On the mechanism of cell internalization of chrysotile fibers: an immunocytochemical and ultrastructural study

Human breast carcinoma cells (CG5) and human laryngeal carcinoma cells (HEp-2) were exposed to 10 and 50 micrograms/ml of small (about 5 microns) chrysotile asbestos fibers. Morphological and ultrastructural changes were evaluated by means of immunocytochemistry and by scanning and transmission electron microscopy. Our attention was focused on the mechanisms of cell internalization and on transport of chrysotile fibers. The fibers appeared to penetrate the cell cytoplasm and to be translocated in proximity of the nucleus. Small chrysotile fibers could also be found inside the nucleus of interphase cells. Involvement of the main cytoskeletal components, i.e., microfilaments, intermediate filaments, and microtubules, in the cytotoxicity of chrysotile fibers was also evaluated. Our findings suggest that after fiber penetration, a rearrangement of the cytoskeletal apparatus occurs. It has also been observed that small fibers remain associated with the cytoskeletal framework, which can thus play a role in asbestos intracytoplasmic translocation in epithelial cells. Furthermore, after the cell has completely recovered its morphology, fiber internalization ultimately seems to lead to the formation of giant multinucleated cells. These data could be indicative of an interaction occurring between asbestos fibers and the normal mitotic process. The disturbance of the cell cytoskeleton and the close morphologic contact between asbestos fibers and the cell's nuclear region may be of importance in explaining the well-known carcinogenic effects of asbestos mineral fibers.     

Effect of chrysotile and crocidolite on the morphology and growth of rat pleural mesothelial cells

The effects of the UICC asbestos fibers chrysotile A and crocidolite on the morphology and growth of rat pleural mesothelial cells were examined. The response was different according to the fiber used. Within 4 hr of treatment with 5, 10, 20, or 50 micrograms/ml of chrysotile fibers, cell morphology showed intense vacuolization, in both logarithmic and confluent cells. Following treatment of growing cells with 20 or 50 micrograms/ml of chrysotile fibers, cell spreading was also observed. Four hours after treatment of cells with 5, 10, or 20 micrograms/ml of crocidolite fibers, no such changes were seen. Vacuolization appeared later and was much less marked than for chrysotile fibers. The mesothelial cell population-doubling time of about 30 hr was not significantly altered by addition of 5 to 20 micrograms/ml of crocidolite fibers, but a concentration of 50 micrograms/ml lengthened doubling time to about 90 hr. Treatment with 5 or 10 micrograms/ml chrysotile fibers usually prolonged this time, but 20 or 50 micrograms/ml caused cell lysis. After 48 hr of incubation with both types of fiber, the proportion of mitosis was the same as in control cultures, whatever the fiber concentration, and asbestos fibers were often seen inside dividing cells.     

Effects of asbestos fibers on cell division, cell survival, and formation of thioguanine-resistant mutants in Chinese hamster ovary cells

The ability of crocidolite fibers to induce point mutations and mitotic abnormalities in Chinese hamster ovary (CHO) cells was examined in cell cultures. The purpose has been to study the possibilities for establishing in vitro test methods to quantify genetic damage induced by asbestos and other mineral fibers. Results obtained with the CHO/hypoxanthine guanine phosphoribosyl transferase system indicated that crocidolite fibers per se do not significantly increase the number of thioguanine-resistant mutants. Crocidolite fibers also failed to potentiate the mutagenicity of benzo[a]pyrene. Time-lapse cinematography and microscopy showed that asbestos (crocidolite) fibers were markedly cytotoxic. Among surviving cells some underwent abnormal cell divisions which resulted in multi- and micronucleate cells. Many cells that contained a few asbestos fibers, however, underwent mitosis and successfully formed two mononucleate daughter cells capable of further divisions. Individual, fiber-containing cells were examined by time-lapse television recordings for 4-5 days. During this time period some cells underwent six divisions and generated an almost normal number of daughter cells. Cells which contained fibers that were longer or equivalent to the diameter of the mitotic cell (20 microns), showed different forms of mitotic abnormalities. The frequency of multinucleate cells was drastically increased following exposure to asbestos fibers. Only rarely, however, did these cells divide to produce viable daughter cells capable of continued cell multiplication. The frequency of multinucleate cells was dependent on the dose of exposure to asbestos fibers and could possibly be used as an index of the degree of mitotic disturbances induced by mineral fibers.     

