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Possible mechanism of chrysotile asbestos-stimulated superoxide anion production in guinea pig alveolar macrophages.

Excessive production of active oxygen radicals by macrophages is proposed to play an important role in asbestos-related diseases. The purpose of this study was to examine the capacity and mechanisms of action of various forms of asbestos to stimulate superoxide anion production by guinea pig alveolar macrophages. Chrysotile, but not the amphiboles (crocidolite, anthophyllite, or amosite), stimulated a rapid (less than 1 min) and dose-dependent (2.5-50 micrograms/ml) production of superoxide anion at noncytotoxic doses (2.5 to 25 micrograms/ml). The stimulation of superoxide anion production by chrysotile could be blocked by putative protein kinase C inhibitors (staurosporine, sphingosine, and fluphenazine). Chrysotile also stimulated phosphatidylinositol turnover as measured using 32Pi incorporation into phospholipids, [3H]-diacylglycerol levels, and intracellular calcium mobilization as measured using fura-2 and 45Ca. In addition, pertussis toxin partially blocked chrysotile-stimulated superoxide anion production. We conclude that the mechanism of guinea pig alveolar macrophage stimulation by chrysotile, but not the amphibole asbestos forms, is consistent with a mechanism which is similar to that used by agonists such as N-formyl-Nle-Leu-Phe resulting in stimulated phosphatidylinositol turnover, calcium mobilization, and activation of protein kinase C.

Animals↗

Malignant peritoneal mesothelioma in a 17-year-old boy with evidence of previous exposure to chrysotile and tremolite asbestos.

We describe a case of malignant peritoneal mesothelioma arising in a 17-year-old boy. The diagnosis was based on a comprehensive study including light microscopy, histochemistry, immunohistochemistry, evaluation of the clinical course, and autopsy examination. Analytical transmission electron microscopy showed a concentration of 510,000 asbestos fibers/g dry lung tissue. The fibers were represented by chrysotile (62%) and tremolite (38%) asbestos. About 40% of the total fibers were longer than 5 microns. The presence of tremolite fibers was probably due to environmental exposure to contaminated cosmetic talc. This is the first reported case of pathologically proven exposure to asbestos dust in malignant mesothelioma of childhood and adolescence.

Adolescent↗

Crocidolite asbestos suppresses the differentiation of HL-60 cells induced by DMSO.

This study was undertaken to determine if the biological function of inducers for cell differentiation is affected by asbestos fibers, which are sometimes deposited in human tissues. Protein kinase C activity, c-myc protein expression and cell surface CR3 expression were used as the markers of cell differentiation. The function of dimethylsulfoxide (DMSO), an inducer of cell differentiation, was suppressed by the co-culturing of crocidolite asbestos, because DMSO reacted with the hydroxyl radical released after the stimulation with crocidolite and spent itself. Superoxide dismutase (SOD) inhibited the effect of crocidolite, reacting rapidly with .O2- before the secondary release of .OH. Asbestos fibers deposited in tissues may inhibit the function of inducers which stimulate immature cells to differentiate, because such inducers frequently are also radical scavengers.

Asbestos↗

Enhanced lipid peroxidation in lung lavage of rats after inhalation of asbestos.

Exposure of phagocytic cells to asbestos in vitro results in an augmented production of reactive oxygen metabolites and increased peroxidation of lipids. The aim of this investigation was to assess the extent of lipid peroxidation both in cells and fluid obtained from bronchoalveolar lavage (BAL), and in lungs of rats exposed to crocidolite asbestos or titanium dioxide (TiO2), a nonfibrous particulate control. In comparison to sham and TiO2-exposed rats, the BAL fluid and cells of crocidolite-exposed animals contained significantly elevated levels of malondialdehyde (MDA), a breakdown product of lipid peroxidation detected using high-pressure liquid chromatography (HPLC). In contrast, no significant differences in MDA were detected in lavaged lung tissue from these animals. Inhalation of crocidolite caused an early inflammatory response characterized by elevated numbers of polymorphonuclear leukocytes and lymphocytes, as well as enhanced total protein in BAL. Pulmonary fibrosis and increased lung hydroxyproline also were observed after 20 days of exposure. Exposure to TiO2 did not cause inflammation, pulmonary fibrosis, or elevated amounts of hydroxyproline in the lung. Our results show that exposure to the fibrogenic and inflammatory mineral, crocidolite, results in an enhanced lipid peroxidation in BAL cells and fluid not observed after inhalation of the particulate TiO2. These novel observations suggest that MDA in BAL may be useful as a biomarker of exposure to inhaled asbestos or other oxidants.

