Effects of asbestos on initiation of DNA damage, induction of DNA-strand breaks, P53-expression and apoptosis in primary, SV40-transformed and malignant human mesothelial cells.

Human mesothelial cells (HMC), the progenitor cells of asbestos-induced mesothelioma, are particularly sensitive to the genotoxic effects of asbestos, although the molecular mechanisms by which asbestos induces injury in HMC are not well known. The high susceptibility of HMC to simian virus 40 (SV40)-mediated transformation is assumed to play a causative role in the pathogenesis of mesothelioma. The aim of this study was to investigate the asbestos-induced DNA damage in cultured HMC and SV40-transformed HMC (MeT-5A) compared with their malignant counterparts, i.e. human mesothelioma cells (MSTO). The time-dependent initiation of DNA-strand breaks as well as the induction of oxidative DNA base modifications were key factors for investigation. HMC, MeT-5A and MSTO cells were exposed to chrysotile and crocidolite asbestos (3 microg/cm2) during different time periods (1-72 h). DNA damage was investigated by use of the Comet assay and alkaline unwinding, the latter in combination with the Fpg protein. The P53 level was analyzed by immunofluorescence, and measurement of apoptosis was conducted by flow cytometry. We found a significant induction of DNA damage in asbestos-treated HMC already after an exposure time of 1.5 h. This effect could not be observed in treated MeT-5A and MSTO cells. Also, a time-dependent significant increase in DNA-strand breaks was observed by alkaline unwinding in asbestos-treated HMC, but not in treated MeT-5A and MSTO cells. In none of the three cell lines we could detect oxidative DNA damage recognized by the Fpg protein (e.g. 8-oxo-guanine), up to 24 h after exposure to asbestos. In contrast to what was found in HMC, P53 was over-expressed in untreated MeT-5A and MSTO. The induction of apoptosis by asbestos fibers was suppressed in MeT-5A and MSTO cells. Crocidolite fibers induced the higher genotoxic effects and chrysotile the more pronounced apoptotic effects. We conclude that asbestos induces DNA damage in HMC already after a very short exposure time in the absence of 8-oxo-guanine formation. The presence of SV40-Tag in MeT-5A and MSTO cells results in an increased expression of P53, but not in additive genotoxic effects after exposure to asbestos. The deregulation of the apoptotic pathway may lead to proliferation of genomically damaged cells and finally to the development of mesothelioma.

Induction of mitotic cell division distrubances and mitotic arrest by pyrethroids in V79 cell cultures.

Five pyrethroids (fenvalerate, deltamethrin, cypermethrin, permethrin, cyfluthrin) differing in their chemical purity were investigated on their cytotoxic effects, especially on their ability to induce mitotic cell division disturbances using Chinese hamster lung cells of line V79. The colony forming ability (CFA) resulted in distinct differences of the cytotoxic effect of the tested pyrethroids, whereby permethrin was found to be most toxic. With the exception of fenvalerate all tested pyrethroids gave rise to inhibition of cell cycle progression as shown by G2/M-arrest of synchronized V79 cells by flow cytometry as well as by the increase of the mitotic index as evaluated by light microscopy. The mitotic arresting activity could be attributed to the occurrence of abnormal mitotic figures such as initial and full C-metaphases. The results however indicate, that pyrethroids per se do not contribute to the cytotoxic effects but that other factors such as chemical impurities, source as well as manufacturing process and isomer composition may be responsible for the observed cytotoxic effects.

Supernatants from quartz dust treated human macrophages stimulate cell proliferation of different human lung cells as well as collagen-synthesis of human diploid lung fibroblasts in vitro.

Silicosis is a chronic lung disease, which is caused by inhalation of silica-containing dusts, leading to pulmonary fibrosis. Alveolar macrophages play a key-role in defence against these particles entering the lung. As a result of phagocytosis, the macrophages release mediators, which are involved in various processes of inflammation and immunological defence mechanisms. We established an in-vitro test system composed of human macrophages, human pneumocyte type II cells (line A-549), human diploid lung fibroblasts (line Wi38) and human tracheobronchial epithelial cells (line BEAS-2B). With this model, we were able to study the influence of various cytokines, produced by the macrophages, on cell proliferation and collagen synthesis (only fibroblasts) of the cells in our test-system. In this report, we will summarize data obtained from our in-vitro test system on two cytokines, which are thought to be important in pathogenesis of lung fibrosis: insulin-like growth factor-1 (IGF-1) and transforming growth factor-beta (TGF-beta).

