The pathogenicity of long versus short fibre samples of amosite asbestos administered to rats by inhalation and intraperitoneal injection

For many years it has been accepted that fibre dimensions are the most important factor in the development of asbestos related disease with long fibres being more dangerous than short for all types of asbestos. This information has been derived from in vitro experiments and injection or implantation experiments since the kilogramme quantities of specially prepared dusts that are necessary for long term inhalation have not been available. The present study has taken advantage of the availability of a sample of amosite produced so that almost all fibres were less than 5 micron in length. The effects of this dust were compared to dust prepared from raw amosite that contained a very high proportion of long fibres. Previous data from studies with UICC amosite, which was intermediate in length, were also available for comparison. At the end of 12 months of dust inhalation, significantly more short fibre amosite was present in the lung tissue compared to the long but while the long fibre dust caused the development of widespread pulmonary fibrosis, no fibrosis at all was found in animals treated with short fibre. One third of animals treated with long fibre dust developed pulmonary tumours or mesotheliomas but no pulmonary neoplasms were found in animals treated with short fibre dust. Following intraperitoneal injection, the long fibre amosite produced mesotheliomas in 95% of animals with a mean induction period of approximately 500 days. With short fibre dust, only a single mesothelioma developed after 837 days. In previous inhalation studies with UICC amosite, relatively little pulmonary fibrosis had developed and only two benign pulmonary tumours. This would suggest that to produce a significant carcinogenic response in rat lung tissue amosite fibres must be longer than those in the UICC preparation. Following the injection of UICC amosite, however, mesotheliomas developed in the same proportion of animals and with the same mean induction period as with long fibre dust. From this it would appear that while very short fibres exhibit little carcinogenicity to either lung or mesothelial tissues, mesotheliomas can be produced by dust preparations consisting of shorter fibres than are needed to produce tumours.     

Comparisons of the pathogenicity of long and short fibres of chrysotile asbestos in rats.

Long-term inhalation and intraperitoneal injection studies were undertaken with laboratory rats treated with a specially prepared short-fibre sample of Canadian chrysotile asbestos. This was compared, at an equal mass dose, to dust generated from the same chrysotile batch so as to contain the highest possible number of long fibres. The long-fibre cloud contained roughly five times more fibres greater than 5 micron in length as seen by phase contrast optical microscopy (PCOM). For increasing lengths, the ratio between the dust clouds increased progressively, reaching over 80: 1 for fibres greater than 30 microns in length. Rats treated with long-fibre chrysotile developed six times more advanced interstitial fibrosis (asbestosis) than animals treated with short-fibre chrysotile and three times more pulmonary tumours. At the end of the 12-month dusting period, three times more short chrysotile than long had been retained in the rat lung tissues. During the following 6 months, however, the short-fibre chrysotile was removed from the lungs much more rapidly than the long. Following intraperitoneal injection at a mass dose of 25mg of dust, both long and short chrysotile produced mesotheliomas in more than 90% of rats. At a dose level of 2.5mg of dust, the short-fibre chrysotile produced mesotheliomas in only one-third as many rats as the long-fibre dust which still produced mesotheliomas in more than 90% of animals injected. At a dose level of 0.25mg of dust, the short-fibre chrysotile produced no mesotheliomas while the long-fibre chrysotile still produced these tumours in 66% of rats. In the two highest doses, where short-fibre chrysotile produced mesotheliomas, the mean tumour induction period was significantly longer than for tumours produced by long chrysotile.     

Comparisons of the pathogenicity of long and short fibres of chrysotile asbestos in rats

Long-term inhalation and intraperitoneal injection studies were undertaken with laboratory rats treated with a specially prepared short-fibre sample of Canadian chrysotile asbestos. This was compared, at an equal mass dose, to dust generated from the same chrysotile batch so as to contain the highest possible number of long fibres. The long-fibre cloud contained roughly five times more fibres greater than 5 micron in length as seen by phase contrast optical microscopy (PCOM). For increasing lengths, the ratio between the dust clouds increased progressively, reaching over 80: 1 for fibres greater than 30 microns in length. Rats treated with long-fibre chrysotile developed six times more advanced interstitial fibrosis (asbestosis) than animals treated with short-fibre chrysotile and three times more pulmonary tumours. At the end of the 12-month dusting period, three times more short chrysotile than long had been retained in the rat lung tissues. During the following 6 months, however, the short-fibre chrysotile was removed from the lungs much more rapidly than the long. Following intraperitoneal injection at a mass dose of 25mg of dust, both long and short chrysotile produced mesotheliomas in more than 90% of rats. At a dose level of 2.5mg of dust, the short-fibre chrysotile produced mesotheliomas in only one-third as many rats as the long-fibre dust which still produced mesotheliomas in more than 90% of animals injected. At a dose level of 0.25mg of dust, the short-fibre chrysotile produced no mesotheliomas while the long-fibre chrysotile still produced these tumours in 66% of rats. In the two highest doses, where short-fibre chrysotile produced mesotheliomas, the mean tumour induction period was significantly longer than for tumours produced by long chrysotile.     

