The biological reactivity of calcium silicate composites: in vivo studies

The biological reactivity of three respirable samples of calcium silicate composites was compared with that of chrysotile and titanium dioxide 1 and 4 weeks after intratracheal instillation of different doses of the minerals into rat lungs. The parameters chosen for investigation were numerical, cytological, and biochemical (RNA, DNA, and hydrolytic enzyme activities) alterations induced in the free cells, biochemical changes in the remaining lavaged lung tissue, and the amount of the lipoprotein-rich fraction (pulmonary surfactant) lavaged from the alveolar surface. Differential, dose-dependent (by mass) effects were produced by the composites which were all more reactive than titanium dioxide initially (1 week) although after clearance (4 weeks) many of the induced alterations produced by two composites were practically reversible. The relationship between the reactivity of the calcium silicates in simple in vitro model systems with their activity in vivo is discussed.     

Cell toxicity, hemolytic action and clastogenic activity of asbestos and its substitutes.

The cell toxicity, hemolytic and clastogenic activity were examined in various kinds of asbestos and some asbestos substitutes with reference to the their mineralogical and physicochemical characteristics. There were thirty-five fibrous and non-fibrous samples including UICC chrysotile, size-selected samples of UICC chrysotile, chrysotile altered by heating and grinding, Yamabe (Japan) chrysotile with long and short fibers, Coalinga (U.S. A.) chrysotile with short fibers, UICC crocidolite, amosite, and 19 non-asbestos samples such as, glass fibers, calcium silicates, sepiolites and some clay minerals. The cell toxicity and the hemolytic and clastogenic activity of asbestos were the strongest for chrysotile among all of the asbestos samples tested, and their strengths varied with fiber length and with the conditions of grinding and heating. These cellular effects of Yamabe chrysotile with long fibers and size-selected UICC chrysotile with long fibers were stronger than those of chrysotile of the same origin but with short fibers. These effects were weaker in chrysotile altered by heating and grinding. Among the asbestos substitutes, the cell toxicity, hemolytic and clastogenic activities of thin glass fibers were more marked than those of thick glass fibers. The four types of sepiolite were strongly hemolytic, but their cell toxicity and clastogenicity varied according to their grade of crystallinity and/or fiber size. These effects of calcium silicates and some clay minerals were generally low but varied with mineral species. In general, the cell toxicity, hemolytic and clastogenic activities of the asbestos substitutes tested here were mild compared with those of asbestos.     

Rat lung reactivity to natural and man-made fibrous silicates following short-term exposure

The inflammatory and fibrogenic potential of three naturally occurring and two man-made industrial minerals were compared. Groups of five rats each received respectively a single intratracheal instillation of saline (control), UICC chrysotile B asbestos, short chrysotile 4T30, attapulgite, xonotlite (a calcium silicate), and Fiberfrax (an aluminum silicate) at doses of 1, 5, and 10 mg. One month after the treatment, assessment of lung morphology and bronchoalveolar lavage were performed on each animal. Under these conditions, UICC chrysotile B at all doses tested (1, 5, and 10 mg) induced fibrotic lesions in bronchiolar tissues while short chrysotile 4T30 (1, 5, and 10 mg) caused focal accumulation of inflammatory cells in the alveolar structures but no apparent fibrosis. Compared to these positive reactions with different fibrogenicity, xonotlite caused minimal inflammatory reactions detectable only at high dose (10 mg) and by bronchoalveolar analysis. By contrast, the rat lung reacted more significantly to attapulgite and Fiberfrax although the tissue reaction differed considerably for these two materials. While attapulgite, at doses up to 10 mg caused minimal reactions characterized by mononuclear cell infiltration mainly in the alveolar structures, Fiberfrax at 1 mg and higher caused significant granulomatous reactions and the appearance of early fibrosis. Overall the order of lung biological reactivity observed for the various silicates was xonotlite much less than attapulgite less than short chrysotile 4T30 less than Fiberfrax less than UICC chrysotile B. These observations indicate that Fiberfrax, attapulgite, and, to a lesser extent, xonotlite are biologically active within the time span studied and potentially deleterious for lung tissue.     

