The effect of asbestos cement, UICC asbestos samples and quartz on the peritoneum of the mouse.]

Aqueous suspensions of asbestos cement powder injected experimentally into the peritoneal cavity of mice act as a fibrogenic agent, as do chrysotile asbestos or chrysotile asbestos-containing soil samples. The fibrotic nodules caused by the dust resemble morphologically silicosis granulomas. In addition, asbestos cement has a characteristically strong cytotoxic effect during the first 2 weeks of the experiment. It is suggested that this is due to the chrysotile asbestos and/or the calcite component of the powder. Amosite and crocidolite, on the other hand, induce a diffuse peritoneal fibrosis with the appearance of numerous foreign body giant cells and asbestos bodies. Dust particles displaced to the regional lymph nodes are frequent in the animals treated with quartz, asbestos cement and asbestos-containing soil samples. A spindle cell type sarcoma arising from the visceral peritoneum is observed in animals injected with crocidolite or asbestos cement. In addition, dusts containing chrysotile asbestos induce considerable amyloidosis of the liver and spleen.     

Morphological aspects of interactions between asbestos fibers and human mesothelial cell cytoskeleton

The interaction of chrysotile and amphibole asbestos fibers with the cytoskeleton of cultured human mesothelial cells from nontumoral pleural effusions was studied using scanning electron and immunofluorescence microscopy. Asbestos-exposed mesothelial cells show a massive annular condensation of cytokeratin filaments, forming a concentric ring enveloping the nucleus and the phagocytosed asbestos fibers. By detergent extraction of the cells it could be shown that the asbestos fibers are in close contact with the nuclear membrane and associated with the cytoskeletal framework of the cells. An association of cytokeratin filaments with the asbestos could be observed during phagocytosis of the fibers. The disturbance of the cell cytoskeleton and the close morphologic contact between asbestos fibers and the nuclear membrane may have some relevance in explaining the well-recognized carcinogenic effects of asbestos mineral fibers.     

Mesotheliomas and asbestos type in asbestos textile workers: a study of lung contents.

The asbestos contents of the lungs of former employees of an asbestos textile factory were determined at necropsy using a transmission electron microscope. Those who had died of mesothelioma were compared with a matched sample of those who had died of other causes. The predominant fibre processed in the factory was chrysotile, but crocidolite had also been used. The lung content was consistent with the known exposure to chrysotile, but the crocidolite content was also high, being about 300 times that of the general population of the United Kingdom. The lungs of those with mesothelioma did not contain more of either chrysotile or crocidolite than the lungs of the controls, so no particular type of asbestos could be implicated in causing the mesotheliomas. The evidence of substantial exposure to crocidolite means that the mesotheliomas that occurred in this factory could not be attributed with any certainty to the exposure to chrysotile.     

Inability of chrysotile asbestos fibers to modulate the 2-acetylaminofluorene-induced UDS in primary cultures of rat hepatocytes

There is now growing evidence that asbestos fibers could act in association with genotoxic compounds, either as cocarcinogens or promoters, in the process of carcinogenesis. The hepatocyte/UDS assay system has been taken to advantage to investigate the capacity of fibers to modulate the effects of genotoxic compounds on the cell, as we previously demonstrated the hepatocytes can engage in phagocytosis of chrysotile fibers. Measurement of UDS was performed by a biochemical procedure involving liquid scintillation counting (LSC) of a purified DNA fraction as well as by radioautography. Both LSC and radioautography revealed that chrysotile asbestos fibers UICC B at concentrations up to 100 micrograms/ml do not elicit UDS, whereas 2-acetylaminofluorene (2-AAF) at low concentrations (0.05-0.625 micrograms/ml) significantly induces it in parallel positive controls. In an attempt to test the cocarcinogen hypothesis, cultures of hepatocytes were simultaneously exposed for 20 h to 2-AAF (0.05 and 0.25 micrograms/ml) and asbestos fibers (1 and 10 micrograms/ml) given as simple mixtures. It was found that the 2-AAF-induced UDS activity was the same whether fibers were present or not. This was observed with both UDS evaluation procedures at all concentration combinations selected. An analysis of variance applied to the data collected from several experiments confirmed that there was no significant 2-AAF-fiber interaction. Our data suggest the absence of intrinsic genotoxic properties for chrysotile fibers. They also indicate that the modulation of the cellular response to genotoxic agents by asbestos fibers is not detected under our test conditions and may require longer-term exposures to be expressed.     

