Evaluation of the dose-response and fate in the lung and pleura of chrysotile-containing brake dust compared to chrysotile or crocidolite asbestos in a 28-day quantitative inhalation toxicology study.

This study provides an understanding of the biokinetics and potential toxicology in the lung and pleura following inhalation of brake-dust (brakes manufactured with chrysotile). The design included a 28-day repeated multi-dose inhalation exposure (6 h/d, 5 d/wk, 4 wks) followed by 28-days without exposure. Fiber control groups included a similar grade chrysotile as used in the brakes and a commercial crocidolite asbestos. Aerosol fiber distributions of the chrysotile and crocidolite were similar (fiber-length > 20 µm/cm3: Chrysotile-low/high 42/62; Crocidolite-low/high 36/55; WHO-fibers/cm3: Chrysotile-low/high 192/219; Crocidolite-low/high 211/255). The total number of aerosol particles/cm3 in the brake-dust was similar to that in the chrysotile (Brake-dust 710-1065; Chrysotile 532-1442). Brake-dust at particle exposure levels equal to or greater than chrysotile or crocidolite caused no indication of microgranulomas, epithelial hyperplasia, or fibrosis (Wagner score < 1.7) or changes in bronchoalveolar lavage (BAL) indices from the air control. Chrysotile BAL indices did not differ from the air control. Pathologically, there was low level of inflammation and epithelial hyperplasia, but no fibrosis (Wagner score ≤ 3). Crocidolite induced elevated neutrophils and cell damage (BAL), persistent inflammation, microgranulomas, and fibrosis (Wagner scores 4) which persisted through the post exposure period. Confocal microscopy of snap-frozen chestwalls showed no difference between control, brake-dust and chrysotile-HD groups or in thickness of visceral or parietal pleural. The crocidolite exposure resulted in extensive inflammatory response, collagen development and adhesions between the visceral and parietal surfaces with double the surface thickness. These results provide essential information for the design of a subsequent subchronic study.

DNA methylation profiling of asbestos-treated MeT5A cell line reveals novel pathways implicated in asbestos response.

Occupational and environmental asbestos exposure is the main determinant of malignant pleural mesothelioma (MPM), however, the mechanisms by which its fibres contribute to cell toxicity and transformation are not completely clear. Aberrant DNA methylation is a common event in cancer but epigenetic modifications involved specifically in MPM carcinogenesis need to be better clarified. To investigate asbestos-induced DNA methylation and gene expression changes, we treated Met5A mesothelial cells with different concentrations of crocidolite and chrysotile asbestos (0.5 ÷ 5.0 µg/cm2, 72 h incubation). Overall, we observed 243 and 302 differentially methylated CpGs (≥ 10%) between the asbestos dose at 5 µg/cm2 and untreated control, in chrysotile and crocidolite treatment, respectively. To examine the dose-response effect, Spearman's correlation test was performed and significant CpGs located in genes involved in migration/cell adhesion processes were identified in both treatments. Moreover, we found that both crocidolite and chrysotile exposure induced a significant up-regulation of CA9 and SRGN (log2 fold change > 1.5), previously reported as associated with a more aggressive MPM phenotype. However, we found no correlation between methylation and gene expression changes, except for a moderate significant inverse correlation at the promoter region of DKK1 (Spearman rho = - 1, P value = 0.02) after chrysotile exposure. These results describe for the first time the relationship between DNA methylation modifications and asbestos exposure. Our findings provide a basis to further explore and validate asbestos-induced DNA methylation changes, that could influence MPM carcinogenesis and possibly identifying new chemopreventive target.

MicroRNA expression in lung tissues of asbestos-exposed mice: Upregulation of miR-21 and downregulation of tumor suppressor genes Pdcd4 and Reck.

