UICC Asbestos Research Articles

Low dose effects of fibrous and non-fibrous mineral dusts on the proliferation of mammalian cells in vitro

This report presents preliminary results of studies on the growth stimulating properties of mineral dusts. The studies were carried out with Syrian hamster diploid embryonic fibroblasts and Chinese hamster fibroblasts, line B14F28. Toxicity testing was done by the determination of the plating efficiency as a measure of growth and viability. The following dusts were used: the modified UICC asbestos fibres amosite, (AFF) crocidolite (KFF), chrysotile (KFF); in addition glass fibre (GFF), corundum and quartz DQ 12. Concentrations >2 μg/cm 2 of AFF, CFF, KFF and GFF depressed the plating efficiency of B14F28 cells in a dose-dependent manner, but all of the fibres and corundum increased colony sizes at concentrations of 0.16–0.33 μg/cm 2, in the case of corundum, AFF, KFF, and CFF also at up to 0.66 or 1.32 μg/cm 2. DQ 12 did not enhance colony growth. The stimulation of proliferation could be demonstrated both in terms of colony size (diameter) and cell numbers. The factor(s) responsible for proliferation stimulation reside in the supernatant, since the medium of dust-treated cell cultures was able to stimulate colony growth after removal of the dusts by filtration. The results indicate the induction of growth factors (cytokines) by low concentrations of the mineral dusts. Experiments concerning the effect of dusts on embryonic golden hamster fibroblasts yielded similar results. The plating efficiency was inhibited by concentrations of GFF and CFF >0.25 μg/cm 2 and by AFF, KFF, GFF and corundum at concentrations >5 μg/cm 2, but colony counts were significantly increased by AFF, KFF and corundum at concentrations of 0.25–3 μg/cm 2. This biological reaction which was observed in different cell types appears to be especially relevant in the context of environmental exposure where low dust concentrations prevail.

The lung biological activity of american attapulgite

Attapulgite is a fibrous mineral industrially consumed at the rate of over a million tons per year but the biological activity of the material is not fully known. To evaluate the in vivo toxicity of the fibrous material, we exposed the tracheal lobe of 16 sheep to a single exposure of either 100 ml saline, 100 mg UICC asbestos fibers in 100 ml saline, 100 mg short asbestos fibers in 100 ml saline, or 100 mg attapulgite in 100 ml saline. The animals were studied by bronchoalveolar lavage (BAL) at Days 2, 12, 24, 40, and 60 and by autopsy at Day 60. In the saline-exposed sheep, BAL and lung histology did not change. In the UICC asbestos-exposed animals, we reproduced the BAL changes previously reported (R. Bégin, M. Rola-Pleszczynski, S. Massé, Y. Berthiaume, and G. Drapeau, 1985, Environ. Res. 36, 389-404). In the short asbestos-exposed sheep, there were no significant BAL changes. In the attapulgite sheep, we found significant and sustained increases in total BAL cells (X2, P less than 0.05), macrophages (X2, P less than 0.05), neutrophils (X5, P less than 0.01), fibronectin (X2-3, P less than 0.05), lactate dehydrogenase (X2, P less than 0.05), beta-glucuronidase (X2, P less than 0.05), but BAL cellularity returned to control levels by Day 60 whereas in the UICC asbestos-exposed sheep, it remained significantly above control. Lung histology demonstrated the characteristic peribronchiolar fibrosing alveolitis in the UICC asbestos-exposed sheep, whereas macrophagic alveolitis with minimal airway distortion was seen in the short asbestos-exposed sheep and in all of the attapulgite-exposed sheep but three which had typical peribronchiolar alveolitis quite similar to that observed in UICC-exposed sheep, but of lower intensity. In conclusion, the study documents that attapulgite is not an inert material for lung tissue; it does produce a macrophagic and neutrophilic alveolitis in the early inflammatory phase of lung reaction with peribronchiolar involvement in some sheep. Whether or not this initial attapulgite induced alveolitis leads to lung fibrosis remains to be evaluated in a longer follow-up study of attapulgite-exposed animals.

Asbestos-induced Modulation of Release of Regulatory Molecules from Alveolar and Peritoneal Macrophages