Hydrophobic Bile Acid-Induced Micronuclei Formation,Mitotic Perturbations,and Decreases in Spindle Checkpoint Proteins: Relevance to Genomic Instability in Colon Carcinogenesis

We show, for the first time, that hydrophobic bile acids cause aberrations of the mitotic machinery of colon cells that can give rise to aneuploidy, the chromosomal perturbations common in colon tumors. First, we show that DOC induces a statistically significant fourfold increase in the number of micronuclei in NCM-460 cells (a noncancerous colon cell line) and a threefold increase in the number of micronuclei in binucleated HT-29 colon cancer cells using the cytokinesis block micronucleus assay. Second, we observed mitotic aberrations after DOC treatment, including improper alignment of chromosomes at the metaphase plate, lagging chromosomes during anaphase, anaphase/telophase chromatin bridges, multipolar divisions, and formation of polynucleated cells. It was determined that there was a statistically significant threefold increase in the number of aberrant metaphases after short-term and long-term exposure of HT-29 and HCT-116 cells, respectively. Third, we showed with Western blots and immunohistochemistry that a likely basis for these mitosis-related perturbations included decreased expression of the spindle checkpoint proteins, Mad2, BubR1, and securin. Fourth, results of DOC treatment on nocodazole-challenged cells further indicated deficiencies in activation of the spindle assembly checkpoint. This study provides mechanisms by which hydrophobic bile acids can induce genomic instability in colon epithelial cells.     

Induction of micronuclei in cultured mammalian cells by fume condensates of roofing asphalt

A considerable number of workers in the United States are employed in asphalt industries and are potentially exposed to asphalt fumes. The information regarding the potential carcinogenic hazards of such fumes to exposed workers is still limited. Studies have been conducted to determine the cytogenetic effects of roofing asphalt fume using cultured mammalian cells. Exponentially growing Chinese hamster lung fibroblasts (V79 cells) were exposed to different concentrations of condensates of type I and type III roofing asphalt fumes, generated at temperatures similar to actual roofing operation (316 ± 10°C). The frequencies of micronucleated cells in the treated and control cultures were determined. Additionally, immunofluorescent staining of kinetochore with human anti-kinetochore primary antibody and flouresceinated goat anti-human IgG was used to investigate the potential mechanism of micronucleus formation. The results show that both types of roofing asphalt fume condensates caused a significant increase in the frequency of micronucleated cells, and that 70% of micronucleated cells induced by asphalt fume condensates carried kinetochore-positive micronuclei. These findings indicate that both type I and type III roofing asphalt fumes are capable of causing principally cytogenetic damage by spindle apparatus alternations in cultured mammalian cells. © 1996 Wiley-Liss, Inc.     

Effects of methylmercury on mitotic mouse glioma cells.

The effects of methylmercury on cultured mammalian cells were studied by light and electron microscopy. At 5 x 10(-6) M concentration methylmercury depressed the proliferation of mouse glioma cells almost completely and increased the mitotic indices. In the electron micrographs of the cells treated with 5 x 10(-6) M methylmercury for 4 hr, microtubules as mitotic spindle fiber were absent and chromosomes were scattered in a disorderly fashion in the cytoplasm. From these observations we concluded that exposure to methylmercury inhibits the cell mitosis by blocking the polymerization of tubulin to microtubules and results in the accumulation of the cells on the way of mitosis. Changes in behaviors of other mitotic apparatus and of fibrillar structures other than microtubules are also described.     