OBJECTIVES: Asbestos causes lung cancer and malignant mesothelioma in humans, but the precise mechanism has not been well understood. MicroRNA (miRNA) is a short non-coding RNA that suppresses gene expression and participates in human diseases including cancer. In this study, we examined the expression levels of miRNA and potential target genes in lung tissues of asbestos-exposed mice by microarray analysis. METHODS: We intratracheally administered asbestos (chrysotile and crocidolite, 0.05 or 0.2 mg/instillation) to 6-week-old ICR male mice four times weekly. We extracted total RNA from lung tissues and performed microarray analysis for miRNA and gene expression. We also carried out real-time polymerase chain reaction (PCR), Western blotting, and immunohistochemistry to confirm the results of microarray analysis. RESULTS: Microarray analysis revealed that the expression levels of 14 miRNAs were significantly changed by chrysotile and/or crocidolite (>2-fold, P < .05). Especially, miR-21, an oncogenic miRNA, was significantly upregulated by both chrysotile and crocidolite. In database analysis, miR-21 was predicted to target tumor suppressor genes programmed cell death 4 (Pdcd4) and reversion-inducing-cysteine-rich protein with kazal motifs (Reck). Although real-time PCR showed that Pdcd4 was not significantly downregulated by asbestos exposure, Western blotting and immunohistochemistry revealed that PDCD4 expression was reduced especially by chrysotile. Reck was significantly downregulated by chrysotile in real-time PCR and immunohistochemistry. CONCLUSIONS: This is the first study demonstrating that miR-21 was upregulated and corresponding tumor suppressor genes were downregulated in lung tissues of asbestos-exposed animals. These molecular events are considered to be an early response to asbestos exposure and may contribute to pulmonary toxicity and carcinogenesis.

In vitro toxicity of fibrous glaucophane.

The health hazard represented by the exposure to asbestos may also concern other minerals with asbestos-like crystal habit. One of these potentially hazardous minerals is fibrous glaucophane. Fibrous glaucophane is a major component of blueschist rocks of California (USA) currently mined for construction purposes. Dust generated by the excavation activities might potentially expose workers and the general public. The aim of this study was to determine whether fibrous glaucophane induces in vitro toxicity effects on lung cells by assessing the biological responses of cultured human pleural mesothelial cells (Met-5A) and THP-1 derived macrophages exposed for 24 h and 48 h to glaucophane fibres. Crocidolite asbestos was tested for comparison. The experimental configuration of the in vitro tests included a cell culture without fibres (i.e., control), cell cultures treated with 50 µg/mL (i.e., 15.6 µg/cm2) of crocidolite fibres and 25-50-100 µg/mL (i.e., 7.8-15.6-31.2 µg/cm2) of glaucophane fibres. Results showed that fibrous glaucophane may induce a decrease in cell viability and an increase in extra-cellular lactate dehydrogenase release in the tested cell cultures in a concentration dependent mode. Moreover, it was found that fibrous glaucophane has a potency to cause oxidative stress. The biological reactivity of fibrous glaucophane confirms that it is a toxic agent and, although it apparently induces lower toxic effects compared to crocidolite, exposure to this fibre may be responsible for the development of lung diseases in exposed unprotected workers and population.

Characteristics and modifying factors of asbestos-induced oxidative DNA damage.

Respiratory exposure to asbestos has been linked with mesothelioma in humans. However, its carcinogenic mechanism is still unclear. Here we studied the ability of chrysotile, crocidolite and amosite fibers to induce oxidative DNA damage and the modifying factors using four distinct approaches. Electron spin resonance analyses revealed that crocidolite and amosite containing high amounts of iron, but not chrysotile, catalyzed hydroxyl radical generation in the presence of H(2)O(2), which was enhanced by an iron chelator, nitrilotriacetic acid, and suppressed by desferal. Natural iron chelators, such as citrate, adenosine 5'-triphosphate and guanosine 5'-triphosphate, did not inhibit this reaction. Second, we used time-lapse video microscopy to evaluate how cells cope with asbestos fibers. RAW264.7 cells, MeT-5 A and HeLa cells engulfed asbestos fibers, which reached not only cytoplasm but also the nucleus. Third, we utilized supercoiled plasmid DNA to evaluate the ability of each asbestos to induce DNA double strand breaks (DSB). Crocidolite and amosite, but not chrysotile, induced DNA DSB in the presence of iron chelators. We cloned the fragments to identify break sites. DSB occurred preferentially within repeat sequences and between two G:C sequences. Finally, i.p. administration of each asbestos to rats induced not only formation of nuclear 8-hydroxy-2'-deoxyguanosine in the mesothelia, spleen, liver and kidney but also significant iron deposits in the spleen. Together with the established carcinogenicity of i.p. chrysotile, our data suggest that asbestos-associated catalytic iron, whether constitutional or induced by other mechanisms, plays an important role in asbestos-induced carcinogenesis and that chemoprevention may be possible through targeting the catalytic iron.