Comparison of cytotoxicity of man-made vitreous fibres

The purpose of the study was to compare the cytotoxicity of man-made vitreous fibres (MMVFs): four refractory ceramic fibres (RCFs 1–4), two glasswool fibres (MMVF 10 and 11), a rockwool fibre (MMVF 21) and a slagwool fibre (MMVF 22). The ability of the fibres to induce haemolysis in sheep erythrocytes, to release lactate dehydrogenase (LDH) from rat alveolar macrophages (AM) and to increase the production of reactive oxygen metabolites (ROMs) in human polymorphonuclear leukocytes (PML) was studied. To assess the relative cytotoxicity of MMVFs, their toxicity was compared with that induced by quartz, chrysotile or titanium dioxide. MMVFs induced a modest, but dose-dependent, increase of haemolysis at doses of 0.5, 2.5 and 5.0 mg ml⁻¹. The amount of haemolysis and LDH release induced by MMVFs was generally similar to that induced by titanium dioxide. Glasswool fibre MMVF 10 induced less LDH release from rat AM than rockwool MMVF 21 or slagwool MMVF 22 fibres, whereas glasswool fibre MMVF 11 induced less LDH release than slagwool fibre MMVF 22 (P<0.05). All fibres also dosedependently increased the production of ROMs at doses between 25 and 500 μg ml⁻¹. The shapes of the time-courses of MMVF-induced production of ROMs suggest that the mechanisms whereby the different fibres induce ROM production may exhibit similar features. There are clear-cut differences in the potency of various MMVFs to induce cytotoxicity and oxidative burst. The present results also emphasize the importance of using several measures of toxicity when assessing the biological activity of various fibres in vitro.     

Antibody producing cells in the spleens of mice treated with pathogenic mineral dust.

Experiments were carried out to assess the effect of intraperitoneal injection of the mineral dusts, titanium dioxide, quartz, or asbestos, on splenic lymphocyte antibody forming cells in immunised mice. Titanium dioxide and quartz caused similar, about one third, reductions in plaque forming cells; asbestos caused substantial reduction to about a quarter of the number found in control spleens. The inhibition of antibody forming cells in the spleen found with chrysotile was dose dependent and both chrysotile and crocidolite asbestos were similar in activity. Systemic immunomodulation after local deposition of mineral dust may be important to the development of disease.     

Antibody producing cells in the spleens of mice treated with pathogenic mineral dust

Experiments were carried out to assess the effect of intraperitoneal injection of the mineral dusts, titanium dioxide, quartz, or asbestos, on splenic lymphocyte antibody forming cells in immunised mice. Titanium dioxide and quartz caused similar, about one third, reductions in plaque forming cells; asbestos caused substantial reduction to about a quarter of the number found in control spleens. The inhibition of antibody forming cells in the spleen found with chrysotile was dose dependent and both chrysotile and crocidolite asbestos were similar in activity. Systemic immunomodulation after local deposition of mineral dust may be important to the development of disease.     

Mount St. Helens' volcanic ash: hemolytic activity

Volcanic ash samples from four Mount St. Helens' volcanic eruptions were subjected to mineralogical, analytical, and hemolytic studies in order to evaluate their potential for cytotoxicity and fibrogenicity. Plagioclase minerals constituted the major component of the ash with free crystalline silica concentrations ranging from 1.5 to 7.2%. The in vitro hemolytic activity of the volcanic ash was compared to similar concentrations of cytotoxic and inert minerals. The ash was markedly hemolytic, exhibiting an activity similar to chrysotile asbestos, a known fibrogenic agent. The hemolysis of the different ash samples varied with particle size but not with crystalline silica concentration. The results of these studies taken in conjunction with the results of our animal studies indicate a fibrogenic potential of volcanic ash in heavily exposed humans.     

The electric properties of fiber surface and the toxicity of asbestos

Thermal treatment, autoclaving and their combination yielded 3 samples of chrysotile-asbestos. With these, as compared with parent Type PRZh-1-50 Bazhenov chrysotile, the structure of fibers deteriorated and the number of negative discharges increased in the first two samples; the superficial structure substantially increased and the number of positive centers greatly decreased in the third sample. The ratio of positive to negative centers was less in all treated samples than that in the parent one, it decreased particularly during combined treatment. In this sample, hemolytic and mutagenic activity and cytotoxicity were substantially decreased; carcinogenic activity was greatly decreased (by 4 times) (as shown by the pleural mesothelioma indication test during intrapleural administration to rats). Thermal treatment and autoclaving insignificantly lowered the hemolytic activity and mutagenicity; carcinogenicity of these samples was much less (by 2.3 and 2 times) than that of parent chrysotile.     