Vitronectin adsorption to chrysotile asbestos increases fiber phagocytosis and toxicity for mesothelial cells

Biological modification of asbestos fibers can alter their interaction with target cells. We have shown that vitronectin (VN), a major adhesive protein in serum, adsorbs to crocidolite asbestos and increases fiber phagocytosis by mesothelial cells via integrins. Because chrysotile asbestos differs significantly from crocidolite in charge and shape, we asked whether VN would also adsorb to chrysotile asbestos and increase its toxicity for mesothelial cells. We found that VN, either from purified solutions or from serum, adsorbed to chrysotile but at a lower amount per surface area than to crocidolite. Nevertheless, VN coating increased the phagocytosis of chrysotile as well as of crocidolite asbestos. VN coating of both chrysotile and crocidolite, but not of glass beads, increased intracellular oxidation and apoptosis of mesothelial cells. The additional apoptosis could be blocked by integrin-ligand blockade with arginine-glycine-aspartic acid peptides, confirming a role for integrins in the fiber-induced toxicity. We conclude that VN increases the phagocytosis of chrysotile as well as of crocidolite asbestos and that phagocytosis is important in fiber-induced toxicity for mesothelial cells.     

Adsorption of proteins by artificial mineral fibers: in vitro experiments

Since previous studies reported that in vitro some proteins and phospholipids were absorbed by asbestos fibres, namely chrysotile, in this study, man made filamentous glass fibers are been tested in vitro at the presence of proteins. The objective was to obtain evidence to evaluate whether continuous glass fibers have a behaviour similar to asbestos fibres. A sample of chrysotile fibres was used as control. Uptake of bovine serum albumin and horse spleen ferritin on these continuous glass fibres has been observed. However on glass fibres adsorbed less proteins per weight unit (22 mg/g and 12 mg/g respectively for albumin and ferritin) than asbestos chrysotile fibres (42 mg/g and 49 mg/g respectively for albumin and ferritin).     

Carcinogenicity of chrysotile asbestos: A case control study of textile workers

Chrysotile is the predominant type of asbestos used in the United States and thus represents the most important source of exposure to asbestos already in place. While the steepest exposure-response observed for lung cancer has been in workers exposed to chrysotile in textile operations, some argue that chrysotile is less carcinogenic than amphibole asbestos types. Mineral oil exposures have been hypothesized to be responsible for the highly elevated lung cancer risk seen in textile workers. A lung cancer case-control analysis among a cohort of South Carolina chrysotile asbestos textile workers was conducted. Only a modest reduction in the slope of the lung cancer exposure-response relationship was observed after controlling for mineral oil exposures. These data do not support mineral oil exposure as a plausible explanation for the elevated lung cancer risk seen in chrysotile asbestos textile workers. The possible role of longer, thinner, more carcinogenic fibers in textiles is one plausible hypothesis needing further investigation.     

Modulation of genotoxic effects in asbestos-exposed primary human mesothelial cells by radical scavengers, metal chelators and a glutathione precursor