Minimal asbestos exposure in germline BAP1 heterozygous mice is associated with deregulated inflammatory response and increased risk of mesothelioma.

Germline BAP1 mutations predispose to several cancers, in particular malignant mesothelioma. Mesothelioma is an aggressive malignancy generally associated with professional exposure to asbestos. However, to date, we found that none of the mesothelioma patients carrying germline BAP1 mutations were professionally exposed to asbestos. We hypothesized that germline BAP1 mutations might influence the asbestos-induced inflammatory response that is linked to asbestos carcinogenesis, thereby increasing the risk of developing mesothelioma after minimal exposure. Using a BAP1(+/-) mouse model, we found that, compared with their wild-type littermates, BAP1(+/-) mice exposed to low-dose asbestos fibers showed significant alterations of the peritoneal inflammatory response, including significantly higher levels of pro-tumorigenic alternatively polarized M2 macrophages, and lower levels of several chemokines and cytokines. Consistent with these data, BAP1(+/-) mice had a significantly higher incidence of mesothelioma after exposure to very low doses of asbestos, doses that rarely induced mesothelioma in wild-type mice. Our findings suggest that minimal exposure to carcinogenic fibers may significantly increase the risk of malignant mesothelioma in genetically predisposed individuals carrying germline BAP1 mutations, possibly via alterations of the inflammatory response.

Asbestos induces aberrant crypt foci in the colon of rats.

The carcinogenicity of asbestos to the gut is controversial. The aberrant crypt focus (ACF) assay is proposed as a test for colon carcinogens. We have scored ACF in the colon of rats and mice, one month after per os gavages with suspensions of asbestos fibers. Crocidolite asbestos induced ACF in the colon of rats in two independent experiments (P = 0.02 and P < 0.01 compared to controls given water), and was ten times less effective than the carcinogen azoxymethane. Chrysotile asbestos also induced ACF in rats. Neither crocidolite nor chrysotile induced ACF in mice. The data suggest that ingested asbestos may be carcinogenic to the colon.

Inhaled crocidolite mutagenicity in lung DNA.

We used transgenic mice carrying the lacI reporter gene to study the mutagenesis potential of asbestos crocidolite. The animals were exposed by nose-only inhalation to an aerosol containing 5.75 mg/m(3) crocidolite dust for 6 hr/day and 5 consecutive days. After 1, 4, and 12 weeks, we examined four end points: the cytology of bronchoalveolar lavage, the lung load of crocidolite, the hydrophobic DNA adducts, and the mutations in the lacI reporter gene. Twelve weeks after exposure, nearly 10% of the inhaled fibers remained in the lung (227 +/- 103 ng/mg lung). There was evidence of a typical inflammatory response consisting of multinucleate macrophages at weeks 4 and 12, whereas immediately after the exposure, we observed numerous polymorphonuclear neutrophils. The mutant frequency significatively increased during the fourth week after the exposure: 13.5 [time] 10(-5) in the exposed group versus 6. 9 10(-5) in the control group. The induction factor, defined by the ratio of checked mutants of exposed mice to checked mutants of control mice, was 1.96. The mutation spectrum of control lung DNA and exposed lung DNA was similar, suggesting the possible involvement of a DNA repair decrease in crocidolite-treated animals. We used the (32)P-postlabeling method and did not detect any increase of either 5 mC or bulky adduct in treated mice. This is the first study that demonstrates asbestos mutagenicity in vivo after a nose-only inhalation.

Short-term inhalation and in vitro tests as predictors of fiber pathogenicity.