Evidence that ultrafine titanium dioxide induces micronuclei and apoptosis in Syrian hamster embryo fibroblasts

Inhaled ultrafine titanium dioxide (UF-TiO2) particles cause pronounced pulmonary inflammation, in contrast to fine TiO2. Previous studies provide evidence for the production of reactive oxygen species by alveolar macrophages, after overloading with UF-TiO2 particles and cytotoxicity of UF-TiO2 in rat lung alveolar macrophages. UF-TiO2 also causes pulmonary fibrosis and lung tumors in rats. UF-TiO2 particles are photogenotoxic, but in general, information on the genotoxicity of UF-TiO2 is still limited. We studied the potential of UF-TiO2 (particle size less than or equal to 20 nm) and fine TiO2 (particle size > 200 nm) to induce chromosomal changes, which can be monitored by the formation of micronuclei (MN) in Syrian hamster embryo (SHE) cells. We also analyzed UF-TiO2-treated cells for apoptosis induction. The MN assay revealed a significant increase in MN induction (p less than or equal to 0.05) in SHE cells after treatment with UF-TiO2 (1.0 micro g/cm2) for 12 hr (mean, 24.5 MN/1,000 cells), 24 hr (mean, 31.13 MN/1,000 cells), 48 hr (mean, 30.8 MN/1,000 cells), 66 hr (mean, 31.2 MN/1,000 cells), and 72 hr (mean, 31.3 MN/1,000 cells). Bisbenzimide staining of the fixed cells revealed typical apoptotic structures (apoptotic bodies), and the apoptosis-specific "DNA ladder pattern" resulting from internucleosomal cleavage was identified by gel electrophoresis. Furthermore, transmission electron microscopy of the exposed cells revealed the typical chromatin compaction of apoptosis.     

On the release of asbestos fibers from weathered and corroded asbestos cement products

The controversy on whether weathered and corroded asbestos cement products are emitting biologically significant asbestos fiber concentrations in ambient air has not been resolved. Nor is it known if the weathered and corroded asbestos cement products release asbestos fibers which have the same carcinogenic potency as "standard" chrysotile. The purpose of this research project was to develop a method for sampling and measuring asbestos fiber emissions from solid planar surfaces (i.e., roofs and facades) consisting of asbestos cement products and to develop methods for studying the physical and chemical changes and the carcinogenic potency of the emitted fibers. Using this method asbestos fiber emissions in ambient air have been measured in the FRG during 1984/1986. The emissions of asbestos fibers longer than 5 microns were in the range 10(6) to 10(8) fibers/m2.hr. The ambient air concentrations of these asbestos fibers were for the most part less than 10(3) fibers/m3. It was shown that the emitted asbestos fibers were chemically changed and it was shown with animal experiments that their carcinogenic potency did not differ from the carcinogenicity of "standard" chrysotile fibers.     

Cytogenetic studies on chrysotile asbestos

The cytogenetic effects of chrysotile asbestos (chrysotile A) in vivo in Rhesus monkeys and Swiss albino mice, and in vitro in embryonic hamster cells were investigated. Single oral dosages of chrysotile, 100 or 500 mg/kg, failed to induce chromosome aberrations in bone marrow cells of Rhesus monkeys. Similarly, oral or intraperitoneal administration of chrysotile over a dose range from 0.4 to 400 mg/kg, failed to induce micronuclei formation in bone marrow cells of mice. However, chrysotile induced a significant and dose-related increase in chromosome aberrations and a dose-related inhibition of the mitotic index of cultured Syrian hamster embryo cells.     

Micronuclei formation induced by X-ray irradiation does not always result from DNA double-strand breaks

X-ray induced formation of micronuclei is generally thought to result from DNA double-strand breaks (DSBs). However, DNA DSBs inhibit the cell cycle progression that is required for micronucleus formation. In order to reconcile this apparent discrepancy, we investigated whether DNA DSBs induced during the G1 phase could lead to micronucleus formation. We irradiated human embryonic (HE17) cells that had been treated with a radical scavenger, either DMSO or ascorbic acid (AsA), and determined the level of suppression of DNA DSBs or micronuclei. When DNA DSBs were evaluated using 53BP1 foci, treatment with 5 mM AsA did not inhibit the numbers of foci at various intervals after X-ray irradiation; however, treatment with 5 mM or 256 mM DMSO did have a significant inhibitory effect. By contrast, an assay of micronucleus numbers showed that treatment with 5 mM or 256 mM DMSO before X-ray irradiation resulted in almost no inhibition of micronucleus formation, but treatment with 5 mM AsA did have a significant inhibitory effect. These results clearly showed that AsA could suppress micronucleus formation, although it was not effective for suppression of DNA DSBs. Therefore, we conclude that DNA DSBs induced in the G1 phase do not directly lead to micronucleus formation.     

