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.     

Characterization of asbestos fibers in lungs and mesotheliomatous tissues of baboons following long-term inhalation

Changes in the dimensions of inhaled asbestos fibers in the lung and translocation of intrapulmonary asbestos fibers into mesothelial tissues were investigated in 17 baboons (5 exposed to amosite, 4 to chrysotile, 5 to crocidolite, and 3 unexposed). The animals received different cumulative doses of asbestos by inhalation, followed by varying recovery periods (0–69 months). All asbestos types induced pulmonary asbestosis with severity directly related to the cumulative dose. There were a larger number of asbestos bodies in the lung of the amphibole-exposed animals than in those exposed to chrysotile. A tissue burden study, using transmission electron microscopy on 25-μm paraffin sections, ashed in a low-temperature asher, was performed. Intrapulmonary amosite fibers were shorter in geometric mean length compared with a standard amosite sample (UICC) (3.3 μm). In explanation, it was considered that long fibers might not be able to reach the lower respiratory tract and/or long fibers might be fragmented into shorter fibers. Further, in the amosite-exposed group, the mean length of intrapulmonary fibers increased with the extension of recovery period, suggesting that shorter fibers had been cleared from the lung. The chrysotile standard sample (UICC) had a shorter geometric mean length (1.1 μm) than amosite. The mean length of intrapulmonary chrysotile did not noticeably change with the extension of inhalation and recovery periods; however, the mean width decreased with the extension of these periods. This finding strongly suggested that separation of thick chrysotile fibers had occurred in the lung. The crocidolite standard sample (Transvaal) had a shorter geometric mean length (1.4 μm) than amosite. The mean length of intrapulmonary crocidolite fibers increased with the extension of inhalation and recovery periods, suggesting that shorter fibers had been cleared from the lung during both the inhalation and recovery periods. There was no specific tendency of size distribution among four distinct interstitial locations (peribronchiolar, alveolar septal, subpleural, and interlobular connective tissue) within the same lung exposed to either amosite, chrysotile or crocidolite. In four animals, malignant mesothelioma developed in the pleura (2 amosite and 1 UICC crocidolite) and the peritoneum (1 UICC crocidolite). Asbestos fibers were found in the mesotheliomas. Their size distribution in mesotheliomatous tissue and lung was not significantly different in two animals, but the fibers were shorter and thinner in another two. The presence of fibers in the neoplasms was confirmed, and translocation of fibers from the lung into the pleura or the peritoneum was strongly suggested. © 1993 Wiley-Liss, Inc.     

An assessment of the fibrogenic potential of very short 4T30 chrysotile by intratracheal instillation in rats

Three groups of five rats each received, respectively, a single intratracheal instillation of saline (control), 5 mg of UICC chrysotile B asbestos, and 5 mg of a preparation of very short chrysotile fibers (4T30, 100% less than 8 micron) isolated by a sedimentation procedure. At various intervals after the treatment (1 to 60 days), assessment of lung morphology was performed on each animal. Although the two types of chrysotile fibers have similar chemical composition, structure, and surface charge, the lung tissue reaction differed considerably. Lungs of animals exposed to UICC chrysotile B showed significant pathological alterations as early as 7 days following treatment. The lesions were localized in and around terminal bronchioles and consisted of inflammatory cells, fibroblasts and collagen deposition which distorted and obstructed small airways. Reaction to very short 4T30 chrysotile fibers was quite distinct. Seven days after treatment, lungs of these animals showed alveolar and interstitial accumulation of inflammatory cells. The alveolitis persisted 60 days after treatment and no fibrosis was apparent. It appears that very short 4T30 chrysotile fibers are much less fibrogenic than UICC chrysotile B and that intratracheal instillations in rats may represent a useful mean of rapidly assessing the fibrogenic potential of various dusts. These observations support the concept that fiber length is an important factor for fibrogenicity of asbestos.     

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.     