Induction of sister chromatid exchanges (SCE) in human tracheal epithelial cells by the fractions PM-10 and PM-2.5 of airborne particulates.

Recent epidemiological studies in the United States and in Europe indicate, that the coarse fraction (PM-10) of airborne particulates, smaller than 10 microm and particularly the fine fraction (PM-2.5) smaller than 2.5 microm are responsible for adverse health effects, causing an increasing morbidity and mortality. Furthermore, an association was reported between air pollution, especially the levels of the fine respirable particles and death from lung cancer. The epithelium of the respiratory tract is the major target of airborne particulates and the location of the most common cancer in man, bronchogenic carcinoma. The genotoxic activity of the coarse (PM-10) and the fine fraction (PM-2.5) of airborne particulates leading to mutation and cancer can be analyzed using in vitro models of human bronchoepithelial cells. In our study collection of the coarse (PM-10) and the fine fraction (PM-2.5) of airborne particulates was conducted in the winter of 1996 in the highly industrialized Rhine-Ruhr region (Germany). For collection we selected an urban area (Düsseldorf), an industrialized area Duisburg and a rural area (Borken). Airborne particulates were collected with a Low Volume M-10 dichotomous sampler (Graseby-Andersen) equipped with glass fiber filters. Chemical substances were extracted from filters with di-chloromethane and quantitatively transferred to dimethylsulfoxide (DMSO). As target cells for testing the genotoxic activity we used cultures of the human bronchioepithelial cell line (BEAS-2B). As a sensitive cytogenetic endpoint for evaluation of the genotoxic activity of extracts of airborne particulates we utilized the induction of 'sister chromatid exchanges' (SCE). The coarse fraction PM-10 and especially the fine fraction PM-2.5 of airborne particulates from all three locations caused a strong dose-related induction of 'sister chromatid exchanges'. The fine fractions PM-2.5 from the three locations exerted a stronger genotoxic activity than the corresponding coarse fractions PM-10. While airborne particulates from Düsseldorf and Duisburg revealed a comparable genotoxic activity, the samples from Borken disclosed a lower genotoxicity. It is important that especially the fine fraction PM-2.5, exerted a strong genotoxicity equivalent to substances of airborne particulates from less than 0.5 m3 of air. Results of this study and earlier reports demonstrate that the human tracheobronchial epithelial cell line (BEAS-2B) in vitro offer a reliable and sensitive in vitro model for genotoxicity testing of airborne particulates, especially of the coarse (PM-10) and fine fraction (PM-2.5).

Malignant transformation of Syrian hamster kidney cells in vitro by interaction of airborne particulates and simian virus (SV-) 40.

Permanent inhalation of great variety of air pollutants raises serious problems concerning adverse effects on human health. Especially in airborne particulates more than 1000 inorganic and organic compounds have been detected, among them also carcinogens and mutagens, such as polycyclic aromatic hydrocarbons, polycyclic-aza-compounds, carbonic acids, phenols, ketons, heavy metals, etc. The role of airborne particulates in genesis of the most common cancer in man, the bronchogenic carcinoma, is still unresolved. Samples of airborne particulates were collected at locations of the highly industrialized Rhine-Ruhr region (Germany) employing a high volume sampler HVS-150 (Ströhlein Instruments) prepared with glass fibre filters. Samples were extracted in a Soxhlet-apparatus with dichloromethane. Using a rotating evaporator the solvent was evaporated and the residual substances dissolved in dimethylsulfoxide for cell culture experiments. Carcinogenic activity of extracts of airborne particulates was evaluated by the bioassay of 'enhancement' of malignant cell transformation in vitro. In this bioassay, exponentially growing cell cultures from kidneys of the Syrian golden hamster were treated with various concentrations of extracts of airborne particulates for 18 h. Thereafter, exposed and control cultures were infected with the papovavirus simian virus (SV-) 40 (Strain RH 911) at a multiplicity of infection (M.O.I.) of 300-500 ID50. A strong dose-dependent 'enhancement' of cell transformation frequency occurred in kidney cultures of the Syrian golden hamster pretreated with extracts of airborne particulates and thereafter infected with the simian virus 40. It has to be emphasized, that very low quantities of extractable substances corresponding to particulates collected from 0.5 m3 of air, induced a strong highly significant increase in cell transformation frequency. Inoculation of transformed cells into syngeneic animals produced in a high percentage malignant tumors, mostly sarcomas.