Psychological Complications in Industrial Injuries

The dehydroxylated product obtained by heating hemolytic asbestos fibers of the chrysotile variety at 1,000 C still possesses hemolytic properties. A comparison of the lytic processes of these two materials indicates that they are not identical, as is shown by the fact that they are antagonized by the selective action of different polymers such as sodium carboxymethyl cellulose, in the case of the unheafed chrysotile, and polyvinyipyridine-N-oxide, in the case of the heated product. The nature of the surface of the two materials and the polymer structure may explain the different interactions.     

Effects of asbestos on epithelioid cell lines.

The in vitro effects of asbestos fibers on the growth and viability of CHO cells, an epithelioid cell line derived from Chinese hamster ovary, and on K-22 cells, an epithelial cell line derived from rat liver, have been studied. Relatively low concentrations of asbestos (10 micrograms/ml) were quite cytotoxic to both cell types. A sample of chrysotile asbestos was more toxic than samples of crocidolite or amosite. The toxic factor could not be extracted from the asbestos and toxicity occurred only if there was physical contact between the fibers and the cells. Although the phorbol ester class of tumor promoters induces the synthesis of plasminogen activator in various cell cultures, asbestos did not induce this protease in epithelioid or fibroblast cell cultures. The results obtained are compared to previous in vitro results obtained with fibroblast or macrophage cultures and are discussed in terms of their possible relevance to asbestos-induced fibrosis and tumorigenesis.     

Problems of Immunizing College-Age Adults

Collagenase digestion of lung fibroblast antigen preparations was shown to destroy their antigenic reactivity in most instances when reacted with lung-reactive antibodies. In other instances, elastase digestion rendered the preparation nonreactive. Such a finding demonstrates that collagen and/or elastin are the major components involved in this reactivity. This was also supported by sensitizing the fibroblast with rabbit antihuman lung coUagen and demonstrating an inhibition when these same fibroblasts were used in the antiglobulin consum,ption test with known lungoe reactive sera. The use of fibroblast cells as antigens in other serological tests was also, studied. Cytotoxicity tests showed definite cytopathic effect when conducted with lung-reactive sera. Similar cytopathic changes were not noted with sera from either chronic disease patients or the control sera.     

Identification of carcinogens by measurement of cell-mediated immunity: III. Immunity to asbestos-induced rat peritoneal mesothelioma

Antitumor cell-mediated immune (CMI) responses were detected in rats with asbestos (Canadian chrysotile B)-induced peritoneal mesotheliomas. The CMI was determined by in vitro lymphoid-cell cytotoxicity expressed by committed peripheral blood lymphoid cells (PBLC) obtained from the tumor-bearing animals toward cultured target cells of the tumors. Two apparently different target cell cultures which were developed from tumors of the same rat, one with epitheliod and the other with fibroblastic morphology, were lysed to the same degree by the sensitized PBLC. Specificity was observed for the PBLC reactivity as no cytotoxicity was expressed toward target cells derived from tumors of either ectodermal or entodermal origin. Nor were PBLC obtained from rats bearing tumors of these two tissue types capable of expressing injury to the mesothelioma target cells. These findings indicate that immune responsiveness developed to mesothelioma and suggest that such measurements might be useful for detecting environmental exposures to asbestos fibers.     

Biological effects of chrysotile after SO2 sorption. II. Effects on alveolar macrophages and red blood cells.