The genotoxicity of asbestos fibers is generally mediated by reactive oxygen species (ROS) and by insufficient antioxidant protection. To further elucidate which radicals are involved in asbestos-mediated genotoxicity and to which extent, we have carried out experiments with the metal chelators deferoxamine (DEF) and phytic acid (PA), and with the radical scavengers superoxide dismutase (SOD), dimethylthiourea (DMTU) and the glutathione precursor Nacystelyn trade mark (NAL). We investigated the influence of these compounds on the potency of crocidolite, an amphibole asbestos fiber with a high iron content (27%), and chrysotile, a serpentine asbestos fiber with a low iron content (2%), to induce micronuclei (MN) in human mesothelial cells (HMC) after an exposure time of 24-72 h. Our results show that the number of crocidolite-induced MN is significantly reduced after pretreatment of fibers with PA and DEF. This effect was not observed with chrysotile. In contrast, simultaneous treatment of cells with asbestos and the OH*scavenging DMTU or the O2- -scavenging SOD significantly decreased the number of MN induced by chrysotile and crocidolite. In particular, DMTU almost completely suppressed micronucleus induction by both fiber types. A similar effect was observed in the presence of the H(2)O(2)-scavenging NAL after chrysotile treatment of HMC. By means of kinetochore analysis, it could be shown that the number of clastogenic events is decreased after PA and DEF pretreatment of fibers as well as after application of the above-mentioned scavengers. Our results show that chrysotile asbestos induces an increased release of H(2)O(2) in contrast to crocidolite. Also, the iron content of the fiber plays an important role in radical formation, but nevertheless, chrysotile produces oxy radicals to a similar extent as crocidolite, probably by phagocytosis-mediated oxidative bursting.     

Chrysotile asbestos fibers mediate transformation of Escherichia coli by exogenous plasmid DNA

The ability of chrysotile asbestos fibers to introduce the exogenous plasmid pUC18 into Escherichia coli JM109 cells was tested. Cells were transformed with pUC18 DNA although the frequency of transformation was quite low: 759+/-301 transformants were obtained per microgram of pUC18. Plasmids were purified from E. coli which had been transformed by mediation with chrysotile asbestos. Following this, the plasmids were confirmed to be pUC18 by Southern hybridization. This asbestos-mediated transformation was optimal within 5 min when 10 mg ml(-1) of asbestos was used. Plasmids up to 7.69 kb were introduced by this method.     

In vitro cytotoxicity of chrysotile asbestos to human pulmonary alveolar macrophages is decreased by organosilane coating and surfactant

Human pulmonary alveolar macrophages were used to quantitate the cytotoxic effect of surface-altered chrysotile asbestos. Little difference was observed in mortality between chrysotile asbestos that was surface-treated to a 42% extent by a hydrophobic organosilane or untreated chrysotile. Little or no effect on mortality was observed when human pulmonary alveolar macrophages were cultured with untreated chrysotile or acid-leached asbestos in the presence of 10 mM dipalmitoyl lecithin. However, when human pulmonary alveolar macrophages were cultured with a hydrophobically-treated (to a 42% or 95% extent) chrysotile asbestos in the presence of 10 mM dipalmitoyl lecithin, a statistically significant decrease in mortality was observed compared to untreated chrysotile. No mutagenic activity was observed when V79 cells were cultured with acid-leached, or 42% hydrophobically-treated chrysotile asbestos, even when human pulmonary alveolar macrophages were included as an activation source. The 95% hydrophobically-treated and acid-leached chrysotile also exhibited decreased binding of benzo[a]pyrene compared to untreated chrysotile asbestos.Abbreviations AHH aryl hydrocarbon hydroxylase - B(a)P benzo[a]pyrene - CA chrysotile asbestos - CHO Chinese hamster ovary - DL dipalmitoyl lecithin - DMEM Dulbecco's Modified Eagle's Medium - FBS fetal bovine serum - O^r resistance to ouabain - PAH polycyclic aromatic hydrocarbon - PAM pulmonary alveolar macrophage - SCE sister chromatid exchange Deceased.     

AKT/mTOR and c-Jun N-terminal kinase signaling pathways are required for chrysotile asbestos-induced autophagy