Asbestos inhalation causes several immunologic abnormali ties in exposed individuals and experimental animals, 1 Pernis B Vigliani EC Selikoff JJ Rheumatoid factor in serum of individuals exposed to asbestos. Ann NY Acad Sci. 1965; 132: 112 Crossref PubMed Scopus (52) Google Scholar , 2 Kagan E Solomon A Cochrane JC Bleissner EI Glukman J Rocks PH et al. Immunological studies of patients with asbestosis. Clin Exp Immunol. 1977; 28: 261 PubMed Google Scholar , 3 Bozelka BE Sestini P Gaumer HR Hammad Y Heather CJ Salvaggio JE A murine model of asbestosis. Am J Pathol. 1983; 112: 326 PubMed Google Scholar and it has been suggested that these phenomena have a role in the induction of pulmonary fibrosis and could be related to alterations of macrophage-mediated regulatory activities. 4 Pernis B Vigliani EC The role of macrophages and immunocytes in the pathogenesis of pulmonary diseases due to mineral dusts. Am J Ind Med. 1982; 3: 133 Crossref Scopus (22) Google Scholar To investigate whether asbestos could affect the release of immunoregulatory molecules from macrophages, we have studied the effect of the exposure in vitro of mouse resident alveolar (AMø) and peritoneal macrophages (PMø) to nontoxic concentrations of asbestos on their suppressive capacity on the mitogen-induced proliferation of syngeneic spleen lymphocytes, on their ability to release arachidonic acid metabolites and superoxide anion (O$$) in response to zymosan and on the spontaneous release of interleukin 1 (Il-1). PMø purified by adherence and AMø obtained by bronchoalveolar lavage from 8-16-week-old C3H/HeN mice were cultivated in flat-bottom wells in the presence of control medium, latex beads, or UICC asbestos amosite, 80 µg/ml, for 21 hours. The medium was then carefully aspirated and the Mø further processed. Suppressive capacity was evaluated as previously described 5 Sestini P Bozelka BE deShazo RD Salvaggio JE Murine alveolar macrophage-mediated lymphocyte cytostasis: kinetics and mechanisms. Cell Immunol. 1982; 30: 108 Google Scholar by adding syngeneic spleen cells stimulated with an optimal dose of Con-A and evaluating the incorporation of tritiated thymidine after 72 hours of culture. The release of O$$ was measured by ferricytochrome c reduction after stimulation with opsonized zymosan. Prostaglandin E2 (PCE2) and F2α, (PGF2α), and leukotriene C4 (LTC4) were evaluated by radioimmunoassay after stimulation with zymosan 200 µg/rnl in medium without serum. Il-1 was evaluated by the C3H/HeJ thymocyte proliferation assay. The presence of interleukin-2 (Il-2) activity in the supernatants was assayed by the ability to support the proliferation of the CTLL cell line. In agreement with previous experiments 6 Bozelka BE, Sestini P, Hammad Y, Salvaggio JE. Effects of asbestos fibers on alveolar macrophage-mediated cytostasis. Environ Res (in press) Google Scholar the exposure of AMø and PMø in vitro to amosite caused a dose-dependent decrease in their suppressive capacity, whereas phagocytosis of latex had no effect. This phenomenon was not due to loss of cell viability as judged by LDH release and trypan blue exclusion, and was paralleled by a marked reduction of the ability to produce Ol. Asbestos exposure caused a small increase of spontaneous PGE2 production from both Mø populations; however, it had different effects on the zymosan-induced release of PGE2 and PGF2α from AMø and PMø. In fact, while PMø showed a reduction of the release of these metabolites after exposure to asbestos compared with untreated cells, AMø showed a marked increase. The release of LTC4, however, was decreased in both Mø populations. Latex beads had minimal effects on these activities. In addition, amosite but not latex beads induced the release of Il-1 in cultures of AMø and PMø. Il-1 activity was present in the supernatants during the first 24 hours of culture, and the release continued during further incubation, even in serum-free medium. No Il-2 activity or dialyzable inhibitors were found in the supernatants.

A spin label study of the effects of asbestos, quartz, and titanium dioxide dusts on the bovine erythrocyte membrane.

The effects of five UICC asbestos samples, titanium dioxide, and quartz on the bovine red cell membrane have been studied in erythrocyte ghosts by the spin labelling technique. Analysis of the electron paramagnetic resonance (EPR) spectra of two sulphydryl reactive spin labels and one fatty acid spin label in red cell ghosts showed modifications in membrane protein after asbestos treatment but no alterations in membrane lipids. In experiments with quartz no membrane changes were noted but titanium dioxide altered the proteins bound with the protein reactive spin label used in the present study. The possible mechanism for these effects is discussed.

Effects of nonfibrous minerals in the V79-4 cytotoxicity test.

The use of the V79-4 system as a primary screen for fiber carcinogenicity is dependent on the observed correlation between cytotoxicity of the test material in the system and mesothelioma following intrapleural injection in animals. This correlation has been established for relatively pure samples of fibrous minerals. The wider application of the system as a screen for industrial dusts may depend on the ability of the system to show an unequivocal response to small yet significant percentages of fibrous dust in the presence of a large excess of nonfibrous material. Some materials tested, including platy minerals, show a response in the test which, while clearly less than that from UICC asbestos samples, could be construed as a positive result. Some, though not all, of these minerals show a nonlinear dose response in the test. This anomalous dose response may aid in the identification of some false positive results but the variety of responses to nonfibrous minerals places a limit on the predictive value of the test mixtures. Results obtained from mixtures of "standard" dust samples suggest this limit is reached at or above 10% fibrous content. Such levels would be significant in terms of human exposure. Extension of the concentration range tested does not improve the predictive value of the test.

Assessment of progression of asbestosis in the sheep model by bronchoalveolar lavage and pulmonary function tests.