Comparative analysis of cyto- and genotoxic effects of airborne particulates on human and rodent respiratory cells in vitro.

In our highly industrialized world air pollution has become an important topic. Beside gaseous pollutants airborne particulates are of great medical concern, containing several hundred mostly organic substances. They are incriminated to cause an excess mortality. Airborne particulates were collected in the heavily industrialized Ruhr region utilizing a high volume sampler HVS 150 (Ströhlein Instruments) equipped with glass fibre fiiters. Chemical substances were extracted with dichloromethane and quantitatively transferred to dimethyl sulfoxide for tissue culture experiments. Cytotoxicity of extracts was determined by reduction of 'plating efficiency' of human cell line A-549 (pneumocyte type II). The induction of 'sister chromatid exchanges' was used as a sensitive bioassay for detection of genotoxic activity of airborne particulates. As target cells we utilized tracheal epithelial cells of the Syrian golden hamster and the rat, human bronchial epithelial cells of line BEAS-2B and human lymphocytes. Quantities of substances equivalent to airborne particulates from 4 and more m(3) air exerted cytotoxic effects, while quantities of substances from 0.5 m(3) of air were markedly genotoxic.

Induction of cell division disturbances in chinese hamster lung cell line V79 by a complex mixture of polycyclic aromatic hydrocarbons and aza-heterocyclic compounds.

A complex chemical mixture of heavy oil (Z1003), composed of polycyclic aromatic hydrocarbons (PAHs) and aza-heterocyclic compounds (AHCs), has been analysed for its ability to induce cell division disturbances on Chinese hamster lung cell line V79. In addition to the complex chemical mixture (Z1003), separated fractions of PAHs and AHCs (including four subfractions according to their polarity) were investigated. The complex mixture Z1003, PAH and AHC fractions and subfractions (excluding the weak polar subfraction) caused a strong dose-dependent mitotic arrest in cell cultures of V79 cells. The increase of metaphases was accompanied by a decrease of anaphases and telophases. In low concentrations of Z1003, PAH and AHC fractions, predominantly initial metaphases (dislocated chromosomes and/or chromosomes arranged in groups) were observed. In higher concentrations, additionally full C-metaphases and metaphases with condensed chromatin were detected. The results indicate a partial or total loss of the spindle apparatus with the result of an aneuploidy-inducing potential. According to the linkage of aneuploidy to malignant cell transformation and cancer development, a carcinogenic potency of the complex chemical mixture Z1003 and its fractions by a non-genotoxic or aneugenic mechanism should be taken into consideration.

Sister chromatid exchanges in rodent tracheal epithelium exposed in vitro to environmental pollutants.

