic effects which may lead to inflammations.

Genotoxic and Cytotoxic Effects in Exfoliated Buccal and Nasal Cells of Chromium and Cobalt Exposed Electroplaters.

ABSTRACT Results of a number of studies indicate that electroplaters have increased cancer risks as a consequence of exposure to genotoxic metals such as chromium (VI) and nickel. These effects may be due to induction of damage of the genetic material which plays a key role in the etiology of cancer, and it was found that workers in galvanization factories exhibited increased levels of DNA damage. The aim of the present study was to investigate genetic stability in workers of a bright plating factory who are exposed to chromium (Cr) and cobalt (Co). Exfoliated cells were collected from the buccal and nasal mucosa of workers (n = 42) and matched controls (n = 43) and analyzed for induction of micronuclei (MN) which are formed as a consequence of chromosomal aberrations. In addition, other nuclear anomalies namely nuclear buds (Nbuds) which are formed as a consequence of gene amplification and markers indicating different stages of cell death (condensed chromatin, karyorrhexis, karyolysis, and pyknosis) were also assessed. No evidence was noted for induction of MN, but significantly increased rates of Nbuds in cells from both, buccal and nasal mucosa, were found. Parameters which are indicative for cytotoxic effects were more pronounced in nasal cells and rose with duration of employment period. Overall, our findings indicated that no apparent chromosomal damage occurred in bright electroplaters. However, data demonstrated that acute cytotoxic effects may lead to inflammations and/or lesions in epithelia of the respiratory tract of the workers.

Work Intensity, Low-Grade Inflammation, and Oxidative Status: A Comparison between Office and Slaughterhouse Workers

Limited knowledge exists about the impact of physical workload on oxidative stress in different occupational categories. Thus, we aimed to investigate the oxidative and inflammatory status in employees with different physical workloads. We enrolled a total of 79 male subjects, 27 office workers (mean age 38.8 ± 9.1 years) and 52 heavy workers, in a slaughterhouse (mean age 40.8 ± 8.2 years). Fasting blood was drawn from an antecubital vein in the morning of the midweek before an 8-hour or 12-hour work shift. The antioxidative capacity was assessed measuring total antioxidant capacity (TAC), uric acid, total polyphenols (PPm), and endogenous peroxidase activity (EPA). Total peroxides (TOC), malondialdehyde (MDA), and myeloperoxidase (MPO) were analyzed as prooxidative biomarkers, and an oxidative stress index (OSI) was calculated. In addition, hsCRP, interleukin-6 (IL-6), MDA-LDL IgM antibodies, galectin-3, adrenocorticotropic hormone (ACTH), and the brain-derived neurotrophic factor (BDNF) were measured as biomarkers of chronic systemic inflammation and emotional stress. TOC (p = 0.032), TAC (p < 0.001), ACTH (p < 0.001), OSI (p = 0.011), and hsCRP (p = 0.019) were significantly increased in the heavy workers group, while EPA, BDNF (p < 0.001), and polyphenols (p = 0.004) were significantly higher in office workers. Comparison between 8 and 12 h shifts showed a worse psychological condition in heavy workers with increased levels for hsCRP (p = 0.001) and reduced concentration of BDNF (p = 0.012) compared to office workers. Oxidative stress and inflammation are induced in heavy workers and are particularly pronounced during long working hours, that is, 12-hour versus 8-hour shifts.