Chrysotile asbestos is closely associated with excess mortality from pulmonary diseases such as lung cancer, mesothelioma, and asbestosis. Although multiple mechanisms in which chrysotile asbestos fibers induce pulmonary disease have been identified, the role of autophagy in human lung epithelial cells has not been examined. In this study, we evaluated whether chrysotile asbestos induces autophagy in A549 human lung epithelial cells and then analyzed the possible underlying molecular mechanism. Chrysotile asbestos induced autophagy in A549 cells based on a series of biochemical and microscopic autophagy markers. We observed that asbestos increased expression of A549 cell microtubule-associated protein 1 light chain 3 (LC3-II), an autophagy marker, in conjunction with dephosphorylation of phospho-AKT, phospho-mTOR, and phospho-p70S6K. Notably, AKT1/AKT2 double-knockout murine embryonic fibroblasts (MEFs) had negligible asbestos-induced LC3-II expression, supporting a crucial role for AKT signaling. Chrysotile asbestos also led to the phosphorylation/activation of Jun N-terminal kinase (JNK) and p38 MAPK. Pharmacologic inhibition of JNK, but not p38 MAPK, dramatically inhibited the protein expression of LC3-II. Moreover, JNK2^−/− MEFs but not JNK1^−/− MEFs blocked LC3-II levels induced by chrysotile asbestos. In addition, N-acetylcysteine, an antioxidant, attenuated chrysotile asbestos-induced dephosphorylation of P-AKT and completely abolished phosphorylation/activation of JNK. Finally, we demonstrated that chrysotile asbestos-induced apoptosis was not affected by the presence of the autophagy inhibitor 3-methyladenine or autophagy-related gene 5 siRNA, indicating that the chrysotile asbestos-induced autophagy may be adaptive rather than prosurvival. Our findings demonstrate that AKT/mTOR and JNK2 signaling pathways are required for chrysotile asbestos-induced autophagy. These data provide a mechanistic basis for possible future clinical applications targeting these signaling pathways in the management of asbestos-induced lung disease.     

Induction of angiogenesis by intraperitoneal injection of asbestos fibers

Tumors and activated macrophages release angiogenic factors that stimulate migration and proliferation of capillaries. We studied the development of angiogenesis before the appearance of mesotheliomas in C57B1/6 mice. Weekly i.p. injections of crocidolite asbestos fibers produced mesotheliomas after 30-50 wk. The initial histologic response to asbestos fibers was a nodular lesion on the peritoneal lining composed of clusters of fibers, activated macrophages, and proliferating mesenchymal cells. The earliest visible evidence of angiogenesis was seen surrounding 7% of these lesions 14 days after a single injection of 200 micrograms of crocidolite asbestos fibers. After six weekly injections, 30% of the lesions containing asbestos fibers were surrounded by a capillary network radiating toward the center of the lesion. Other mineral fibers, including chrysotile asbestos and fiberglass, also induced angiogenesis after six weekly injections. In contrast, only 8% of the lesions containing short asbestos fibers (90.6% less than or equal to 2.0 microns) and 9% of the lesions containing silica particles showed evidence of angiogenesis. We conclude that tumorigenic mineral fibers induce angiogenesis in the peritoneal lining, whereas nontumorigenic mineral particles or short asbestos fibers are less effective. Ingrowth of new blood vessels around clusters of asbestos fibers may facilitate the later emergence of mesotheliomas at these sites.     

Elemental allelopathy by an arsenic hyperaccumulating fern,Pteris vittata L.

Aims The functional advantages of arsenic (As) hyperaccumulation by plants are poorly understood. One proposed benefit, termed elemental allelopathy, occurs when hyperaccumulated As is cycled from the plant back into the top layer of soil, allowing As hyperaccumulators to gain an advantage over intolerant species by increasing soil As concentrations ([ As]) underneath their canopy. To date, there are no studies that detail the presence of increased soil [ As] associated with As hyperaccumulators. In this study, we documented variation in the soil [ As] associated with the Chinese brake fern, Pteris vittata L. and also compared the effects of environmentally relevant soil and solution [ As] on competitor plant growth.Methods Four populations of P. vittata were identified in central Florida, USA. P. vittata tissue samples and soil samples were collected at the base of and at 3 m away from ferns in each population (n = 36). Five sample locations were randomly selected from each site, and soils from the base and 3 m away from each fern were collected to examine the effects of naturally occurring soil [ As] on the germination and growth of a potential competitor plant (Oxalis stricta). Solutions with increasing [ As] were also used to examine the threshold for negative effects of [ As] on O. stricta growth. [ As] were measured using inductively coupled plasma mass spectrometry (ICP-MS).Important findings Overall, soil [ As] from the base of ferns was nearly twice that of soil 3 m away indicating that ferns hyperaccumulate As. However, ferns and their associated soil, contained different [ As] depending on their collection site, indicating that these populations accumulate and use [ As] differently. O. stricta growth decreased and germination was delayed as solution and soil [ As] increased. However, the relative distance from the fern that the soil was collected from did not affect growth, which would be expected with elemental allelopathy. Our results show that P. vittata is associated with higher soil [ As] and these concentrations are sufficient to inhibit growth of competitors. However, the absence of a strong inhibitory relationship associated with proximity to the fern across all locations suggests that the possible functional advantages of elemental allelopathy may depend on site specific characteristics.     