In our highly industrialized world, air pollution has become a major topic. The human respiratory tract is constantly exposed to air pollutants by inhalation. Besides gaseous pollutants airborne particulates are of great importance, containing a complex mixture of several hundred substances. The tracheobronchial epithelium is the major target site of airborne particulates as well as the origin of the most common cancer in man, the bronchogenic carcinoma. In our study we collected samples of airborne particulates in winter 1991 in the highly industrialized Rhine-Ruhr area (Germany) with a high-volume sampler on glass fiber filters. Airborne particulates were extracted with di-chloromethane and quantitatively transferred to dimethyl sulfoxide (DMSO) for tissue culture experiments. As target cells for genotoxicity testing we used cultures of rodent tracheal epithelial cells from the Syrian golden hamster and from the rat. Induction of "sister chromatid exchanges" (SCE) was utilized as a sensitive cytogenetic endpoint for evaluation of the genotoxic activity of extracts of airborne particulates. In presence of global extracts (GEX) we observed a dose-dependent, highly significant increase of SCE in tracheal epithelial cells of the Syrian golden hamster and of the rat. It is remarkable that even quantities of chemical substances equivalent to airborne particulates from less than 1 m3 of air were genotoxic. Results of this study and earlier reports demonstrate that rodent tracheal epithelial cells offer a reliable and sensitive in vitro model for genotoxicity testing of airborne particulates. Therefore, tracheal epithelial cells in vitro appear a meaningful alternative to other human and rodent cell culture systems which have been used for genotoxicity testing of air pollutants.

Rodent tracheal epithelial cells in vitro: a comparative study of normal cells, enhanced growth variants, SV-40 transformed cells and their interactions.

Tracheal epithelial cells of the golden Syrian hamster can be successfully cultivated in vitro and applied as a model system of respiratory tract epithelium. By morphology and growth characteristics of tracheal epithelial cells, "normal cells", "enhanced growth variants" and "transformed cells" were distinguished in vitro. Hamster tracheal epithelial cells, transformed by Simian virus (SV)-40 expressed in cell nuclei the specific tumor (T-) antigen and showed an accumulation of tumor suppressor protein p53 by immunofluorescence. Cocultivation of "enhanced growth variants" and of "transformed cells" on a "feeder layer" of normal hamster tracheal epithelial cells revealed remarkable differences in loss of "contact inhibition of growth". A "cytokine" released by NIH-3T3 cells stimulated cell proliferation and seems to be important for cell growth of hamster tracheal epithelial cells. Preliminary characterization of the "cytokine" disclosed a molecular weight of more than 30 kDa, a relative thermostability and a loss of activity by treatment with mercaptoethanol indicating disulfide bridges in a molecule.

A rapid method for detection of nongenotoxic carcinogens of environmental pollutants using synchronized V79 cells and flow cytometry.

Synchronized V79 cells were treated before entering mitosis with known and suspicious mitotic arrestants and analyzed by flow cytometry and by light microscopy. Colcemid, nocodazole, vinblastine, diethylstilbestrol, triethyl lead and cadmium sulfate caused a dose dependent mitotic arrest of up to 80%, in comparison with 6% for the controls. Mixtures of polycyclic aromatic hydrocarbons and heterocyclic compounds induced a mitotic arrest of 50%-60%. Extracts of airborne particulates revealed a mitotic arrest of 10%-40%. In contrast, benzoquinone and hydroquinone led to a G2-block rather than to a mitotic arrest. Results of flow cytometry measurements correlated well with those obtained by light microscopy. Cell synchronization in combination with flow cytometry seems to be of considerable value as a rapid method for testing nongenotoxic agents with mitotic arresting activity.

Assessment of cancer hazard from environmental pollution in Silesia.

New concepts of cancer risk estimation have been developed during the past decade. Short-term bioassays dealing with mutagenicity and carcinogenicity of environmental samples are being replaced by more relevant molecular epidemiology studies. The general idea of using a battery of bioassays remains unchanged while the origin of tested samples is different. Instead of testing samples collected from the environment, body fluids or human cells from exposed populations are under investigation. This paper reviews the collaborative study on cancer risk assessment in highly polluted industrial region of Silesia in which both approaches had been employed during the 1985-1995 period. A potent carcinogenic activity of airborne pollutants was indicated in a battery of in vitro and in vivo short-term assays. These studies were followed by the molecular epidemiology study performed on human populations inhabiting the region of Silesia. An elevated damage of genetic material on the chromosome and/or DNA levels was observed in the Silesian populations as compared with proper rural controls.

Tracheal epithelial cells in vitro as a model to study genotoxicity of airborne particulates.