An experimental study has been carried out using different biological and biochemical in vivo and in vitro tests for assessing the toxicity of natural UICC (A) chrysotile and SO2-sorbed UICC (A) chrysotile and for detecting a possible synergistic effect. For in vivo studies, rabbits received an intratracheal injection of chrysotile fibers suspended in physiological saline (PS). The control group received only PS; all animals were sacrificed after 68 hr. The alveolar free cells were harvested by pulmonary lavage. The results have shown that chrysotile induces a decrease in the free cell population but there was no significant difference in the number of viable cells or in the nature of cells harvested between the two chrysotile groups. Enzymatic activities of the alveolar macrophages from animals injected with SO2-sorbed chrysotile showed a significant increase of the enzymes LDH and acid phosphatases. The LDH increase could be related to the affinity of the enzyme regarding some chemical forms of SO2. In vitro studies using alveolar macrophages harvested by pulmonary lavage have shown no differences between the two chrysotile groups when cell viability and enzyme release were studied. The toxic effect was due to chrysotile fibers (decrease of 38% in cell viability and increase in cellular enzyme release when compared with control). When rabbit red blood cells were used, both natural and SO2 chrysotile fibers showed the same hemolytic activity. The failure to detect high differences between the two chrysotile groups may be related to the chemical form of sorbed SO2.     

Leaching of chrysotile asbestos in human lungs: Correlation with in vitro studies using rabbit alveolar macrophages

The chemical microanalysis of chrysotile fibers obtained from human lungs and from chrysotile phagocytosed in vitro by rabbit alveolar macrophages (AM) was carried out with the use of an energy dispersive spectrometer mounted on a scanning electron microscope (SEM). This microanalysis was compared to natural fibers in order to investigate the chemical stability of chrysotile in biologic residence. The Mg versus Si content was estimated by the ratio of the peaks intensity Si:Mg. Lung samples were obtained from workmen with definite asbestos exposure, and chrysotile asbestos was extracted by chemical digestion. Phagocytosed chrysotile fibers were obtained after incubating standard chrysotile (A UICC) with rabbit alveolar macrophages for 24 hours or 5 days. Both chrysotile fibers from human lungs and from in vitro incubation with AM have shown an increase of the Si:Mg ratio in respect to standard fibers. Moreover, the results have shown that the magnesium leakage was not constant along the fiber axis and was different from one fiber to another.     

纳米TiO2毒性的试验研究

目的：对纳米TiO2使用的安全性进行评价。方法：对纳米TiO2的急性毒性、蓄积毒性、刺激性、致突变性进行动物试验观察。结果：纳米TiO2对小鼠急性经口毒性LD50〉10 000mg/kg体重，属实际无毒级；小鼠蓄积毒性K〉5，为弱蓄积性；一次完整皮肤刺激试验、急性眼刺激试验和阴道黏膜刺激试验结果为无刺激性；3个剂量诱导PCE微核细胞率与阴性对照组比较差异无统计学意义（P〉0．05），对小鼠骨髓PCE无诱变作用。结论：纳米TiO2是一种较安全的抗菌剂。     

Effects of chrysotile and acid-treated chrysotile on macrophage cultures

Beck, E. G., Holt, P. F., and Nasrallah, E. T. (1971).Brit. J. industr. Med.,28, 179-185. Effects of chrysotile and acid-treated chrysotile on macrophage cultures. The addition of chrysotile asbestos to monolayer cultures of peritoneal and alveolar macrophages produces an increase in membrane permeability, as measured by eosin uptake and lactic dehydrogenase activity of the supernatant fluid. The lactate synthesis is increased, however. It is suggested that the permeability of the cell membrane is increased while dust particles are being phagocytosed, which may take several hours when the particles are fibrous, but that this does not imply cell damage.

Treatment of chrysotile with acid, which leaves a silica surface, results in a product that reduces lactate synthesis, implying cytotoxicity. This change is counteracted by poly(2-vinyl-pyridine 1-oxide). The polymer does not affect the properties of the native chrysotile.

     

Binding of environmental carcinogens to asbestos and mineral fibres.

A rapid method has been developed for measuring the binding capacity of asbestos and other mineral fibres for environmental carcinogens. Benzo(alpha)pyrene (B(alpha)P), nitrosonornicotine (NNN), and N-acetyl-2-aminofluorene (NAAF) were assayed in the presence of Canadian grade 4T30 chrysotile, chrysotile A, amosite, crocidolite, glass microfibres, glasswool, attapulgite, and titanium dioxide. Chrysotile binds significantly more carcinogens than the other mineral fibres. This binding assay is reproducible with coefficients of variation of less than 8% and 6% respectively for inter and intra assay. The influence of pH was also studied, and there is good correlation between the carcinogen binding and the charge of the tested mineral fibres. The in vitro cytotoxicity on macrophage like cell line P388D1 and the haemolytic activity of various mineral fibres were also measured; a good correlation was found between the binding capacity and the cytotoxicity of tested mineral fibres on P388D1 cells. These results give some explanations for the reported synergism between exposure to asbestos and the smoking habits of workers.     