Administration, Inhalation↗

Deposition, clearance, and translocation of chrysotile asbestos from peripheral and central regions of the rat lung.

We investigated the pulmonary deposition, clearance, and translocation of chrysotile asbestos in the context of our previously developed model of asbestosis in the rat. Adult male rats were exposed for 3 hr to an aerosol of chrysotile asbestos. Subgroups were sacrificed up to 29 days postexposure and the lungs of the animals fixed. Peripheral and central regions of the left lung were resected, digested, and analyzed for fiber content by scanning electron microscopy. Pulmonary deposition did not differ between peripheral and central regions. There was no evidence of translocation of fibers from central to peripheral regions. The average diameter of retained fibers decreased over time, consistent with longitudinal splitting. The average length of retained fibers increased over time, consistent with slower clearance of longer fibers. We employed a novel counting scheme to ensure accurate fiber number measurements, allowing the calculation of clearance rates for fibers 0.5 to greater than or equal to 16 microns in length. Fibers of length greater than or equal to 16 microns were cleared slowly, if at all. These findings could have important implications for the pathogenesis of asbestos-related pleural disease. Many fibers are deposited in the peripheral region, and the longest (greater than or equal to 16 microns) will persist there for extended periods.

Aerosols↗

Effects of chrysotile asbestos on coho salmon and green sunfish: evidence of behavioral and pathological stress.

The effects of chrysotile asbestos on larval coho salmon (Oncorhynchus kisutch) and juvenile green sunfish (Lepomis cyanellus) were investigated at levels approximating those reported in the Great Lakes basin (10(6) fibers/liter). Behavioral stress effects, such as loss of rheotaxic position and balance, were observed in salmon exposed at 3.0 X 10(6) fibers/liter and in sunfish exposed at 1.5 and 3.0 X 10(6) fibers/liter. Coho larvae at 1.5 X 10(6) fibers/liter were significantly more susceptible to an anesthetic stress test, becoming ataxic and losing equilibrium faster than control cohorts (P less than 0.001). Two of 106 larvae exposed at 3.0 X 10(6) fibers/liter developed tumorous swellings and three additional fish developed coelomic distentions. Cytological examination of ventral epidermal tissue revealed cellular histolysis, and evidence by transmission electron microscopy confirmed the presence of asbestos in the salmon larvae. Distortion of the lateral line region in asbestos-treated coho salmon was linked to behavioral and orientational aberrations. Differential mortality was not observed between control and treated groups of either test species.

Animals↗

Effect of chrysotile asbestos fibers on germ cells of mice.

An Indian form of chrysotile asbestos procured from a local asbestos factory (Hyderabad) was tested for its toxic effects on spermatocytes and sperm of mice. Swiss albino male mice were fed orally with chrysotile asbestos suspended in water. The concentration tested was 20 mg/kg/day. Chronic oral administration of chrysotile failed to induce chromosomal aberrations and abnormal sperms in mice.

Animals↗

On the mechanism of cogenotoxic action between ingested amphibole asbestos fibres and benzo[a]pyrene: I. Urinary and serum mutagenicity studies with rats.

Recently, there has been concern that ingested asbestos may cause an increase in cancer incidence in populations exposed to fibre-contaminated drinking water. Although animal experiments failed to demonstrate carcinogenicity of the oral asbestos exposure, the high adsorption capacity of the fibres creates the possibility of cocarcinogenic action with adsorbed organics. In a simple in vivo model we demonstrated earlier that UICC crocidolite and anthophyllite asbestos fibres were able to adsorb carcinogen molecules from aqueous solutions. When orally administered, these fibres increased the sister chromatid exchange frequency in bone marrow cells of rats. In the present study we tried to follow the desorption and metabolization processes of carcinogenic benzo[a]pyrene molecules transported by the ingested fibres using the highly sensitive Salmonella/Ames mutagenicity assay. The bacterial test was performed on concentrated serum and urine samples of the treated animals by using the TA98 and 100 strains in the presence and absence of liver microsomal and deconjugating enzymes. All sets of urine and serum samples failed to show mutagenic activity indicating a lack of both desorption in the serum and the ability of the liver to metabolize. Considering our results, the cytogenetic impact demonstrated earlier in the bone marrow can be explained by a local action of accumulated and transported carcinogen molecules.