The major target site of airborne particulates is the tracheobronchial epithelium of the respiratory tract. It is also the origin of the most common cancer in man, bronchogenic carcinoma. Rodent tracheal epithelial cells in culture can be used to study the genotoxic activity of airborne particulates leading to mutation and cancer. Airborne particulates were collected in the heavily industrialized Rhine-Ruhr region using a high volume sampler HVS 150 (Ströhlein Instruments) equipped with glass fibre filters. Chemical substances were extracted with di-chloromethane or methanol and quantitatively transferred to dimethyl sulfoxide for tissue culture experiments. Tracheal epithelial cells of the Syrian golden hamster and the Wistar rat were dissociated by pronase treatment and cultivated in a 'complex' medium. The induction of sister chromatid exchanges was used as a sensitive bioassay for detection of genotoxic activity of airborne particulates. Extracts of airborne particulates led to a dose-related highly significant induction of sister chromatid exchanges in cell cultures of tracheal epithelial cells of the hamster and the rat. Even quantities of chemical substances equivalent to airborne particulates from less than 1 m(3) air were markedly genotoxic.

Tumour necrosis factor-alpha induction by endotoxin-containing coal mine dusts in cultures of human macrophages and its effects on pneumocyte type II cells.

Recent results indicate that tumour necrosis factor alpha (TNFalpha) may have an important role in the pathogenesis of silicosis. Supernatants of macrophages exposed to quartz and coal mine dust were tested for the presence of TNFalpha. Monocytes were isolated from peripheral blood and cultured for 10-14 days. After in vitro maturation of monocytes to cells with characteristics of macrophages, they were incubated with quartz dust DQ12 and various coal mine dusts from the Ruhr Valley for 24 hr. TNFalpha bioactivity in the supernatants of dust-treated macrophages was measured in a cytotoxicity bioassay with L929-mouse fibroblasts. Endotoxin, the lipopolysaccharide-containing cell wall component of Gram-negative bacteria, is the most important stimulator of TNFalpha induction in human macrophages. Suspensions of coal mine dusts from the Ruhr Valley and quartz dust DQ12 were therefore analysed for the presence of endotoxin by the very sensitive Limulus amoebocytes lysate test. Only a few suspensions of coal mine dusts from the Ruhr Valley contained endotoxin. Only endotoxin-containing dusts stimulated macrophages to produce TNFalpha. Incubating human pneumocytes type II (line A-549) with TNFalpha as the pure substance led to a transformation of these epithelial cells into spindle-shaped cells. This morphological transformation was accompanied by marked inhibition of pneumocyte type II proliferation.

Inhibition of phagocytosis of human macrophages induced by airborne particulates.

Human monocyte-derived macrophages isolated from peripheral blood were treated with different extracts of airborne particulates collected in the highly industrialized Rhine-Ruhr area. All tested extracts showed a substantial impairment of phagocytosis by inhibition of phagocytic activity as well as phagocytic capacity, while cell viability was rather well maintained. Significant reduction of phagocytosis already appeared at a concentration equivalent to extracted particulates from 3.8 m3 air. Having properties of alveolar macrophages, human monocyte-derived macrophage cultures may offer a reliable in vitro model for assessment of pulmonary toxicity by respirable pollutants.

Comparative genotoxicity testing of airborne particulates using rodent tracheal epithelial cells and human lymphocytes in vitro.

In our study samples of airborne particulates were collected in the heavily industrialized Rhine-Ruhr region utilizing a high volume sampler HVS 150 (Ströhlein Instruments) equipped with glass fibre filters. Chemical substances were extracted from filters with dichloromethane and quantitatively transferred to dimethyl sulfoxide (DMSO) for tissue culture experiments. For detection of genotoxicity of extract of airborne particulates we utilized as a sensitive bioassay the induction of 'sister chromatid exchanges' (SCE) in cultures of human lymphocytes and of tracheal epithelial cells of the Syrian golden hamster. The extract of airborne particulates was added in various concentrations to cell cultures of human lymphocytes and hamster tracheal epithelial cells in presence of bromodeoxyuridine for 72 or 48 h, the last 3 h in presence of demecolcine or nocodazole, respectively. Extract of airborne particulates led in both test systems--human lymphocytes and tracheal epithelial cells of the hamster--to a dose-dependent, highly significant induction of 'sister chromatid exchanges'. Very low quantities of substances corresponding to airborne particulates from less than 1 m3 air were highly effective in both cell systems. In comparison, tracheal epithelial cells of the Syrian golden hamster revealed a higher sensitivity showing a steeper increase of 'sister chromatid exchanges' than human lymphocytes.