Micronucleus test using cultured new born rat astrocytes

Micronuclei is induced in cytoplasm as a consequence of the formation of chromosomal fragments or remaining chromosomes during cell division by the cause of clastogens or spindle poisons, and is used as an indicator of genotoxicity screening tests. There are few short-term genotoxicity screening tests using brain cells. We attempted to establish a new in vitro micronucleus test (MN test) system by use of central nervous system cells. Primary cultured astrocytes were prepared from newborn male Sprague-Dawley (SD) rats. In growth curve of astrocytes, doubling time was determined to be 31 h. In time study, the highest frequency of micronuclei was observed at 48 h, 72 h and 6 h-exposure-66 h-recovery by vincristine (VCR), mitomycin C (MMC) without metabolic activation system and cyclophosphamide (CPM) with metabolic activation system, respectively. Dose-response relationships between micronucleus frequency and concentrations of MMC, VCR and CPM were observed, respectively. It is suggested that the in vitro MN test using new born rat-astrocytes could be used as a screening test of environmental and occupational genotoxic chemicals in the central nervous system cells.     

三种细胞形态学分析方法在体外微核检测中的比较研究

【目的】探讨基于流式细胞术(FCM)和细胞高内涵筛选技术(HCS)的两种高通量体外微核试验方法应用于化学品遗传毒性评价的可行性。【方法】参考经济合作与发展组织(OECD)TG487推荐的程序方法,采用化学品遗传毒性评价的典型阳性剂环磷酰胺(CP)、丝裂霉素C(MMC)为受试物,CP剂量范围为5~20 mg/L,MMC剂量范围为0.25~1.0 mg/L,分别设3个剂量组,短时处理(4 h)体外培养的CHL细胞模型,分别建立基于FCM和HCS的高通量体外微核测试方法。平行采用传统胞质分裂阻断微核法,在加/不加代谢活化系统条件下,检测各剂量组细胞的微核形成情况,并分析微核细胞率。实验同步设置阴性对照组(无血清MEM培养液)。【结果】经常规人工显微镜阅片和高通量方法的FCM及HCS得到的CP、MMC体外微核试验结果均为阳性:经CP诱导产生的微核细胞率(CP浓度由低到高)依次为1.9%、7.6%、10.4%(人工阅片),2.8%、2.6%、7.8%(FCM),2.8%、6.2%、9.1%(HCS);经MMC诱导产生的微核细胞率(MMC浓度由低到高)依次为5.9%、11.4%、16.7%(人工阅片),3.2%、3.7%、5.1%(FCM),7.9%、10.1%、10.2%(HCS)。受试物各剂量组与阴性对照组相比,差异均有统计学意义(P<0.05),且呈现剂量-反应关系。【结论】在本试验条件下,流式细胞术、高内涵体外微核检测的方法与传统人工阅片的方法得到的检测结果均一致,提示体外微核高通量方法可以实现细胞微核形成的自动化检测,提高检测效率,可为将高通量体外微核测试方法纳入化学品遗传毒性评价相关标准提供数据支持。     

New insight into intrachromosomal deletions induced by chrysotile in the gpt delta transgenic mutation assay

BACKGROUND: Genotoxicity is often a prerequisite to the development of malignancy. Considerable evidence has shown that exposure to asbestos fibers results in the generation of chromosomal aberrations and multilocus mutations using various in vitro approaches. However, there is less evidence to demonstrate the contribution of deletions to the mutagenicity of asbestos fibers in vivo. OBJECTIVES: In the present study, we investigated the mutant fractions and the patterns induced by chrysotile fibers in gpt delta transgenic mouse primary embryo fibroblasts (MEFs) and compared the results obtained with hydrogen peroxide (H2O2) in an attempt to illustrate the role of oxyradicals in fiber mutagenesis. RESULTS: Chrysotile fibers induced a dose-dependent increase in mutation yield at the redBA/gam loci in transgenic MEF cells. The number of lambda mutants losing both redBA and gam loci induced by chrysotiles at a dose of 1 microg/cm(2) increased by > 5-fold relative to nontreated controls (p < 0.005). Mutation spectra analyses showed that the ratio of lambda mutants losing the redBA/gam region induced by chrysotiles was similar to those induced by equitoxic doses of H2O2. Moreover, treatment with catalase abrogated the accumulation of y-H2AX, a biomarker of DNA double-strand breaks, induced by chrysotile fibers. CONCLUSIONS: Our results provide novel information on the frequencies and types of mutations induced by asbestos fibers in the gpt delta transgenic mouse mutagenic assay, which shows great promise for evaluating fiber/particle mutagenicity in vivo.     