A wide range of fiber types was tested in two in vitro assays: toxicity to A549 epithelial cells, as detachment from substrate, and the production of the proinflammatory cytokine tumor necrosis factor (TNF) by rat alveolar macrophages. Three of the fibers were also studied in vivo, using short-term inhalation followed by a) bronchoalveolar lavage to assess the inflammatory response and b) measurement of cell proliferation in terminal bronchioles and alveolar ducts, using incorporation of bromodeoxyuridine (BrdU). The amount of TNF produced by macrophages in vitro depended on the fiber type, with the man-made vitreous fibers, and refractory ceramic fibers being least stimulatory and silicon carbide (SiC) whiskers providing the greatest stimulation. In the epithelial detachment assay there were dose-dependent differences in the toxicity of the various fibers, with long amosite being the most toxic. However, there was no clear relationship to known chronic pathogenicity. Fibers studied by short-term inhalation produced some inflammation, but there was no clear discrimination between the responses to code 100/475 glass fibers and the more pathogenic amosite and SiC. However, measurements of BrdU uptake into lung cells showed that amosite and SiC produced a greater reaction than code 100/475, which itself caused no more proliferation than that seen in untreated lungs. These results mirror the pathogenicity ranking of the fibers in long-term experiments. In conclusion, the only test to show potential as a predictive measure of pathogenicity was that of cell proliferation in lungs after brief inhalation exposure (BrdU assay). We believe that this assay should be validated with a wider range of fibers, doses, and time points.

Accelerated progression of asbestos-induced mesotheliomas in heterozygous p53+/- mice.

Asbestos fibers produce diffuse malignant mesotheliomas in chronic rodent inhalation assays or after direct intrapleural or intraperitoneal injection. In vitro models have provided evidence that asbestos fibers are genotoxic carcinogens that can directly or indirectly generate reactive oxygen- and nitrogen-derived species that cause DNA damage. Heterozygous p53+/- mice show an increased incidence and reduced latency of malignant mesotheliomas induced by weekly intraperitoneal injections of crocidolite asbestos fibers. In this study, we investigated whether loss of heterozygosity (LOH) at the p53 tumor-suppressor gene locus contributes to accelerated tumor progression. LOH was found in 50% of the tumors produced in heterozygous p53+/- mice. In contrast to tumors that arise in p53+/+ mice or those that retained one p53 allele, LOH was associated with large tumor masses with central areas of necrosis, local invasion, and penetration of lymphatics. Increased tumor size was not associated with increased levels of cell proliferation as determined by BrdU incorporation, but it was correlated with a reduction in apoptosis as determined morphologically and by the TUNEL assay. Wild-type p53 protein is essential for cell cycle arrest in response to DNA damage and in maintenance of genomic stability. Cell lines established from tumors that showed LOH at the p53 tumor-suppressor gene locus were nearly tetraploid. These results suggest that p53 haplo-insufficiency sensitizes mice to the clastogenic or aneuploidogenic effects of crocidolite asbestos fibers, resulting in a shorter latent period. As solid tumors develop, spontaneous loss of the wild-type allele accompanied by decreased apoptosis and genetic instability is associated with accelerated tumor growth, invasion, and lymphatic dissemination.

Short-term crocidolite inhalation studies in mice: validation of an inhalation chamber.

A nose-only inhalation chamber is described: this chamber being computer automated has been particularly designed for mice on which it was validated using a crocidolite aerosol at a nominal concentration of 13.6 mg/m3, 6 h/day during 5 days. A month later the mice showed typical inflammatory bronchoalveolar liquids with many polynucleated or activated macrophages and asbestos bodies. The burden of crocidolite fibers ranged from 345,000 to 1,300,000 fibers per mg of dried lung. This study demonstrates that during the month that followed a short-term mice exposure to crocidolite fibers, the inflammatory response was still persistent. These toxicological endpoints validate the nose-only inhalation chamber to be useful for common or transgenic mice.

Studies on genotoxicity of orally administered crocidolite asbestos in rats: implications for ingested asbestos induced carcinogenesis.

The early genotoxic action of oral exposure to UICC crocidolite asbestos fibres was studied in different short-term tests. Fischer-344 rats were gavaged with 50 mg/b.w.kg untreated asbestos fibres and fibres which had been allowed to adsorb benzo(a)pyrene molecules from extremely low concentration (0.25-2.5 microg/ml) aqueous solutions. This system can be considered a model for the drinking of potable water contaminated by asbestos fibres together with biologically active organic micro-pollutants. The Ames Salmonella mutagenicity assay was performed on concentrated urine and serum samples of treated animals. The formation of micronuclei and sister chromatid exchanges was also studied in the bone marrow of the exposed rats. The micronucleus analysis indicated marginal genotoxic activity only upon treatment with crocidolite prepared from the solution of 1 microg/ml. A dose-dependent increase was, however, demonstrated in the sister chromatid exchange frequency upon treatment with benzo(a)pyrene coated fibres. These experiments suggest the acute cogenotoxic activity of such fibres in orally exposed animals.