Human monocyte-derived macrophage cultures: An alternative test system for the detection of pulmonary toxicity induced by inhaled particulate pollutants.

Alveolar macrophages are major target cells for toxicity from inhaled particulates. To investigate pulmonary toxicity induced by airborne particulates a phagocytosis assay using human monocyte-derived macrophage cultures was used. Monocytes were isolated from peripheral blood and cultured for 10-14 days. During this period the monocytes differentiated to macrophages. After treatment with various concentrations of different samples of airborne particulate matter, phagocytosis was induced by the addition of Polychromatic Fluoresbrite Microspheres at a cell/particle ratio of 1:10. Phagocytosis was assessed from the determination of phagocytic activity (% cells showing phagocytosis) and of phagocytic capacity (number of phagocytized particles/cell). A concentration-dependent reduction of phagocytic activity and capacity was observed, while cell viability was not greatly affected as compared with the control. These results were in good agreement with those obtained from in vivo inhalation experiments with rodents as well as with those using rat alveolar macrophages obtained after bronchoalveolar lavage and treated in vitro with extracts of particulate matter. Therefore, it is suggested that the use of human monocyte-derived macrophage cultures represents a promising in vitro test system for the evaluation of pulmonary toxicity induced by particulate pollutants.

Influence of airborne suspended matter on mitotic cell division.

The effect of organic extracts of airborne suspended matter collected in the highly polluted industrial region of Silesia (Poland) on mitotic cell division was evaluated in the Chinese hamster V79 cell line. Crude benzene extracts as well as sequential elution solvent chromatography (SESC) fractions were investigated for their ability to affect the mitotic index, the proportion of anaphases-telophases to metaphases (AT/M ratio), the cloning efficiency and to produce aneuploid cells. The incidence of cell division disturbances in V79 cells exposed to extracts increased in a concentration-dependent manner. Mitotic arrest, manifested as a highly increased mitotic index and a concomitant decrease in the AT/M ratio, was found for the crude extract at a dose corresponding to 0.75 m3 of air. Comparable effects were noticed for SESC fraction 4, probably containing monophenol compounds. A strong dose-dependent reduction of cloning efficiency of V79 cells demonstrated cytotoxic activity of both the crude extract and fraction 4.

In vitro cytogenetic assays for the detection of mitotic aneuploidy by particulate pollutants.

The ability of particulate pollutants to act on the mitotic cell division process and to induce aneuploidy in V79 cell cultures has been investigated. Extracts of airborne particulates and particulate car exhaust caused mitotic arrest connected with a dose-dependent increase in the incidence of initial and full C-metaphases. Furthermore, numerical chromosome alterations such as hyperdiploidy and polyploidy in subsequent cell divisions were induced with increasing concentrations. These findings indicate that particulate pollutants from the ambient air and from car exhaust contain potent spindle poisons with the ability to produce mitotic aneuploidy in mammalian cells. The significance of these results is that induced aneuploidy by particulate pollutants represents a genetic and somatic risk to exposed populations.

Genotoxicity of particulate emissions from gasoline-powered engines evaluated by short-term bioassays.

Atmospheric pollutants, especially airborne particulates, contain a large number of genotoxic substances capable of inducing human health effects via inhalation. One important source of air pollutants are exhaust particles from automobile traffic. This concerns mainly diesel exhaust for which genotoxic properties are evident. With respect to particle emissions produced by gasoline-powered cars little information on a genotoxic potential is available. In this study 3 particulate emission extracts from different gasoline-powered cars driven with leaded or unleaded gasoline were investigated for cytotoxic and genotoxic activities by means of short-term bioassays using mammalian cell culture systems. All tested extracts were found to induce a broad spectrum of cytotoxic and genotoxic effects suggesting that gasoline exhausts are under high suspicion of contributing to health effects in human populations via air pollution.