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.     

Genetic effects of crocidolite asbestos in Chinese hamster lung cells

Chinese hamster lung cells cultured in the presence of crocidolite asbestos displayed inhibition of cell growth. Cell death was directly associated with the phagocytosis of larger fibres as observed with the aid of trypan blue. Water soluble components of crocidolite did not appear to inhibit cell growth. Chromosomal aberrations were induced by the asbestos. The aberrations were confined mainly to structural aberrations--breaks and fragments. Electron-microscopic preparation indicated that asbestos was readily contained in phagosomes. Phagocytosed asbestos appeared to be a weak mutagen in its ability to induce gene mutation at the hypoxanthine-guanine phosphoribosyl transferase locus.     

Biological durability and oxidative potential of man-made vitreous fibres as compared to crocidolite asbestos fibres

In this study we investigated relationships between redox properties and biodurability of crocidolite asbestos fibres and three different man-made vitreous fibres (MMVF): traditional stone wool fibres (MMVF 21), glass fibres (MMVF 11) and refractory ceramic fibres (RCF). Each fibre type was incubated up to 22 weeks in four different incubation media: gamble solution (GS) pH 5.0 and pH 7.4, representing blood plasma without proteins, and surfactant-like solution (SLS) pH 5.0 and pH 7.4. During incubation time aliquots of incubation mixtures were removed and analysed in a biochemical model reaction, mimicking activated phagocytes. In addition, changes of fibre morphology and chemical composition were examined using SEM- and EDX-technology. In the presence of crocidolite asbestos fibres and MMVF 21 the formation of OH*-radicals according to the Haber-Weiss sequence could be demonstrated, whereas MMVF 11 and RCF showed no reactivity. Crocidolite asbestos fibres exhibited a significant higher activity compared with the stone wool fibres at the onset of incubation. The oxidative capacities of these fibre types were shown to depend on both specific surface area and iron content. The oxidative potentials of crocidolite asbestos fibres as well as MMVF 21 were not constant during incubation over several weeks in each incubation medium. The reactivities showed sinoidal curves including reactivities much higher than those at the onset of incubation time. These irregular changes of oxidative capacity may be explained by changes of the redox state of fibre surface-complexed iron. Furthermore our results showed clear differences between incubation of fibres in GS and SLS, respectively, indicating that phospholipids play an important part in fibre dissolution behaviour and oxidative reactivity. In conclusion we suggest, that biodurability testing procedures should not exclusively concentrate on dissolution rates of fibres. They should include fibre characteristics concerning known pathogenic mechanisms to evaluate the real toxic potential of the fibre type looking at. Secondly we suggest, that phospholipids should be constituents of incubation liquids used for standardised fibre biodurability test procedures thus representing more realistic incubation conditions.     

Induction of 8-hydroxydeoxyguanosine by man made vitreous fibres and crocidolite asbestos administered intraperitoneally in rats

Inhaled fibres with certain physico-chemical properties are known to induce mesothelioma in humans. The induction of reactive oxygen (ROS) or nitrogen species (RNS) have been suggested as molecular mechanism of fibre induced carcinogenesis. In earlier studies we were able to demonstrate that crocidolite asbestos in vivo induces mutations in transgenic rats with a specific molecular spectrum that indicates the involvement of 8-hydroxydeoxyguanosine (8-OHdG) as pre-mutagenic adduct. 8-OHdG may be induced by primary (direct) and/or secondary (cellular mediated) mechanisms. Therefore, the induction of 8-OHdG as well as the inflammatory response of animals treated with fibre samples significantly differing in their physico-chemical characteristics was investigated. As appropriate system to study mesothelioma carcinogenesis, intraperitoneal injection in rats was used with samples of UICC crocidolite, crocidolite with reduced iron content, and a vitreous fibre (MMVF 11). Equal numbers of carcinogenic fibres from each sample revealed significant comparable increases in 8-OHdG induction. Parameters of inflammation (percentage of macrophages and TNF-alpha secretion) correlated significantly with the induction of 8-OHdG, 10 weeks after treatment.     

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.     