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.

Differential diagnosis of malignant tumours in the abdominal cavity of rats after intraperitoneal injection of crocidolite or benzo[a]pyrene.

In our investigation (i.p. test), crocidolite and benzo[a]pyrene, both caused a progression from initially reactive, then autonomously transformed proliferation of myofibroblasts and undifferentiated mesenchymal cells to malignant, multidirectionally differentiated (desmin and ED-1 positive) fibro-histiocytic tumours. Immunohistochemically these tumours showed no morphological characteristics (for example co-expression of vimentin and keratin in spindle-shaped tumour cells) of human asbestos-associated malignant mesotheliomas. On the other hand many tumour cells induced by crocidolite and benzo[a]pyrene had an ultrastructural appearance resembling fibroblasts and myofibroblasts. These have been demonstrated in only a few desmoplastic and sarcomatous mesotheliomas in human beings. None of the tumours revealed the typical ultrastructural features of epitheloid or transitional mesotheliomas. Apparently, both carcinogenic substances induce the transformation of undifferentiated pluripotent mesenchymal cells in rat peritoneum, regardless of their localization in the submesothelial compartment or perivascular connective tissue (preferentially after crocidolite application) or in the connective tissue pseudocapsule of major benzo[a]pyrene containing beeswax/tricaprylin depots in the mesometrium and mesenterial fatty tissue. In this way asbestos fibres in this animal experiment do not seem to induce an arrest in differentiation of intermediate or immature mesothelial cells as supposed formerly, but rather affect undifferentiated mesenchyme cells and myofibroblasts. This is an explanation for the immunohistochemical expression of markers of muscular differentiation in these tumour cells, which is known to occur in human malignant fibro-histiocytic tumours. If supplementary immunohistochemical investigations with different keratin antibodies also fail to confirm the mesothelial differentiation of the tumours induced in our i.p. test, the decision to call them "mesotheliomas" should be reconsidered. Further immuno-transmission-electron microscopical investigations with intermediate filament or macrophage antibodies are needed to clarify whether the term malignant "fibrohistiocytic sarcoma", "mesenchymoma" or "mesothelioblastoma" would be more correct from the morphological point of view.

P53 mutations in tumours induced by intraperitoneal injection of crocidolite asbestos and benzo[a]pyrene in rats.

Mutation analysis of the tumour suppressor gene p53 in tumours induced in the peritoneal cavity of rats revealed differences in the mutational pattern with regard to the carcinogenic substances applied. In tumours induced by benzo[a]pyrene a considerable amount of p53 mutations resulting in an altered protein structure could be detected. For the development of these tumours an escape from the p53 mediated cell cycle control can be assumed. However, in tumours of the same tumour type induced by crocidolite asbestos no mutations could be observed. Since there were even no spontaneous p53 mutations detectable in this tumour group, it is obvious that in these tumours the escape from cell cycle control does not take place via inactivation of p53. Therefore, it is concluded that the molecular mechanisms of carcinogenesis and tumour development in this tumour type depend on the type of carcinogen applied.

ESR investigation of the oxidative damage in lungs caused by asbestos and air pollution particles.

Exposure to asbestos and air pollution particles can be associated with increased human morbidity and mortality. However, the molecular mechanism of lung injuries remains unknown. It has been postulated that the in vivo toxicity results from the catalysis of free radical generation. Using electron spin resonance (ESR) in conjunction with the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) we previously investigated in vivo free radical production by rats treated with intratracheal instillation of asbestos (crocidolite fibers) and an emission source air pollution particle (oil fly ash). In this report we compare the effect of two different exposures on the type of free radicals they induce in in vivo animal model. Twenty-four hours after the exposure, ESR spectroscopy of the chloroform extract from lungs of animals exposed to either asbestos or oil fly ash gave a spectrum consistent with a carbon-centered radical adduct (aN = 15.01 G and aH = 2.46 G). To test whether free radical formation occurred in vivo and not in vitro, a number of control experiments were performed. Combinations (both individually and together) of asbestos or oil fly ash and 4-POBN were added to lung homogenate of unexposed rats prior to chloroform extraction. No detectable ESR signal resulted. To exclude the possibility of ex vivo free radical generation, asbestos or oil fly ash was added to lung homogenate of an animal treated with 4-POBN. Also, 4-POBN was added to lung homogenate from rats instilled with asbestos or oil fly ash. Neither system produced radical adducts, indicating that the ESR signal detected in the lung extracts of the treated animals must be produced in vivo and not ex vivo or in vitro. In conclusion, ESR analysis of lung tissue demonstrated that both exposures produce lipid-derived radical metabolites despite their different composition and structure. Analogously, both exposures provide evidence of in vivo enhanced lipid peroxidation. Furthermore, it is concluded that without the presence of a spin-trapping agent, no free radical metabolites could be detected directly by ESR in either exposure.