The fate of chrysotile-induced multipolar mitosis and aneuploid population in cultured lung cancer cells

Chrysotile is one of the six types of asbestos, and it is the only one that can still be commercialized in many countries. Exposure to other types of asbestos has been associated with serious diseases, such as lung carcinomas and pleural mesotheliomas. The association of chrysotile exposure with disease is controversial. However, in vitro studies show the mutagenic potential of chrysotile, which can induce DNA and cell damage. The present work aimed to analyze alterations in lung small cell carcinoma cultures after 48 h of chrysotile exposure, followed by 2, 4 and 8 days of recovery in fiber-free culture medium. Some alterations, such as aneuploid cell formation, increased number of cells in G2/M phase and cells in multipolar mitosis were observed even after 8 days of recovery. The presence of chrysotile fibers in the cell cultures was detected and cell morphology was observed by laser scanning confocal microscopy. After 4 and 8 days of recovery, only a few chrysotile fragments were present in some cells, and the cellular morphology was similar to that of control cells. Cells transfected with the GFP-tagged α-tubulin plasmid were treated with chrysotile for 24 or 48 h and cells in multipolar mitosis were observed by time-lapse microscopy. Fates of these cells were established: retention in metaphase, cell death, progression through M phase generating more than two daughter cells or cell fusion during telophase or cytokinesis. Some of them were related to the formation of aneuploid cells and cells with abnormal number of centrosomes.     

Ecological studies of Eastern Australian fruit flies (Diptera: Tephritidae) in their endemic habitat

Fortnightly fruit fly captures for a 2-year period at Cooloola (south-east Queensland) contained 11 species. Three species, viz., D. tryoni, D. neohumeralis and D. endiandrae predominated. The peak trap catches of 7 species corresponded with the peak fruiting times of their major hosts. There was no direct relationship between temperature and rainfall and the variations in population numbers. The host plants of some species do not grow in the Cooloola area and there is evidence that large numbers of flies migrate into the region from other breeding areas up to 100 km away. Pockets of tropical rainforest such as Cooloola could be important adult fruit fly feeding areas even in the absence of larval host plants.     

Oxygen radical-mediated mutagenic effect of asbestos on human lymphocytes: suppression by oxygen radical scavengers.

The mutagenic effect of chrysotile asbestos fibers and zeolite and latex particles on human lymphocytes in whole blood has been studied. It was concluded that their mutagenic activities were mediated by oxygen radicals because they were inhibited by antioxidant enzymes (SOD and catalase) and oxygen radical scavengers (rutin, ascorbic acid, and bemitil). It was proposed that oxygen radicals were released by phagocytes activated upon exposure to mineral dusts and fibers. The study of lucigenin- and luminol-amplified chemiluminescence of peritoneal macrophages stimulated by chrysotile fibers and zeolite and latex particles has shown that their mutagenic action is probably mediated by different oxygen species, namely, by the iron-oxygen complexes (perferryl ions) plus hydrogen peroxide, hydrogen peroxide, and superoxide ion, respectively. From the oxygen radical scavengers studied, rutin was the most effective inhibitor of the mutagenic effect of mineral fibers and dusts.     