To study the relationship between the results of bronchoalveolar lavage and pulmonary function tests during induction and progression of asbestosis, three groups of six sheep were exposed repeatedly by intratracheal injection to either saline (controls), low doses of Canadian chrysotile UICC asbestos (cumulative exposure 328 mg) (low-dose group), or high doses of the same fibres (cumulative dose 2282 mg) (high-dose group) until there was clear evidence of alveolitis from the lung biopsy specimens of all sheep of the high-dose group. During the course of this induction and for the following eight months lung biopsies, bronchoalveolar lavage and pulmonary function tests were performed at two-month intervals. At the time of initial alveolitis in the high-dose group there was no significant change in the cellularity of the bronchoalveolar lavage fluid, but static lung compliance (Cst), vital capacity (VC), arterial oxygen tension (Pao2), and diffusion capacity (DL/VA) were significantly lower than in the other groups. In the following months, as the alveolitis evolved into a fibrosing process, macrophages and neutrophils from the bronchoalveolar lavage fluid increased significantly and pulmonary function deteriorated. Proteins and enzymes in the bronchoalveolar lavage fluid also increased significantly in the high-dose group. These data show that in the sheep model of asbestosis simple tests of pulmonary function correlate well with histological changes and changes in the bronchoalveolar lavage fluid in the course of the disease and can be used to assess progression of asbestosis.

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

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

Absence of mutagenic activity of three forms of asbestos in liver epithelial cells

The amosite, crocidolite, and chrysotile forms of UICC asbestos were assayed for their ability to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase locus. Using adult rat liver-derived (ARL-6 and ARL-18) cells in culture, no consistent or significant increase occurred in the number of mutants resistant to the purine analog, 6-thioguanine, after exposure to toxic levels of amosite, crocidolite, or chrysotile. These results support the concept that asbestos is an epigenetic carcinogen that does not alter DNA.

In vitro reactivity of alveolar macrophages and red blood cells with asbestos fibres treated with oxalic acid, sulfur dioxide and benzo-3,4-pyrene

The effects of 3 UICC asbestos fibres (A chrysotile, crocidolite, amosite) were observed in vitro on red blood cells (RBC) and alveolar macrophages (AM). The reactivity of the fibres after leaching with 0.1 N oxalic acid or adsorption of SO 2 or benzo-3,4-pyrene (BP) was studied. The haemolytic activity of crocidolite and amosite was very low. A cytotoxic effect on AM occurred when the fibres were present in high concentration (100 μg/ml), this was characterized by a release of both cytoplasmic (LDH) and lysosomal (ß-galactosidase) enzymes. The leached fibres were more haemolytic than the unleached ones, and more ß-galactosidase (ß-Gal) than lactic dehydrogenase (LDH) was released from the AM. In contrast to the amphiboles, chrysotile fibres were highly haemolytic and induced a selective release of ß-Gal from AM. Leached fibres were less haemolytic and were cytotoxic for AM (both enzymes were released). Their in vitro reactivity was similar to that observed with quartz. The results showed that SO 2 changed the reactivity very little. BP sorption on acid-leached chrysotile decreased the LDH release from

Explanation for the insensitivity to tilts of the electron diffraction patterns of amphibole asbestos fibers

The previously reported observation that the electron diffraction patterns of single amphibole UICC asbestos fibers are insensitive to tilts of ± 20° along and perpendicular to the fiber axis (Skikne, M. I., et al. (1971). Electron diffraction patterns of UICC asbestos sample Environ. Res. 4, 141–145) are experimentally confirmed. An explanation for this effect is proposed based on thin sample effects for small tilts and on the pseudohexagonal symmetry of these crystals for larger tilts. Because of this effect it is difficult to distinguish the three monoclinic forms of single amphibole asbestos fibers without previous study of standard (naturally occurring) single crystals.

The generation and evaluation of UICC asbestos clouds in animal exposure chambers.

When carrying out animal inhalation experiments using UICC asbestos it was found necessary to modify the dust cloud generation technique described by Timbrell, to obtain a stable, well-dispersed cloud at concentrations from 1 to 10 mg/m3. The MRE gravimetric dust sampler type 113A recommended by Timbrell as a routine monitoring instrument, was found to undersample at high chrysotile concentrations, and an alternative system was developed using a vertical elutriator. Fibre number counts of the clouds were obtained and correlated with mass concentrations. A Royco particle counter model 225 and Sibata digital dust indicator model AP.3 were tested, and their usefulness at the prevailing high concentrations assessed. Fibre length distributions for clouds of UICC chrysotile A, amosite and crocidolite asbestos are reported.

Effect of quartz and asbestos on erythrocyte surface charge

Experiments in vitro have been conducted to determine the surface-charge density of human red cells incubated with 7 quartz varieties as well as the UICC asbestos dusts: chrysotiles A and B, anthophyllite, amosite, and crocidolite. Red cell mobility was measured by capillary migration on Whatman paper strips and by electrophoresis. All dusts tested significantly decreased the erythrocyte surface electric charge as compared to control samples that were free of dust. Pretreatment of quartz dust with Polyvinylpyridine-N-Oxide restored the electronegativity of erythrocytes. The results obtained provide support for the cytotoxic effect of quartz and asbestos on the external cell membrane.