Comparison of the pulmonary responses to chrysotile and amosite asbestos administered intratracheally. I) Early phase of cellular reactions.

To clarify whether serpentine and amphibole asbestos have different effects on the lung, UICC chrysotile and amosite asbestos were injected intratracheally into hamster lung, and the lungs were examined at 30 min, 1, 4, 8, and 24 h, and 2 and 4 days after asbestos application by light and scanning electron microscopy. A leukocyte and macrophage reaction appeared at 4 h and increased up to 2 days, and the cell reaction was stronger and more prolonged after application of chrysotile than after application of amosite asbestos. Furthermore, chrysotile induced more prominent cell (leukocytes or/and macrophages) necrosis and alveolar wall thickening. These findings indicate that chrysotile asbestos induces more stronger cell reactions in the alveolar wall, and is more noxious than amosite.     

Inhalation and injection studies in rats using dust samples from chrysotile asbestos prepared by a wet dispersion process.

Long term inhalation studies and intraperitoneal injection studies in rats were undertaken with a series of chrysotile asbestos dusts. Three dust samples were generated from chrysotile modified by the wet dispersion process (WDC) and one was from unmodified chrysotile. Following a 1 year inhalation period, all the chrysotile samples proved extremely fibrogenic and carcinogenic and there were no significant differences between the WDC dusts and normal chrysotile. In all experimental groups approximately 25% of animals developed pulmonary carcinomas and in the oldest rats advanced interstitial fibrosis occupied on average 10% of all lung tissue. In the injection studies all the dust samples produced mesotheliomas in over 90% of animals. Very little chrysotile remained in the lungs of the animals that survived longest following dust inhalation and what there was was present as individual chrysotile fibrils. It is suggested that chrysotile is potentially the most harmful variety of asbestos as shown in these and other animal studies but that it is removed from lung tissue quite rapidly. In the long lived human species this may mean that except where exposure levels are very high and of long duration, chrysotile should be less hazardous than other asbestos types.     

Pulmonary reaction to long and short asbestos fibers is independent of fibroblast growth factor production by alveolar macrophages.

The role of alveolar macrophage (AM)-derived secretory products in fibroblast stimulation after the instillation of long and short asbestos to rat lungs is now investigated. A pure sample of 1 mg long crocidolite (greater than 2.5 mu) induced pulmonary fibrosis in 8 weeks, but secretions of lavaged AM from these lungs did not enhance growth or collagen synthesis in cultured rat lung fibroblasts. In contrast, the same dose of short fibers did not produce pulmonary fibrosis, although AM lavaged from these lungs were increased in number, had more phagocytized fibers, and when incubated, secreted factors that stimulated fibroblasts in culture. When normal AM were exposed to these fiber samples for 24 hours in vitro, greater phagocytosis of particles occurred and each asbestos fiber sample induced secretion of an AM-derived growth factor for cultured fibroblasts. The results indicate that both long and short fibers are capable of inducing AM to secrete fibrogenic factors in vitro, but in vivo, cytokine secretion by AM into the alveolar spaces in response to short fibers is not associated with stimulation of the interstitial fibroblast. In contrast, pulmonary fibrosis after long fiber administration appears unrelated to an AM secretion and is probably caused by fiber penetration into the peribronchiolar tissue, where interstitial macrophage activation may occur over several weeks.     

Inhalation and injection studies in rats using dust samples from chrysotile asbestos prepared by a wet dispersion process

Long term inhalation studies and intraperitoneal injection studies in rats were undertaken with a series of chrysotile asbestos dusts. Three dust samples were generated from chrysotile modified by the wet dispersion process (WDC) and one was from unmodified chrysotile. Following a 1 year inhalation period, all the chrysotile samples proved extremely fibrogenic and carcinogenic and there were no significant differences between the WDC dusts and normal chrysotile. In all experimental groups approximately 25% of animals developed pulmonary carcinomas and in the oldest rats advanced interstitial fibrosis occupied on average 10% of all lung tissue. In the injection studies all the dust samples produced mesotheliomas in over 90% of animals. Very little chrysotile remained in the lungs of the animals that survived longest following dust inhalation and what there was was present as individual chrysotile fibrils. It is suggested that chrysotile is potentially the most harmful variety of asbestos as shown in these and other animal studies but that it is removed from lung tissue quite rapidly. In the long lived human species this may mean that except where exposure levels are very high and of long duration, chrysotile should be less hazardous than other asbestos types.     