Mineral phases and some reexamined characteristics of the international union against cancer standard asbestos samples

Standard asbestos samples to be used for biomedical research were first prepared by the International Union Against Cancer (UICC) in 1966 in the United Kingdom and South Africa. Using modern techniques, X-ray diffractometry, analytical transmission electron microscopy, and thermal analysis, we have now analyzed these UICC samples to determine the mineral compositions (mineral phases) and their respective quantities. UICC chrysotile A (from Zimbabwe) contains 2% fibrous anthophyllite as impurity; chrysotile B (from Canada) does not contain any fibrous impurities, only non-fibrous minerals. UICC amosite and crocidolite are almost pure. UICC anthophyllite has 20–30% talc as impurity. The chemical compositions and fiber size distributions of the UICC asbestos samples have also been determined. The mean widths of the fibers of chrysotile A and B are smaller than those of the amphibole fibers. This agrees well with the earlier results which showed the two chrysotile samples to have a larger respirable fraction than the amphiboles. © 1996 Wiley-Liss, Inc.     

A trial on the quantitative risk assessment of man-made mineral fibers by the rat intraperitoneal administration assay using the JFM standard fibrous samples

We tried to evaluate the carcinogenic risk of man-made mineral fiber based on the mesothelioma incidence in female F344 rats after intraperitoneal administration. Rats (female F344/ Nslc, 5-week-old, n=330) were observed for 2 years after the intraperitoneal administration of 5 to 20 mg of 9 types of the JFM (Japan Fibrous Material Research Association) standard fiber samples (glass wool, rock wool, micro fiber glass, three types of refractory fiber, potassium titanate whisker, silicon carbide whisker, titanium oxide whisker), wollastonite (natural fiber) and UICC chrysotile B. All rats administered 10 mg of silicon carbide whisker had developed peritoneal mesothelioma within a year. The cumulative incidence of peritoneal mesothelioma at the end of the experiment was 85% for 10 mg UICC chrysotile B, 77% for 10 mg of potassium titanate whisker, 70% for 5 mg of silicon carbide whisker, 20% for 5 mg of potassium titanate whisker, 20% for 20 mg of refractory fiber 2 and 10% for 20 mg of refractory fiber 1. Carcinogenicity was estimated 2.4 times for silicon carbide whisker and 0.23 for potassium titanate whisker in comparison with UICC chrysotile B. It has been well documented from several experimental studies that man-made fibers are safer than asbestos because of the different durability in the lung. Present results consistently suggest that man-made fibers with high durability have similar or higher risk as carcinogen than asbestos.     

Biological effects of man-made mineral fibers (I)--Reactive oxygen species production and calcium homeostasis in alveolar macrophages

10 types of standard mineral fiber samples (JFM fibers) were tested for their cytotoxicity in alveolar macrophages (AM) in vitro experiments, in which UICC chrysotile B was used as a positive control. The cytotoxicity included the production of superoxide anion radical and hydrogen peroxide, depletion of glutathione (GSH) and increase of intracellular free calcium. The results showed that chrysotile and most of the 10 mineral fibers could increase the production of superoxide anion and hydrogen peroxide, deplete the concentration of GSH and increase the level of free intracellular Ca2+ in AM. But all the effects of JFM fibers were lower than that induced by UICC chrysotile B. Although the cytotoxicity of JFM fibers were lower than that of asbestos, these mineral fibers should be used with highly care for workers in industries.     

Characterization of three types of chrysotile asbestos after aerosolization

Jeffrey Mine and Coalinga Mine chrysotile, two asbestos samples prepared for experimental research by the National Institute of Environmental Health Sciences, and the UICC B chrysotile reference sample have been characterized in the aerosolized state using gravimetric measurements, light microscopy, scanning electron microscopy, and x-ray energy spectrometry. These methods revealed (1) a greater "respirable" mass fraction in the Jeffrey and UICC B preparations compared to the Coalinga sample, (2) for fibers greater than 5 microns in length and less than 3 microns in diameter, Jeffrey Mine chrysotile contained a significantly greater fraction of fibers longer than 40 microns in length compared to the UICC B or Coalinga Mine chrysotiles, and (3) Jeffrey and UICC B chrysotile contained no fibers or fiber clusters which exceeded 2 microns in diameter while Coalinga chrysotile contained numerous fibers and fiber clusters which were greater than 2 microns in diameter. The characterization of these chrysotile preparations in the aerosolized state, in particular the Coalinga Mine chrysotile, demonstrated different fiber length and fiber width distributions when compared with previous characterizations of samples that had been dispersed in a liquid medium by ultrasonification. These observations emphasize the importance of determining size distribution of fibers in the aerosolized state for inhalation studies and the size distribution of fibers in a liquid suspension for oral ingestion, instillation, or injection studies. Because of differences in length-width distributions, each of the studied chrysotile preparations would be expected to have different patterns of deposition in the alveolar regions of the lung after an inhalation exposure.     