Gamma-ray- and fission neutron-induced micronuclei in PHA stimulated and unstimulated human lymphocytes

Two groups of normal human blood cells, one stimulated with phytohaemagglutinin (PHA) for 24 hr (G1-S phase of the cell cycle) and one unstimulated (G0 phase), were irradiated with 60Co gamma rays or 252Cf radiation. A comparison of radiation-induced micronucleus frequencies showed that the high-dose-rate gamma rays were more effective in inducing micronuclei than low-dose-rate gamma rays. In the cells exposed to low-dose-rate irradiation, there was little difference between the frequency of micronuclei in the G0 phase and the G1-S phase. However, cells in the G1-S phase were more sensitive than G0-phase cells to high-dose-rate gamma rays. The relative biological effectiveness of 252Cf neutron irradiation measured in micronucleus assays was consistent with the value obtained for the lethal effect of 252Cf on cultured cells.     

A comparative study on the hemolytic action of short asbestos fibers on human, rat, and sheep erythrocytes

The hemolytic activity of short asbestos fibers isolated by a sedimentation procedure was studied using human (HRBC), rat (RRBC), and sheep (SRBC) red blood cells. The initial velocity (Vi) of hemolysis is proportional to the concentration of fibers with HRBC and RRBC, but not with SRBC. Initial velocity (Vi) is 14.30 for HRBC, 5.75 for RRBC, and 3.60 for SRBC at a concentration of 1,000 micrograms of fibers/ml. Maximum velocity (Vm) is reached at 400 micrograms/ml with HRBC and its value is 16.7. With RRBC and SRBC, Vm is reached at 600 micrograms/ml and its value is 10 and 3.6, respectively. The 50% hemolytic concentration (HC50) at 60 min of incubation is 75 micrograms/ml for HRBC, 240 micrograms/ml for RRBC, and 260 micrograms/ml for SRBC. It appears that the sensitivity against the short asbestos fibers of the three types of RBC used is in the following order: HRBC greater than RRBC greater than SRBC.     

Impact of modification of the fiber surface of chrysotile on its biological activity

Samples of commercial chrysotile-asbestos and asbestos cement, which were equal in number, were prepared. The content of fibers, up to 80 microm in length, was 87.4 and 85.0% in the first and second samples, respectively. Chemical analysis confirmed that there were cement components onto the surface of fibrils in the second sample. Onto the surface of native asbestos fibers, there were considerable distribution bands of active centers in the range of pH values of 5, 6.4, and 7.3; their largest number was at pH 6.4. Asbestos cement fibers had a band at pH 7.3, i.e. there was displacement towards the neutral region. Thus, their capacity for oxidative processes is likely to be lower than that in the fibers from the first sample. The mutagenic activity of the commercial chrysotile, examined in the micronucleus test, was substantially higher (p < 0.01) than that in the asbestos cement sample wherein it did not differ from that seen in the control experiment (saline solution). Mutagenicity was not found in cement and asbestos cement dust (2-3% of fibers) either. It is probable that the absence of mutagenicity in the cement-coated asbestos fibers may be attributable to a considerable reduction in their potencies for the formation of active radicals (oxygen, lipid peroxidation, and others).     

Effect of static magnetic field on the induction of micronuclei by some mutagens

Objectives It is important to assess the risk of static magnetic fields (SMFs) on human health, because epidemiological studies have indicated that SMFs play a role in the development of diseases such as leukemia and brain tumor. In our environment, we have numerous chances to be exposed to not only SMFs but also many chemicals containing mutagens. The aim of this study is to investigate the effect of SMFs on the induction of micronuclei induced by some mutagens. Methods BALB/c mice were exposed to 4.7 tesla (T) SMF for 24 hr immediately after the injection of carboquone (alkylating agent), colcemid (spindle poison), mitomycin C (cross-linking agent), vincristine (spindle poison), sodium fluoride (a byproduct of aluminum plants under strong SMF) or 1-ethyl-1-nitrosourea (brain tumor-, gliomas- and thymic lymphoma-inducing chemical). Results The frequency of micronuclei induced by six mutagens increased after co-exposure to SMF. Conclusions An additive/synergistic effect of SMF and chemicals was observed from the results of increased frequency of micronuclei induced by mutagens in mouse bone marrow erythrocytes.     