Iron mobilization from asbestos by chelators and ascorbic acid

The ability of chelators and ascorbic acid to mobilize iron from crocidolite, amosite, medium- and short-fiber chrysotile, and tremolite was investigated. Ferrozine, a strong Fe(II) chelator, mobilized Fe(II) from crocidolite (6.6 nmol/mg asbestos/h) and amosite (0.4 nmol/mg/h) in 50 mM NaCl, pH 7.5. Inclusion of ascorbate increased these rates to 11.4 and 4.9 nmol/mg/h, respectively. Ferrozine mobilized Fe(II) from medium-fiber chrysotile (0.6 nmol/mg/h) only in the presence of ascorbate. Citrate and ADP mobilized iron (ferrous and/or ferric) from crocidolite at rates of 4.2 and 0.3 nmol/mg/h, respectively, which increased to 4.8 and 1.0 nmol/mg/h in the presence of ascorbate. Since ascorbate alone mobilized iron from crocidolite (0.5 nmol/mg/h), the increase appeared to result from additional chelation by ascorbate. Citrate also mobilized iron from amosite (1.4 nmol/mg/h) and medium-fiber chrysotile (1.6 nmol/mg/h). Mobilization of iron from asbestos appeared to be a function not only of the chelator, but also of the surface area, crystalline structure, and iron content of the asbestos. These results suggest that iron can be mobilized from asbestos in the cell by low-molecular-weight chelators. If this occurs, it may have deleterious effects since this could result in deregulation of normal iron metabolism by proteins within the cell resulting in iron-catalyzed oxidation of biomolecules.     

Cytology of experimental mesotheliomas induced with crocidolite asbestos

The cellular features of 12 pleural and 5 peritoneal effusions, derived from experimental mesotheliomas induced with crocidolite asbestos in white rats, are described. The fluids were obtained 11 to 18 months after the introduction of asbestos into the body cavity. The morphologic characteristics of the cytoplasm, nuclei and nucleoli in the neoplastic mesothelial cells were studied using the Pappenheim, periodic acid-Schiff and Smetana stains. Nearly all effusions examined contained numerous normal and abnormal mitoses. Cell configurations suggestive of amitotic divisions were also observed. The study of the morphologic features of mesothelial cells in effusions in experimental asbestos-induced mesotheliomas may contribute to the understanding of the neoplastic transformation of the mesothelium.     

Cytokine and free radical responses of alveolar macrophages in vitro to asbestos fibres

A method for culturing primary rat alveolar macrophages (AMs) for 14 days was used to compare their responses to crocidolite and chrysotile asbestos fibres. Exposure to crocidolite increased production of tumour necrosis factor-alpha (TNF-alpha) and interleukin 1beta (IL-1beta), whereas exposure to chrysotile did not; neither fibre altered the production of interleukin 6 (IL-6). IL-1beta production increased progressively, while TNF-alpha was fully elevated from day 1. Conversely, chrysotile, but not crocidolite, increased production of superoxide anion and nitric oxide (NO) radicals. These differential responses were only observed by extending the culture beyond the usual 1-3 days.     

Free radical generation in the toxicity of inhaled mineral particles: the role of iron speciation at the surface of asbestos and silica

Free radical generation at the particle/biological fluid interface is one of the chemical processes that contributes to pathogenicity. In order to investigate the role played by iron, fibres of crocidolite asbestos have been modified by thermal treatments to alter their surface iron content. Two radical mechanisms, HO* from H2O2 and cleavage of a C-H bond, which are both active on the original fibres, have been tested on the modified fibres. C-H cleavage is dependent on Fe(II) abundance and location and is suppressed by surface oxidation while HO* release appears independent of the oxidation state of iron. Quartz specimens with different levels of iron impurities have been tested in a similar manner. A commercially available quartz (Min-U-Sil 5) containing trace levels of iron is also active in both tests, but reactivity is not fully suppressed by treatment with desferrioxamine, which should remove/inactivate iron. The radical yield attained is close to the level produced by a pure quartz dust, suggesting the presence of active sites other than iron. Ascorbic acid reacts with both crocidolite and quartz, with subsequent depletion of the level of antioxidant defences when particle deposition occurs in the lung lining layer. Following treatment with ascorbic acid the radical yield increases with quartz, but decreases with asbestos. Selective removal of iron and silicon from the surface may account for the differences in behaviour of the two particulates.     