Ultrastructure of mesothelial regeneration after intraperitoneal injection of asbestos fibres on rat omentum.

In order to describe the ultrastructural features of the early phases of regenerating mesothelium in rat peritoneum, 69 cases were examined after intraperitoneal injection of 0.05-15 mg crocidolite, chrysotile B and other mineral and synthetic fibers. The findings show the presence of intermediate or transition cells between proliferating submesothelial connective tissue cells bearing the ultrastructural phenotype of myofibroblasts and mature fully regenerated mesothelium. Our results and data accumulated in the literature provide strong support for the hypothesis of submesothelial cells origin for regenerating mesothelium.     

Hepatic toxicity and recovery of Fischer 344 rats following exposure to 2-aminoanthracene by intraperitoneal injection.

Humans may be exposed to 2-aminoanthracene (2-AA), a substituted polycyclic aromatic hydrocarbon, and a recognized mutagen and carcinogen, through oral and respiratory routes from contact with a variety of environmental sources. For the present study, we sought to evaluate hepatic damage and recovery in Fischer 344 rats following multiple i.p. injections of 5 mg of 2-AA. Rats were injected weekly for up to 5 weeks. Subgroups were then allowed to recover for 1, 5, or 9 weeks, and biochemical and pathologic changes were evaluated. We observed that weight gains were reduced relative to controls for all groups receiving > or = 2 injections. Serum enzyme levels indicative of liver damage were evident and included alterations in serum aspartate aminotransferase, alkaline phosphatase, total protein, albumin, and globulin. These alterations usually returned to normal by 5 weeks following cessation of 2-AA administration. In contrast, histologic liver changes, including hepatocyte hypertrophy, biliary hyperplasia with oval cell proliferation, altered foci, nodular hyperplasia, and one hepatocellular adenoma became more severe with time. This experiment demonstrates patterns of hepatic damage and recovery in rats exposed to 2-AA.     

Alveolar type II cell responses to chronic inhalation of chrysotile asbestos in rats

The effects of chronic exposure to chrysotile asbestos on alveolar type II cells were examined in the lungs of Fischer 344 rats. Morphometric and three-dimensional analyses were used to characterize the alveolar type II cell and to determine the relationship of asbestos fiber localization to ultrastructural change in these cells. During the 2-yr period of study, type II cell number and volume increased to values more than 4 times those seen in controls. Ultrastructurally, cisternal dilations of the rough endoplasmic reticulum (RER) composed 12% of the total cell volume after 12 mo of exposure to asbestos and was still 15% of the total cell volume 1 yr after fiber exposure had ended compared to less than 1% in control cells. Asbestos fiber density surrounding these cells was directly proportional to the degree of cisternal dilatation in the cell; however, lamellar body volume and number in these cells were not different from that found in control type II cells. The incidence of a subset of type II cells with large lamellated inclusions was 10-fold greater in regions near bronchiolar-alveolar duct junctions, compared to more distal gas exchange regions of the lungs. Normal-sized lamellar bodies were fused to these large lamellated inclusions. These cells also contained significantly greater numbers of lamellar bodies and multivesicular bodies than those type II cells in more distal lung regions. These ultrastructural changes observed in type II cells may be a simple dose response to inhaled asbestos or the manifestation of two distinct populations of cells in the lungs that respond to asbestos in different ways. Asbestos fiber dose, cellular microenvironment, and aberrations of the cell plasma membrane and/or cell cytoskeleton (i.e., microtubules and filaments) are discussed as potential factors in the changes noted in type II cells.     

Fiber localization and its relationship to lung reaction in rats after chronic inhalation of chrysotile asbestos.

Inhalation of chrysotile asbestos fibers causes interstitial lung disease in animals and man. For examination of the anatomic localization of inhaled asbestos and its relationship to alveolar tissue responses of the lung during and after chronic exposure, male and female Fischer 344 rats were exposed to aerosolized chrysotile for 7 hours/day, 5 days/week for 3 or 12 months. A number of exposed animals were kept in filtered air for an additional 12 months. Lung tissue from randomly selected animals in each group was studied by morphometric analysis of electron micrographs. Our findings show that during exposure to asbestos fibers, macrophages and alveolar epithelial cells contain statistically significant amounts of asbestos and are associated with histologic changes indicating marked epithelial injury. Increased amounts of fibers are also localized in the lung interstitium with continued exposure to asbestos and are associated with a progressive interstitial fibrotic reaction. Following cessation of exposure, macrophages and epithelial cells are cleared of fibers and resolve toward normal proportions. However, significant clearance of fibers from the lung interstitium does not occur after cessation of exposure, and there is a continuing process of fibrogenesis. These data provide new insights related to the pathogenesis of diffuse lung disease and the role each alveolar tissue compartment plays in the early and late phases of the disease.     