Activation of the alternative complement pathway and generation of stimulating factors for granulocytes by glass fibers

Continuous-filament glass fibers coated with organic agents, candidate asbestos substitutes, were assessed for their ability to elicit from normal human serum complement-derived cleavage products which are able to stimulate the chemotaxis and the respiratory burst of polymorphonuclear leukocytes. Glass fibers generated chemoattracting and respiratory stimulating factors for polymorphonuclears from human serum. The effect was dose related for chemotaxis from the serum fiber concentration of 75 g/ml to 1,250 g/ml. The serum chemoattracting activity, as well the respiratory stimulation, were dramatically impaired when serum had been preliminarily absorbed with antiC5 antiserum. Since the impairment of chemotactic activity occurred also in the presence of EDTA, but not in the presence of EGTA, we assumed an activation of the alternative complement pathway.Glass fibers were studied in comparison to a UICC sample of Canadian chrysotile asbestos, which is able to activate in vitro the alternative complement pathway.Glass fibers exhibited less ability than asbestos fibers to generate complement cleavage products with chemotactic activity for polymorphonuclears; however, they produced an activity about equal to 80% of a chemotactic standard stimulus such as zymosan-activated plasma.Abbreviations AF asbestos fibers - antiCS-abs-S NHS absorbed with antiserum against C5 - EDTA-CH-S NHS treated with EDTA - EGTA-Ch-S NHS treated with EGTA - GF continuous filament glass fibers coated with a binder of organic substances - NHP normal human plasma - NHS normal human serum - PMN polymorphonuclear luekocytes - ZAP zymosan-activated plasma     

Chrysotile and amosite asbestos induce germ-line aneuploidy in Drosophila

Asbestos toxicity is a problem of considerable public concern and debate, however little is known regarding the biological targets of asbestos fibers. Prompted by reports that asbestos induces aneuploidy in cultured mammalian cells, we have investigated whether asbestos induces germ-line aneuploidy in Drosophila melanogaster. Using the ZESTE genetic test system, we have shown that both chrysotile and amosite asbestos induce sex-chromosome aneuploidy in Drosophila oocytes. Chrysotile appeared to be the more effective agent because it induced approximately equal frequencies of chromosome gain and chromosome loss, while amosite induced chromosome loss only. Two other asbestiform minerals, crocidolite and tremolite, were ineffective in this assay system. These results suggest that possible germ-line effects of asbestos should be considered in evaluating its potential impact on human health.     

Distribution of Phytophthora cinnamomi-Supprcssive Soil in Nature

Phytophthora cinnamomi-suppressivc soils were found to be widely distributed in nature. About 40 % of soil samples collected from locations with different vegetation, soil type or elevation throughout the island of Hawaii were suppressive to chlamydospore germination of Phytophthora cinnamomi. Soil samples collected from the same general areas varied greatly in degree of suppressive-ness to P. cinnamomi, ranging from conducive to strongly suppressive. Among the 155 soil samples tested, those with pH close to 4 or 8 tend to be more suppressive to P. cinnamomi than those with pH close to 6.     

Asbestos bodies in rat lung following intratracheal instillation of chrysotile

Asbestos bodies in rat lung have rarely been reported, and just one previous record of their formation from chrysotile fibres in the rat is known. This paper illustrates the production of numerous true asbestos bodies in the lungs of Lister hooded rats after a single small intratracheal dose of lightly milled chrysotile. The demonstration of these bodies is particularly useful because uncoated chrysotile fibres in lung tissue cannot normally be visualized by light microscopy; the detection of asbestos bodies, and therefore of asbestos fibres, provides a means of directly relating asbestos exposure to observed tissue lesions. The asbestos bodies detected in the present study were nearly always associated with small pulmonary fibrotic lesions. The bodies ranged in length from 5 to 80 micron and were up to 5 micron in diameter. Small spheres, rods and bodies the shape of a comma were common; larger beaded structures were somewhat rarer. The bodies were visible in tissue sections stained routinely with modified Azan stain and with haematoxylin and eosin, but their detection and localization was enhanced by the use of Perls' Prussian blue stain in association with a pale eosin counterstain.