Apoptosis of sea bass (Dicentrarchus labrax L.) neutrophils and macrophages induced by experimental infection with Photobacterium damselae subsp. piscicida

The infection of sea bass (Dicentrarchus labrax L.) by intraperitoneal (i.p.) injection of the agent of fish pasteurellosis Photobacterium damselae subsp. piscicida resulted in the apoptosis of peritoneal neutrophils and macrophages. All the eight virulent and none of the two non-virulent strains tested exhibited apoptogenic activity. A secreted bacterial protein(s) is a likely candidate as the factor(s) responsible for this activity, since no apoptosis was induced by i.p. injected UV-killed virulent strains and the virulent culture supernatants exhibited a thermo-labile apoptogenic activity identical to that of live bacteria. The apoptotic process was characterized by the occurrence of DNA fragmentation detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining and DNA electrophoresis, and of typical ultrastructural alterations namely cell shrinkage, chromatin condensation, nuclear fragmentation and production of blebs with shedding of apoptotic bodies. In the apoptotic process induced by lethal doses of virulent bacteria or culture supernatants both peritoneal macrophages and neutrophils were extensively affected, the majority of these cells being apoptotic and reaching values around 10(7)per peritoneal cavity for each cell type at 24h post-injection. Moreover, the number of non-apoptotic macrophages was always below the initial number in the resting peritoneal cavity. Since macrophages are key cells in the elimination of both bacteria and apoptotic moribund cells and apoptotic bodies, the induction by Ph. damselae subsp. piscicida of simultaneous macrophage and neutrophil apoptosis results, on the one hand, in the destruction of the two phagocytic cell types involved in the restriction of multiplication of the bacteria and, on the other hand, in the uncontrolled progression of the apoptotic process towards secondary necrosis and eventual lysis of high numbers of moribund neutrophils and of neutrophilic apoptotic bodies, with the consequent extensive release of their highly cytotoxic components. Abundant apoptotic cells were also seen in sections of head-kidney from fish dying from experimental pasteurellosis. In contrast, no apoptosis was seen in vitro after the treatment with virulent culture supernatants of sea bass head-kidney macrophage cultures or after the treatment ex vivo of peritoneal exudate leukocytes with virulent bacteria or culture supernatants. The apoptotic process described here appears as a novel and very powerful microbial pathogenic strategy.     

Migration of natural suppressor cells from bone marrow to peritoneal cavity by live BCG

Delayed-type hypersensitivity (DTH) responses were suppressed in mice inoculated with bone marrow cells from mice that had been injected with 10(8) colony-forming units (CFU) of live BCG. Upon analysis of this DTH-suppression by the use of a macrophage migration inhibition (MI) assay, the in vitro correlate of DTH, suppressor macrophages in the peritoneal cavity were found to play an important role in DTH suppression. However, neither suppression of DTH nor production of suppressor macrophages was observed in mice inoculated with bone marrow cells from mice that had been injected with methotrexate (MTX), a folic acid antagonist, and 10(8) CFU of live BCG. Moreover, suppressor cells against the MI activity of peritoneal exudate cells from BCG cell wall-immunized mice existed in bone marrow cells from normal mice, natural suppressor (NS) cells, and they were sensitive to MTX. In addition, these NS cells phagocytized carbonyl iron particles, were adherent to Sephadex G-10, and had Fc receptors, but they had no B or T cell markers, suggesting that these cells belonged to a macrophage compartment. From this evidence, we hypothesized that the origin of suppressor macrophages in the peritoneal cavity induced by live BCG injection was MTX-sensitive NS cells in bone marrow, and that these NS cells were stimulated by a small dose of live BCG trapped in bone marrow after i.v. injection of a high dose of live BCG and migrated from bone marrow to the peritoneal cavity.     

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.     

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.     

Asbestos catalyzes hydroxyl and superoxide radical generation from hydrogen peroxide

To understand chemical characteristics of the asbestos minerals which might contribute to tissue damage, the catalytic properties of three different varieties were studied. Using spin trapping techniques it was determined that crocidolite, chrysotile, and amosite asbestos were all able to catalyze the generation of toxic hydroxyl radicals from a normal byproduct of tissue metabolism, hydrogen peroxide. The iron chelator desferroxamine inhibits this reaction, indicating a major role for iron in the catalytic process, and suggesting a possible mechanism by which asbestos toxicity might be reduced.     

The stimulatory effects of asbestos on NADPH-dependent lipid peroxidation in rat liver microsomes.