Animals↗

A human cell system for detecting asbestos cytogenotoxicity in vitro.

Crocidolite, a carcinogenic asbestos in humans, specifically induces mesothelioma. We investigated the cytogenotoxic effects of crocidolite in a human mesothelioma cell line, MSTO211H, and a human promyelocytic leukemia cell line, HL60. Using confocal laser scanning microscopy, we found that the MSTO211H cells had phagocytotic activity, whereas the HL60 cells did not. In the MSTO211H cells, crocidolite decreased the cell population and increased the numbers of polynucleated cells (PN) and tetraploid cells, and increased the coefficients of variation (CV) of DNA contents in G0/G1 cells and the formation of 8-hydroxydeoxyguanosine. In contrast, crocidolite showed none of these cytogenotoxic effects in HL60 cells. To investigate the importance of phagocytosis in the cytogenotoxicity of crocidolite, we sorted the crocidolite-phagocytosed cells from less-phagocytosed cells by fluorescence-activated cell sorting, and studied the differences in cytogenotoxicity between these two cell groups. We found significant increases in the numbers of PN and tetraploid cells and the CV in the crocidolite-phagocytosed cells compared to the less-phagocytosed cells. These findings indicate that MSTO211H cells are susceptible to the cytogenotoxic effects of asbestos due to their phagocytotic activity, and that the MSTO211H cell line is suitable for the detection of such effects on human cells by asbestos and other materials which need to be phagocytosed to exert their toxicity.

8-Hydroxy-2'-Deoxyguanosine↗

Mediated, thin-layer cell, coulometric determination of redox-active iron on the surface of asbestos fibers.

Redox-active iron on the surface of asbestos fibers was detected and quantified using a thin-layer cell, coulometric method with soluble mediators to shuttle electrons between the mineral fibers and the solid electrode. The working and counter electrodes consisted of gold films on a glass slide with reference electrodes of silver. Asbestos fibers were entrapped in a thin-layer cell of 25 microns thickness. Hexaammineruthenium(II) or o-dianisidine (dication) was used as the reducing or oxidizing mediator, respectively. Hexaammineruthenium(III) undergoes a one-electron reduction, and protonated o-dianisidine undergoes a sequential two-electron oxidation. The measurement involved determination of the total charge for the oxidation or reduction of surface-immobilized Fe(II) or Fe(III) on the asbestos fibers. Analysis of the results showed that crocidolite and amosite have 4.3 +/- 0.7 and 3.3 +/- 0.7 nmol/mg of total redox-active iron that is accessible to the mediators, respectively. This corresponded to a surface coverage of accessible redox-active iron of approximately 4.3 x 10(-11) mol/cm2 for crocidolite and 9.5 x 10(-11) mol/cm2 for amosite. Furthermore, Fe(II) constituted 76% or 25% of the accessible redox-active iron on the surface of crocidolite or amosite, respectively. The method may be applied to other types of solid materials with redox-active species on their surfaces.

Asbestos, Amosite↗

The in vitro genetic effects of fibrous erionite and crocidolite asbestos.

Epidemiologic evidence has recently identified an association between an endemic outbreak of pleural and peritoneal mesothelioma in the Urgup region of Turkey and exposure to zeolite fibres. This malignancy is usually associated with exposure to asbestos dusts whose mineralogical characteristics differ from those of zeolites. The present study further defines the in vitro biologic activity of erionite, a common zeolite fibre found in the Urgup region of Turkey. Both erionite and crocidolite asbestos fibres were clastogenic in synchronous Chinese hamster ovary (CHO) fibroblasts. Both fibres also altered CHO ploidy. Erionite, unlike crocidolite or Min-U-Sil quartz, caused a slight increase in sister chromatid exchanges in synchronous CHO cells. Neither erionite nor crocidolite was mutagenic in a human lymphoblastoid cell line. Erionite fibres thus produced in vitro cytogenic changes similar to those caused by asbestiform mineral dusts and, like asbestos fibres, did not induce mutations in human lymphoblastoid cells.

Aluminum Silicates↗

The role of catalytic iron in asbestos induced lipid peroxidation and DNA-strand breakage in C3H10T1/2 cells.