Binding of environmental carcinogens to asbestos and mineral fibres

A rapid method has been developed for measuring the binding capacity of asbestos and other mineral fibres for environmental carcinogens. Benzo(alpha)pyrene (B(alpha)P), nitrosonornicotine (NNN), and N-acetyl-2-aminofluorene (NAAF) were assayed in the presence of Canadian grade 4T30 chrysotile, chrysotile A, amosite, crocidolite, glass microfibres, glasswool, attapulgite, and titanium dioxide. Chrysotile binds significantly more carcinogens than the other mineral fibres. This binding assay is reproducible with coefficients of variation of less than 8% and 6% respectively for inter and intra assay. The influence of pH was also studied, and there is good correlation between the carcinogen binding and the charge of the tested mineral fibres. The in vitro cytotoxicity on macrophage like cell line P388D1 and the haemolytic activity of various mineral fibres were also measured; a good correlation was found between the binding capacity and the cytotoxicity of tested mineral fibres on P388D1 cells. These results give some explanations for the reported synergism between exposure to asbestos and the smoking habits of workers.     

A review of inhalation toxicology studies with Para-aramid fibrils

The paper summarizes the results of inhalation toxicology studies associated with para-aramid (p-aramid) fibrils. The review is subdivided into two categories: the results of inhalation toxicity studies and mechanistic inhalation studies. Keratin-associated lesions were observed in the lungs of female rats following chronic exposure to high concentrations of p-aramid. These lesions were originally interpreted as cystic keratinizing squamous cell carcinomas (CKSCC). In recent years, this keratinizing lesion has been observed in the lungs of rats with greater regularity in numerous chronic inhalation studies following exposures to a variety of dusts. In an attempt to reach a consensus on an appropriate diagnosis for this lesion, an international panel of pathologists was convened to evaluate the morphological aspects of this lesion. The panel considered that the most appropriate diagnosis for this lesion was ‘proliferative keratin cyst’ (PKC). the biological potential of the PKC remains controversial, but it appears to be unique to the rat species and has little relevance for humans. Mechanistic studies with p-aramid have demonstrated that acute inhalation of high concentrations of fibrils produces a potent but transient pulmonary inflammatory and cell labelling response. The inhaled fibrils have low durability in the lungs of rats as evidenced by a progressive decrease in median fibre lengths with increasing residence time in the lung. In contrast, in a comparative study, size-separated chrysotile asbestos produced a sustained increase over controls in cellular proliferation responses of terminal airways, parenchyma, subpleural and mesothelial regions. In addition, while the short fibres were cleared at a normal or rapid rate, the long (i.e. > 5 μm) chrysotile fibres were retained in the lung or cleared at a slow rate, suggesting that this subpopulation of fibres is durable in the lungs of exposed rats. These results point out the differences in the lung responses between p-aramid fibrils when compared to chrysotile asbestos fibres, namely, that chrysotile produced enhanced and sustained cell proliferation responses, and that the long chrysotile fibres were retained in the lungs of exposed rats.     

Hemolytic activity of crystalline silica--separated erythrocytes versus whole blood.

Whole blood and 1% erythrocyte suspensions were treated with crystalline silica (quartz DQ12, Min-U-Sil5) at concentrations of 0.5, 1, 2, and 5 mg/ml. Quartz DQ12 and Min-U-Sil5 revealed a strong dose-dependent hemolytic activity in the 1% erythrocyte suspension reaching nearly total hemolysis (> 80%) at the highest tested concentration of 5 mg/ml. This effect may be ascribed to surface reactivity by silanol groups. In contrast, using whole blood cultures the tested silica dusts caused no or only minor hemolytic activity (< 4%). The mechanism by which the hemolytic activity is prevented in whole blood cultures can be attributed to a number of factors such as the presence of metal binding proteins and free radical scavenger, antioxidant mechanisms and to coating of the silica surface by proteins, antibodies and complement. In contrast to separated erythrocytes whole blood represents an independent physiological compartment with functions of host defence and regulatory functions against cell damaging effects produced by oxidative stress.