Teratogenicity of asbestos in mice.

Possible teratogenicity of 3 different asbestos (crocidolite, chrysotile and amosite) was assessed in CD1(ICR) mice. Dams on day 9 of gestation were given a single intraperitoneal administration at dose of 40 mg/kg body weight of asbestos suspended in 2% sodium carboxymethyl cellulose solution in phosphate buffered saline, while dams in the control group were given vehicle (10 ml/kg body weight). Dams and fetuses were examined on day 18 of gestation. To compare with the control group, the mean percentage of live fetuses in implantations in the group given crocidolite and the incidence of dams with early dead fetuses in the groups given chrysotile or amosite were increased. While no external or skeletal malformation was observed in the control group, the incidence of external malformation (mainly reduction deformity of limb) in the group given amosite, and the incidences of skeletal malformation (mainly fusion of vertebrae) in the all dosed groups were significantly increased. The result indicated that asbestos (crocidolite, chrysotile and amosite) have fetotoxicity and teratogenicity in mice.

Expression of heme oxygenase-1 in the lungs of rats exposed to crocidolite asbestos.

Oxidative stress is thought to be the pathogenesis of pulmonary fibrosis induced by asbestos, and heme oxygenase-1 (HO-1) protects lung tissue against oxidative stress. We hypothesized that HO-1 is associated with oxidative lung injury caused by exposure to asbestos. This study was conducted to investigate the time course of HO-1 expression of lungs exposed to crocidolite asbestos in vivo. Male Wistar rats were administered 1 mg or 2 mg crocidolite asbestos suspended in saline by a single intratracheal instillation and were sacrificed at 3 d, 1 wk, 1 mo, 3 mo, and 6 mo of recovery time. The expression of HO-1 was observed by Western blot analysis and immunostaining. Protein levels of HO-1 increased at from 3 d to 6 mo following intratracheal instillation of 2 mg crocidolite asbestos. The levels of HO-1 increased at 1 wk and 1 mo following intratracheal instillation of 1 mg crocidolite asbestos. Many HO-1-positive cells were found, particularly in the alveolar macrophages, during immunostaining. These findings suggest that HO-1 may be related to lung disorder induced by dust and therefore can act as a biomarker of lung injury due to dust exposure.

In vivo accumulation of iron on crocidolite is associated with decrements in oxidant generation by the fiber.

In vivo exposures to fibrous silicates are characterized by the formation of asbestos bodies. These structures consist of the original fiber with a coating of inexact composition, but it will include iron and protein. We tested the hypothesis that this iron, accumulated on asbestos bodies, participates in electron transport and oxidant generation. Thirty-day-old, male guinea pigs were intratracheally instilled with 1.0 mg crocidolite. Six months later, the animals were anesthetized, euthanized, and the fibers were isolated from the lungs. Energy-dispersive x-ray analysis and x-ray photoelectron spectroscopy confirmed an accumulation of metal onto the fiber after in vivo exposure. Stains for iron demonstrated a heterogeneous distribution of the metal on the silicate, while the uptake of a commercially available polyclonal antibody to ferritin localized to beaded enlargements along the coated fibers. Chelatable [Fe3+] associated with the fiber increased after in vivo exposure. However, oxidant generation by asbestos bodies was decreased relative to uncoated fibers despite the elevation in the concentration of metal associated with the crocidolite. We conclude that iron is accumulated onto fibers in the lungs of guinea pigs. Some portion of this accumulation of iron is in the form of ferritin, and this metal is not chemically reactive in oxidant production. Asbestos bodies may represent a successful attempt by the host to sequester the metal adsorbed to the surface of a fiber and diminish the oxidative challenge introduced by a fibrous silicate. Subsequently, the generation of free radicals by the fibrous silicate is diminished.