The in vitro cytotoxicity of asbestos fibers: I. P388D1 cells

In this study, the cytotoxicity of 13 fibrous samples of known fiber number and dimensions has been established in P388D1 cells. The cells were exposed in vitro to dust concentrations of 10 or 50 micrograms/ml and, after incubation for 24 or 48 hours, any changes in cellular viability, lactate dehydrogenase, and glucosaminidase levels were determined. In general, there was a close association between the reduction in cellular viability and the loss of intracellular enzymes induced by each dust, the chrysotile asbestos samples proving more cytotoxic than the amphiboles. The cytotoxicity of the fibrous dusts was shown to be related to the number of fibers greater than 8 micron in length in the samples.     

Asbestos concentration and fiber size in lungs of the urban residents]

Asbestos fiber concentrations and fiber size distribution in lung tissues of 53 urban residents (males: 34, female: 19) were analyzed by low temperature ashing-analytical transmission electronmicroscopy. The following findings were obtained. 1. Pulmonary asbestos fibers were found in 51 out of 53 patients. The types of asbestos fibers were chrysotile, amosite, crocidolite, actinolite and tremolite. 2. Thirty-six of 53 patients had no history of occupational asbestos exposure, and their geometric mean concentration of asbestos fibers was 1.67 x 10(6) fibers/g dry lung. Most of these asbestos fibers are probably attributable to general environmental contamination. Thirteen patients who had a history of occupational asbestos exposure showed a geometric mean of their pulmonary asbestos concentrations (5.82 x 10(6) fibers/g dry lung) which was significantly higher than that of patients without occupational asbestos exposure (p less than 0.01). 3. The geometric mean concentration of asbestos fiber in males (2.70 x 10(6)) was higher than in females (1.59 x 10(6)), probably due to a difference in the occupational asbestos exposure between males and females. 4. Regardless of the patient's sex, the geometric mean concentration of asbestos fibers in patients without a history of smoking (male: 4.91 x 10(6), female: 1.78 x 10(6)) was higher than that in patients with a smoking history (male: 2.76 x 10(6), female: 1.37 x 10(6)). The difference, however, was not statistically significant, and no correlation was seen between the concentration of asbestos fibers and smoking history. 5. Although most asbestos fiber utilized in Japan is chrysotile, the geometric mean concentration of chrysotile (0.87 x 10(6)) was almost identical to that of amphibole asbestos fiber (0.90 x 10(6)). 6. Of the asbestos fibers observed, 95% of chrysotile and 85% of amphibole asbestos were less than 5 microns in length and 93% of the total asbestos fibers were too small to be visible by light microscopy.     

The pathological effects of prolonged asbestos ingestion in rats

The effects of prolonged ingestion of amosite, crocidolite, and chrysotile UICC standard reference asbestos samples were examined in groups of laboratory rats. Animals were given over 250 mg per week for periods up to 25 months and were monitored for the remainder of their life span. Animals were examined for evidence of pathological effects and gastrointestinal mucosal cytokinetic disturbances and for signs of penetration and dissemination of fiber. There was no excess of malignant tumors in the experimental groups when compared with control animals, and no gastrointestinal mucosal abnormalities were found. An examination of the cytokinetic status of a subgroup of animals exposed to asbestos similarly showed no evidence of any adverse effects of prolonged ingestion of amosite asbestos fiber. Detailed electron microscopic examination of various tissue residues for the presence of asbestos fibers was also undertaken. No penetration and/or damage to any of the gut tissues was found. Although occasional fibers were found in a variety of tissue residues, there was no evidence of preferential retention of fibers within any specific tissue and no sign of higher fiber burdens in those animals with tumors. It was concluded that there were no significant adverse effects of prolonged asbestos ingestion in healthy laboratory rats. The implications of these findings are discussed in the light of other published work.