Mesothelial cell proliferation after instillation of long or short asbestos fibers into mouse lung.

The relationship of asbestos deposition in the lung to subsequent cell proliferation at the pleural surface is not clear. The present study examines DNA synthesis by various pulmonary cells, particularly those at the pleura after intratracheal injection of 0.1 mg crocidolite to mice using: 1) long fibers (> 20 mu), which are deposited in bronchiolar regions and induce fibrosis; 2) short fibers (< 1 mu), which reach alveoli but do not induce fibrosis. Mice also received 2 microCi/g tritiated thymidine 1 hour before death at intervals to 16 weeks. Short fibers induced only a small increase in labeling of bronchiolar epithelial and interstitial cells, which subsided by 5 days, when a small increase in labeled mesothelial and subpleural cells was seen. In contrast, long fibers damaged the bronchiolar epithelium and became incorporated into connective tissue. During regeneration, 12% of cells were labeled at 3 days and labeling was greater than controls to 4 weeks. Increased peribronchiolar labeling of fibroblasts and interstitial macrophages was seen around long fibers, and increased DNA synthesis by mesothelial and subpleural cells was found. Up to 2% of mesothelial cells were labeled 1 week after long fibers compared to near zero in controls. No long fibers were found at the pleura. Activation of interstitial macrophages in response to long crocidolite fibers is associated with fibroblast proliferation. It is now suggested that mesothelial cells may also be stimulated by cytokines from activated interstitial macrophages that diffuse across the interstitium, without requiring actual fiber translocation to the pleura.     

Effects of long term inhalation of alumina fibres in rats.

Groups of rats were exposed by inhalation to atmospheres containing a refractory alumina fibre (Saffil Fibres, I.C.I.) either as manufactured or in a thermally aged form. Similar groups were exposed to UICC chrysotile A asbestos or clean air to serve as positive and negative controls respectively. Exposures continued for 86 weeks after which the animals were maintained to 85% mortality. Pulmonary reaction to both forms of alumina fibre was minimal; chrysotile asbestos provoked the expected progressive fibrosis. Pulmonary tumours (both benign and malignant) were confined to rats dosed with asbestos. The results support the predicted inert nature of these alumina fibres.     

Comparison of alveolar and interstitial macrophages in fibroblast stimulation after silica and long or short asbestos.

Pulmonary fibrosis in response to silica or asbestos has been attributed to secretion of fibroblast growth factors (FGF) by alveolar macrophages (AM). However, since fibrosis is interstitial, and is associated with particle retention by interstitial macrophages (IM), we have now compared the secretory activity of FGF by rat alveolar (AM) and IM in response to silica and to long or short asbestos fibers. AM were obtained by bronchoalveolar lavage, and IM by collecting macrophages that migrate from explants of a previously lavaged and perfused lung. Six weeks after instilling silica, isolated AM and IM from lungs secreted equal amounts of FGF. Six weeks after instilling short asbestos fibers in vivo, lavaged AM secreted FGF, but there was no change in fibroblast growth and no fibrosis in vivo. After long fibers, that reach the interstitium were instilled, isolated IM secreted FGF, and collagen levels were increased. When IM and AM were isolated from normal rats and exposed to the same silica or asbestos samples in vitro, it was found that all macrophage supernatants contained FGF, and the response of AM and IM was equal. The results indicate that the two macrophage populations respond equally to particles with respect to FGF secretion. The greater fibrotic reaction seen in vivo may be explained by the proximity of fibroblasts to particle-laden macrophages within the interstitium allowing more efficient transfer of FGF.     

Histopathological studies on experimentally induced pulmonary, pleural and peritoneal neoplasms in mice by intraperitoneal injection of chrysotile asbestos and N-methyl-N-nitrosourethane.