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.     

Characteristics of peritoneal macrophages induced by asbestos injection

The effect of single intraperitoneal injections of UICC crocidolite, UICC chrysotile, and a latex control particulate on the induced murine peritoneal macrophage population was measured. Spreading, Fc receptor avidity, and phagocytosis were measured 3, 18, and 70 days after injection. When activation of the induced macrophage populations was found at all three times with asbestos, but not with latex, experiments were undertaken to determine whether the asbestos-activated macrophages had attained the full tumoricidally activated state. An in vitro assay measuring macrophage cytotoxicity to tumor cells revealed that the tumoricidal potential of asbestos-activated macrophages was low at 3 and 5 days and negligible by 35 days after injection. An in vivo assay measuring the effect of asbestos on tumor growth generally supported the contention that asbestos-activated macrophages were not tumoricidal, although one dose of UICC chrysotile did produce a small, significant reduction in growth of a concomitant tumor. It was concluded that a single intraperitoneal asbestos injection in mice induces activated macrophages which do not become fully tumoricidal.     

石英和温石棉致兔肺泡巨噬细胞谷胱甘肽过氧化物酶活性改变的比较研究

为了探讨石英、石棉对抗氧化酶活性的影响,利用改良Ｍｙｖｉｒｋ 法收集兔肺泡巨噬细胞进行体外培养,采用酶－ 底物反应形成有色物来测定巨噬细胞的细胞裂解液中谷胱甘肽过氧化物酶（ＧＳＨＰｘ）的酶活性。结果发现,随着粉尘浓度的增加,石英组和温石棉组巨噬细胞内的ＧＳＨＰｘ的酶活性相应降低,存在显著的剂量反应关系（Ｐ＜ ０．０１）。在不同的粉尘剂量组,温石棉组和石英组的ＧＳＨＰｘ 酶活性均显著低于二氧化钛组（Ｐ＜ ０．０５）,温石棉组低于石英组,但未发现显著的统计学差异。石英组和温石棉组的巨噬细胞死亡率均随粉尘浓度的增加而增大,存在着剂量反应关系,且温石棉组的死亡率高于石英组。石英组、温石棉组巨噬细胞内ＧＳＨＰｘ 酶活性和巨噬细胞的死亡率之间的相关系数分别为－ ０．９３１８ 和－ ０．８９８４,而二氧化钛组只有－ ０．３６３７。这说明石英、温石棉均可致巨噬细胞内ＧＳＨＰｘ 酶活性呈剂量反应关系的降低,使得胞内自由基Ｈ２Ｏ２ 不能正常消除,促使脂质过氧化加重,损害细胞;且温石棉的效应强于石英。提示ＧＳＨＰｘ 酶活性的降低在石英、温石棉致病中具有一定的作用     

Carcinogenic implications of the lack of tremolite in UICC reference chrysotile

Using light and electron microscopy analysis, as well as electron diffraction, and energy-dispersive x-ray analysis, an aliquot of UICC chrysotile B was analyzed with special attention given to any tremolite contamination. Polarized light microscopy, with its limit of detection of approximately 1 μm when using dispersion staining, revealed chrysotile as the only fibrous asbestos component. Analytical electron microscopy at 333,000× of more than 20,000 consecutive fibers showed only the tubular morphology characteristic of chrysotile. These findings highlight that when this sample was used for exposure disease induced in animal models correlates with chrysotile-induced pathology, and does not support an explanation based on the “amphibole hypothesis.” Thus, chrysotile should be considered as having the biologic ability to produce cancers, including mesotheliomas, based on the extensive use of this material as a standard reference material. Am. J. Ind. Med. 34:314–317, 1998. © 1998 Wiley-Liss, Inc.     