The involvement of catalytic iron in the vitro activities of crocidolite asbestos has been investigated. Exposure of C3H10T1/2 cells to either the UICC crocidolite standard reference sample or a non fibrous (milled) derivative resulted in an increase of thiobarbituric acid reactive substances. This catalytic activity was inhibited by pretreatment with the iron chelator desferrioxamine. The effect of this activity on cellular DNA was measured in an assay based on the production of DNA-strand breaks. Increased levels of DNA-strand breaks were detected in cultures treated with both the milled and UICC crocidolite. Inclusion of desferrioxamine with the asbestos inhibited DNA-strand breakage. It is concluded the catalytic iron present on the dust is capable of damaging both lipid and DNA and that this could be an important mechanism in asbestos pathogenicity.

Asbestos↗

Analysis of p16 and p21(Cip1) expression in tumorigenic human bronchial epithelial cells induced by asbestos.

Although asbestos is carcinogenic to humans, the mechanism(s) by which it induces cancer is unknown. Using tumor cell lines generated previously by asbestos treatment of immortalized human bronchial epithelial (BEP2D) cells, we examined alterations in p16 and p21(Cip1) genes together with their protein levels. Results were compared with untreated BEP2D cells, normal human bronchial epithelial cells (NHBE), as well as non-tumorigenic fusion cell lines generated by fusing tumor cells with BEP2D cells. No deletion in the p16 gene was found in any of the tumor cell lines examined. Although p16 protein was expressed at a similar level in tumor and BEP2D cells, there was a fourfold decrease in its expression among NHBE cells. In contrast, both the protein and mRNA expression levels of p21(Cip1) were decreased by about threefold in tumor cell lines when compared with either BEP2D or NHBE cells, which had a similar expression level. Expression of p21(Cip1) mRNA was restored to the control level in all the fusion cell lines examined. The results suggested that down regulation of p21(Cip1) expression is linked to the tumorigenic conversion of BEP2D cells by asbestos.

Asbestos↗

Does asbestos cause laryngeal cancer?

Asbestos is a known carcinogen. Its role in the aetiology of laryngeal cancer has been investigated and debated for the last three decades. Here, we consider much of the published evidence from post-mortem studies, cross-sectional, case-control and cohort studies. We feel that the weight of evidence does not support a causal association for asbestos with laryngeal cancer. The 'positive studies' raise important questions regarding an association; the opportunity may present itself to answer some of these as the incidence of asbestos-related malignancy is increasing in the UK and Europe.

Asbestos↗

Asbestos-associated diseases in a cohort of cigarette-filter workers.

To estimate the effects on health of occupational exposure to crocidolite, a highly toxic form of asbestos, we studied a cohort of 33 men who worked in 1953 in a Massachusetts factory that manufactured cigarette filters containing crocidolite fibers from 1951 to 1957. Twenty-eight of the men have died, as compared with 8.3 deaths expected. This increased mortality was attributable to asbestos-associated diseases. Fifteen deaths were caused by cancer, as compared with 1.8 expected (relative risk, 8.2; 95 percent confidence interval, 4.6 to 13.4), including eight from lung cancer, five from malignant mesothelioma, and two from other types of cancer. There were seven deaths from nonmalignant respiratory disease, as compared with 0.5 expected (relative risk, 14.7; 95 percent confidence interval, 5.9 to 30.3), of which five were due primarily to asbestosis. In contrast, the mortality rates from cardiovascular diseases and all other causes were not increased. Four of the five living workers have pulmonary asbestosis; three of them have recently diagnosed cancers, including two additional lung cancers. We conclude that the extremely high morbidity and mortality in these workers were caused by intense exposure to crocidolite asbestos fibers.

Asbestos↗

Hydrolysis of inositol phospholipids precedes cellular proliferation in asbestos-stimulated tracheobronchial epithelial cells.