The cocarcinogenic effects of asbestos are presented. In lung carcinomas induced in mice, the number of carcinomas and the time of detection of the first carcinoma per tumor-bearing animals were greater and faster in the group with chrysotile plus MNU than either chrysotile or MNU alone. This suggested that chrysotile asbestos had a promoting or cocarcinogenic effect on some carcinogens in the respiratory tract. In the group treated with chryotile alone, a tumor was found in the right pleural cavity at 15 months. This tumor microscopically was similar to the biphasic form of the human diffuse mesothelioma. Microvilli, basement membrane, and junctional apparatus were seen by electron microscope, but other cytoplasmic organelles of the tumor cells were relatively scanty. Two peritoneal tumors developed in gastric and intestinal serosa at 11 and 12 months. Light and electron microscopic studies suggested that the tumors were probably myosarcomas or fibrosarcoms.     

Experimental studies in rats on the effects of asbestos inhalation coupled with the inhalation of titanium dioxide or quartz.

Rats were exposed for 1 year, with a 2-year follow-up, to dust clouds consisting of a mixture of amosite or chrysotile asbestos with either titanium dioxide or quartz. The addition of titanium dioxide to asbestos did not increase levels of pulmonary fibrosis above the amounts produced by chrysotile or amosite alone. Quartz, however, greatly increased fibrosis above that produced by the asbestos types alone. Both particulate dusts caused an increase in the numbers of pulmonary tumours and mesotheliomas compared to asbestos alone but while tumours in animals treated with asbestos and quartz tended to occur earlier than tumours with asbestos alone, in animals treated with dusts containing titanium dioxide, tumour production occurred later than with asbestos alone. In animals treated with mixtures of asbestos and quartz, there was evidence of increased transport of fibres across the visceral pleural surface and this may be associated with the finding of a higher proportion of pleural mesotheliomas than previously reported in experimental inhalation studies from any laboratory using the main asbestos varieties. The presence of particulate dusts made little difference to the amounts of amosite fibre retained in the lung tissue but, with chrysotile, titanium dioxide appeared to increase retention while quartz reduced it.     

Acute inflammation following intraperitoneal injection of antigen into actively sensitised rats.

Rats actively sensitised to bovine serum albumin (BSA) in Freund's complete adjuvant were challenged by intraperitoneal injection of BSA. By washing out the peritoneal cavities at different times it was possible to divide the changes that occurred into two phases. The first was characterised by the release of histamine and slow reacting substance of anaphylaxis (SRS-A) with an associated extravasation of plasma proteins. The second phase was characterised by an increase in neutrophilic polymorphonuclear cells in the peritoneal cavity. Extracellular enzyme activites were also investigated. The kinetics of these events were studied and are discussed in terms of the possible mechanisms involved.     

Effects of cigarette smoke on the clearance of short asbestos fibres from the lung and a comparison with the clearance of long asbestos fibres.

Long asbestos fibres are generally considered to have greater disease-producing potential than short asbestos fibres. However, recent reports have suggested that short fibre asbestos appears to be as effective an inducer of macrophage growth factors and toxic oxygen species as long fibre asbestos, but that short fibres are readily removed from lung and do not gain access to tissues. Because smoke is believed to impair the clearance of asbestos fibres from lung, we examined the clearance of a short (geometric mean length 1.3 microns) amosite preparation administered by intratracheal instillation to guinea-pigs. Half the animals in each group were exposed to the smoke of 10 cigarettes daily. Animals were sacrificed 1 day, 1 week, or 1 month later, the macrophages recovered by lavage, and fibre concentrations and sizes determined by analytical electron microscopy in macrophages and lung tissue. A 30-fold increase was seen in total numbers of fibres retained in macrophages in smokers compared to non-smokers by 1 month, and there was an eightfold increase in retention of short fibres in the lung tissue by 1 month. By contrast, a long fibre amosite preparation (geometric mean length 8.9 microns) showed approximately the same increase in fibre retention in macrophages, but only a twofold increase in tissue retention. We conclude that (1) cigarette smoke markedly impairs the clearance of short amosite fibres from the lung with enhanced retention of fibres in both macrophages and tissue; (2) the effects of smoke on short fibre tissue retention appear to be greater than those on long fibre retention; (3) with the long fibre preparation, smoke causes increased tissue retention of relatively shorter fibres; (4) for both fibre size experiments, the increase in total fibres in macrophages in smoke-exposed animals reflects an increase in the total number of fibre-containing macrophages, rather than an increase in the number of fibres phagocytized per macrophage; (5) enhanced short fibre retention markedly increases total fibre surface area, a parameter which has been suggested as a measure of fibre toxicity, to the point where short fibres might under some circumstances have roughly the same potential toxicity as long fibres. These observations suggest that short asbestos fibres could play an important role in the pathogenesis of some types of asbestos-related disease in cigarette smokers.