Determination of the variability in elemental composition of asbestos fibres by analytical transmission electron microscopy

An investigation was carried out to assess the variability in the elemental composition of asbestos fibres as determined using an energy dispersive X-ray spectrometer (EDS) attached to an analytical transmission electron microscope. UICC reference standards of chrysotile, amosite and crocidolite asbestos were analysed at five locations along the length of each fibre to observe within-and between-fibre variability. A total of 355 analyses were carried out on 71 fibres. No statistically-significant differences were found among the results obtained at the various locations along each fibre.     

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.     

A spin label study of the effect of chrysotile asbestos on erythrocyte membranes.

Alterations in erythrocyte membranes caused by UICC B chrysotile asbestos fibres were studied in red cell ghosts using the spin label technique. The electron paramagnetic resonance (EPR) spectra of two sulphydryl reactive spin labels and one fatty acid spin probe in erythrocyte ghosts showed membrane protein modifications but no changes in lipid fluidity caused by the haemolytic chrysotile asbestos fibres.     

Effect of two particle surface-modifying agents, polyvinylpyridine-N-oxide and carboxymethylcellulose, on the quartz and asbestos mineral fiber-induced production of reactive oxygen metabolites by human polymorphonuclear leukocytes

We studied the capacity of quartz and asbestos fibers to induce the generation of reactive oxygen metabolites in human polymorphonuclear leukocytes (PMNs) with a chemiluminescence (CL) assay. On an equal weight basis, the particulates induced CL in the following order of magnitude: chrysotile, quartz greater than amosite, crocidolite, greater than anthophyllite, wollastonite. The intensity of CL correlated positively with the Alcian blue (a cationic dye) binding capacity of the particles. Polyvinylpyridine-N-oxide (0.5 microgram/ml) inhibited completely the CL induced by quartz but had little effect on the CL induced by asbestos fibers. Carboxymethylcellulose (1.0 microgram/ml), however, reduced the CL caused by chrysotile asbestos but had no effect on the CL induced by the other particulates. Our results suggest that in addition to length and diameter, the effect of quartz and asbestos fibers on inflammatory cells will depend on surface characteristics, including the charge of the particles.     

Effect of quartz and asbestos on erythrocyte surface charge

Summary 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.     

Effect of asbestos on lipid peroxidation in the red cells.

In vitro exposure of red cells to vie International Union against Cancer (UICC) standard reference asbestos samples resulted in an increase of thiobarbituric acid substances. Chrysotiles developed the largest amounts of lipid peroxides, followed by anthophyllite, amosite, and crocidolite in decreasing order. Compared with the control samples erythrocytes free of dusts, all types of the asbestos examined disclosed significant differences. The results obtained provide support for the cytotoxic potential of amosite and crocidolite and, on the other hand, suggest that a lipid peroxidation of unsaturated fatty acids may be involved in the mechanisms(s) of membrane-damaging effects of asbestos dusts.     

Effects of asbestos fibers on alveolar macrophage-mediated lymphocyte cytostasis

Prolonged asbestos inhalation results in pulmonary inflammation and fibrosis. Since alveolar macrophages are active in regulation of immune responses in lung and appear to be involved in the pathogenesis of asbestosis, we evaluated the effects of asbestos exposure on the ability of these cells to regulate lymphocyte function. Alveolar macrophages obtained by lung lavage from BALB/C mice were treated in vitro with either UICC amosite or chrysotile asbestos and the effects on lymphocyte cytostasis compared with those of macrophages incubated with latex beads or zymosan. Macrophages (10%) incubated either alone or with latex beads for 48 hr effectively inhibited lymphocyte mitogenesis. However, alveolar macrophages incubated with either amosite or chrysotile asbestos did not demonstrate intact cytostatic activity. Decreased viability of chrysotile asbestos-treated macrophages correlated with loss of cytostatic effects, but alveolar macrophages exposed to amosite remained viable. We conclude, therefore, that exposure of alveolar macrophages to asbestos can result in loss of their ability to down-regulate lymphocyte proliferation, a finding which may be important in the pathogenesis of asbestos-related disease.