Lipid peroxidation in rat liver microsomes induced by asbestos fibres, crocidolite and chrysotile, is greatly increased in the presence of NADPH, leading to malondialdehyde levels comparable with those induced by CCl4, a very strong inducer of lipid peroxidation. This synergic effect only occurs during the first minutes and could be explained by an increase or a regeneration of the ferrous active sites of asbestos by NADPH, which in turn could rapidly be prevented by the adsorption of microsomal proteins on the surface of the fibres. It is not inhibited by superoxide dismutase, catalase and mannitol, indicating that oxygen radicals are not involved in the reaction. It is also not inhibited by desferrioxamine, indicating that it is not due to a release of free iron ions in solution from the fibres. Lipid peroxidation in NADPH-supplemented microsomes is also greatly increased upon addition of magnetite. This could be linked to the presence of ferrous ions in this solid iron oxide, since the ferric oxides haematite and goethite are completely inactive.     

Cellular composition of spleen and peritoneal exudate in mice after injection of cyclophosphamide-treated L 1210 leukemia cells

The composition of peritoneal exudate and spleen cells of CD2F1 mice after fourfold i.p. administration of L 1210 leukemia cells treated with cyclophosphamide (L 1210-CY cells) were examined. The number of cells in peritoneal cavity did not increase, however, the spleen weight rose after administration of L 1210-CY cells. The per cent of lymphocytes T was increased 2.5 times but the content of macrophages and lymphocytes B was normal in the peritoneal cavity after L 1210-CY cells injections. In the spleen an 1.4 times increase of the per cent of lymphocytes B, but normal level of macrophages and lymphocytes T were observed.     

Apoptosis induced by crocidolite asbestos in human lung epithelial cells involves inactivation of Akt and MAPK pathways

Exposure of human lung epithelial (A549) cells to asbestos fibers causes apoptosis, which is largely attributed to release of iron and generation of reactive oxygen species (ROS) within the cells. To mimic the highly oxidative environment generated by asbestos exposure in the absence of the actual fibers, we used two chemicals; buthione sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis and ferric ammonium citrate (FAC), a source of iron. Here, we report that exposure of A549 cells to crocidolite asbestos led to a significant time-dependent inactivation of signaling proteins, i.e. Akt and all mitogen-activated protein kinases (MAPKs) (p38, ERK1/2 and SAPK/JNK), and subsequently to apoptosis. Unlike crocidolite treatment, the use of BSO and FAC, independently or combined, did not change the phosphorylation status of proteins, nor did it induce apoptosis. Taken together, our results presented herein point to the possibility that crocidolite-induced apoptosis of human lung epithelial cells is not a mere consequence of generation of oxidants but also requires inactivation of major cell growth and differentiation pathways. A. Baldys, P. Pande contributed equally to this publication.     

Leukotriene B4 mediates neutrophil migration induced by heme

High concentrations of free heme found during hemolytic events or cell damage leads to inflammation, characterized by neutrophil recruitment and production of reactive oxygen species, through mechanisms not yet elucidated. In this study, we provide evidence that heme-induced neutrophilic inflammation depends on endogenous activity of the macrophage-derived lipid mediator leukotriene B(4) (LTB(4)). In vivo, heme-induced neutrophil recruitment into the peritoneal cavity of mice was attenuated by pretreatment with 5-lipoxygenase (5-LO) inhibitors and leukotriene B(4) receptor 1 (BLT1) receptor antagonists as well as in 5-LO knockout (5-LO(-/-)) mice. Heme administration in vivo increased peritoneal levels of LTB(4) prior to and during neutrophil recruitment. Evidence that LTB(4) was synthesized by resident macrophages, but not mast cells, included the following: 1) immuno-localization of heme-induced LTB(4) was compartmentalized exclusively within lipid bodies of resident macrophages; 2) an increase in the macrophage population enhanced heme-induced neutrophil migration; 3) depletion of resident mast cells did not affect heme-induced LTB(4) production or neutrophil influx; 4) increased levels of LTB(4) were found in heme-stimulated peritoneal cavities displaying increased macrophage numbers; and 5) in vitro, heme was able to activate directly macrophages to synthesize LTB(4). Our findings uncover a crucial role of LTB(4) in neutrophil migration induced by heme and suggest that beneficial therapeutic outcomes could be achieved by targeting the 5-LO pathway in the treatment of inflammation associated with hemolytic processes.     

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.     

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of naïve macrophages in the peritoneal cavity makes it a preferred site for the collection of naïve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5⁺ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.