Metabolism of inositol phospholipids and phosphatidylcholine was investigated in tracheobronchial epithelial cells exposed to mitogenic concentrations of crocidolite asbestos. Alterations in levels of diacylglycerol, the endogenous activator of protein kinase C, and inositol polyphosphates, presumed mobilizers of intracellular calcium, were examined. Cultures labeled with [3H]glycerol and exposed to proliferative concentrations of crocidolite asbestos demonstrated significant elevations in [3H]diacylglycerol. In contrast, crocidolite-exposed cells labeled with [3H]myristic acid or [3H]choline did not display elevated production of [3H]diacylglycerol or release of [3H]choline metabolites--i.e., evidence of phosphatidylcholine hydrolysis. The soluble tumor promoter phorbol 12-myristate 13-acetate catalyzed both of these changes. myo-[3H]Inositol-labeled cells exposed as briefly as 10 min to mitogenic concentrations of crocidolite demonstrated elevations in [3H]inositol mono-, tris-, and terakisphosphates, phenomena indicating turnover of inositol phospholipids. The detection of diacylglycerol and inositol phosphates in crocidolite asbestos-exposed cells suggests that this fibrous tumor promoter activates phospholipase C as it stimulates cellular proliferation.

Animals↗

Comparison of Calidria chrysotile asbestos to pure tremolite: inhalation biopersistence and histopathology following short-term exposure.

The differences between chrysotile asbestos, a serpentine mineral, and amphibole asbestos have been debated extensively. Many studies have shown that chrysotile is cleared from the lung more rapidly than amphibole. In order to quantify the comparative clearance of chrysotile and the amphibole asbestos tremolite, both fibers were evaluated in an inhalation biopersistence study that followed the European Commission recommended guidelines. In addition, the histopathological response in the lung was evaluated following the short-term exposure. This article presents the results of this study through 90 days after cessation of exposure. Following the termination of the study, a subsequent article will provide the complete results through 12 mo after cessation of exposure. In order to quantify the dynamics and rate by which these fibers are removed from the lung, the biopersistence of a sample of commercial grade chrysotile from the Coalinga mine in New Idria, CA, of the type Calidria RG144 and of a long-fiber tremolite were studied. For synthetic vitreous fibers, the biopersistence of the fibers longer than 20 microm has been found to be directly related to their potential to cause disease. This study was designed to determine lung clearance (biopersistence) and the histopathological response. As the long fibers have been shown to have the greatest potential for pathogenicity, the aerosol generation technique was designed to maximize the number of long respirable fibers. The chrysotile samples were specifically chosen to have 200 fibers/cm3 longer than 20 microm in length present in the exposure aerosol. These longer fibers were found to be largely composed of multiple shorter fibrils. The tremolite samples were chosen to have 100 fibers/cm3 longer than 20 microm in length present in the exposure aerosol. Calidria chrysotile fibers clear from the lung more rapidly (T1/2, fibers L > 20 microm = 7 h) than any other commercial fiber tested including synthetic vitreous fibers. With such rapidly clearing fibers, the 5-day exposure would not be expected to result in any pathological change in the lung, and the lungs of animals that inhaled Calidria chrysotile showed no sign of inflammation or pathology and were no different than the lungs of those animals that breathed filtered air. Following this 5-day exposure to tremolite, the tremolite fibers once deposited in the lung parenchyma do not clear and almost immediately result in inflammation and a pathological response in the lung. At the first time point examined, 1 day after cessation of exposure, inflammation was observed and granulomas were already formed. By 14 days postexposure these microgranulomas had turned fibrotic, and by 90 days postexposure the severity of the collagen deposits had increased and interstitial fibrosis was observed in one of the rats. These findings provide an important basis for substantiating both kinetically and pathologically the differences between chrysotile and the amphibole tremolite. As Calidria chrysotile has been certified to have no tremolite fiber, the results of the current study together with the results from toxicological and epidemiological studies indicate that this fiber is not associated with lung disease.

Aerosols↗

Hemolysis by chrysotile asbestos fibers. I. Influence of the sialic acid content in human, rat, and sheep red blood cell membranes.

Complete removal of accessible sialic acids from human, rat, and sheep red cell membranes does not inhibit the hemolytic properties of chrysotile asbestos fibers. After 60 min of contact, the percentages of hemolysis are comparable between the treated and the nontreated groups. Nevertheless, with low concentrations of fibers the initial velocity of hemolysis of rat and sheep red blood cells is lower in the neuraminidase-treated group. Although sialic acids are not essential for the expression of the hemolytic activity of asbestos, we suggest that these acids could play a role during the first seconds or minutes of the hemolytic process by increasing the electrostatic attraction between the red blood cell membranes and the fibers. However, it is clear that asbestos fibers provoke hemolysis by acting on some other cell membrane component(s).

Animals↗