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Cell lines were established in vitro from five peritoneal mesotheliomas produced in rats by the injection of crocidolite asbestos. These tumours exhibited the previously described diverse range of histological patterns normally associated with mesotheliomas. This diversity of appearance was also evident in culture but cell patterns in vitro did not necessarily correspond to the histology of the original tumour. With increased time in culture most cell lines tended towards a pattern of random orientation associated with the piling up of cells to form discrete masses. Despite this variation in cell pattern in culture, few ultrastructural differences were seen during electron microscope examination. Following varying periods in culture, some cell lines from these tumours were injected into either rats or athymic mice. Only one typical rat mesothelioma was produced but all cell lines produced tumours in athymic mice. The implications of these findings are discussed in relation to the aetiology of asbestos induced mesotheliomas.

The association between occupational asbestos exposure and the development of both pulmonary fibrosis or asbestosis and pulmonary carcinomas is well documented. It has been suggested that the two pathological conditions are associated with asbestos-related carcinomas developing from areas of asbestosis and not occurring when exposure has been too low to produce this type of pulmonary scarring. Experimental inhalation studies so far published have not been designed to examine this association specifically, but many publications have reported that asbestos samples producing high levels of fibrosis is experimental animals are also very carcinogenic. Samples of asbestos or man-made fibers that produce little fibrosis also produce few tumors. These works are reviewed. In order to examine the association between fibrosis and tumor production in more detail, groups of animals with and without pulmonary tumors and with individual fibrosis measurements were assembled from a number of inhalation studies undertaken over a period of years at this Institute. It was found that animals with pulmonary tumors had almost double the amount of pulmonary fibrosis as animals of similar age that did not. In a few of the animals where tumors were found at an early stage of development, their origin from fibrotic areas could be confirmed, although in most cases where tumor deposits were widespread this was not possible. Experimental confirmation of the site of origin of most pulmonary tumors in asbestos-treated rats would require new studies with rats examined specifically at an age when early tumors would be expected.

Rats were exposed to clouds of the following pneumoconiotic dusts: quartz, coal-mine dust, and chrysotile asbestos at 10 or 50 mg/m3 for 8, 32, and 75 days; for comparison, rats were also exposed to the non-pathogenic dust titanium dioxide (TiO2). The bronchoalveolar leukocytes (macrophages and neutrophils) from dust-exposed and control rats were obtained by lavage and tested for their ability to migrate toward zymosan-activated serum. Varying amounts of neutrophils were present depending on the ability of the dust to cause inflammation and the length of exposure. There was a marked loss of chemotactic ability in leukocytes from rats inhaling the pneumoconiotic dusts compared with controls; TiO2-exposed leukocytes had some impairment of chemotaxis, but this was substantially less than that found with the pneumoconiotic dusts. The loss of chemotactic activity did not correlate with the percentage of neutrophils in the lavage cells except when there were very high levels of neutrophils, and there was substantial impairment of chemotaxis with negligible numbers of neutrophils, showing that macrophage chemotaxis was impaired. A phagocytic burden within the leucocytes was not sufficient alone to inhibit chemotaxis, nor was the loss of chemotactic activity due to occupied receptors, since incubation failed to restore chemotaxis. Loss to chemotactic activity by leukocytes from pneumoconiotic dust-exposed lung could be an important factor in the development of pneumoconiosis.

Experimental inhalation in a number of studies has demonstrated that chrysotile asbestos can cause pulmonary fibrosis and both benign and malignant pulmonary tumours, two lesions which are associated in that the studies reporting high tumour rates also found high levels of asbestosis. One comparison reported that animals with malignant tumours had approximately twice the amount of fibrosis in the lung parenchyma as those of similar age without tumours. Many studies have examined the pathogenicity of asbestos administered by ingestion and most of these included chrysotile asbestos: the results have been universally negative apart from one study with amosite that contained no control animals and is best discarded. Only one inhalation study has reported an examination of the larynxes of animals: this found no pathological changes. In many studies, tumours other than the lung had been listed, but significant numbers of kidney tumours have never been recorded. Injection studies inducing mesothelioma have indicated that fibre geometry is important with long thin fibres (> 8 microns in length and < 0.25 microns in diameter) being the most carcinogenic. This has been difficult to confirm for inhaled fibres although fibres less than 5 microns in length appear to cause neither fibrosis nor pulmonary tumours. Similar results have been found with amosite for fibres up to 10-15 microns although longer fibres do produce these conditions. It is suggested that to produce pulmonary fibrosis and neoplasia fibres may need to be longer than 20 microns. Chrysotile has been shown in many studies to be removed from lung tissue much more rapidly than amphibole fibres.(ABSTRACT TRUNCATED AT 250 WORDS)

The in vitro effects due to phagocytosis of asbestos by mouse peritoneal macrophages in various stages of activation have been compared. The amphiboles proved relatively inert; chrysotile, however, expressed a greater degree of cytotoxicity toward those populations of macrophages induced in vivo with asbestos, than toward any of the other populations of cells. These results are compared with data concerning the enzyme release from the different populations of macrophages following phagocytosis of asbestos. The results indicate that those macrophages that have been exposed to a prior stimulation of either amphibole or serpentine asbestos in vivo are particularly sensitive to exposure to a second dose of a toxic fiber.

Samples of the chrysotile taken during and after treatment by the wet dispersion process have been tested for their cytotoxic effect in vitro and the results compared with both a UICC chrysotile A sample and a dust prepared from a standard chrysotile textile yarn. Results were obtained from three different in vitro assay systems utilizing P388D1, V79-4 and A549 cells. A sample which still contained the wetting agent used in the wet dispersion process failed to show activity in any of these assays. The other samples, however, were all active with those dusts obtained by milling the final product and by sampling the air of the factory consistently proving significantly more cytotoxic than the standard chrysotile controls. Preliminary results from a parallel in vivo study suggest that these samples are also more active in producing mesotheliomas in rats.

Three different laboratories independently investigated the ability of ingested mineral fibers to penetrate tissues in rats. All concluded that there was no evidence of tissue penetration by ingested mineral fibers. These experimental observations are supported by the findings in coal and hard-rock miners who swallow, during their lifetime, nearly 100 times the amount of dust that is stored in their lungs. The intestinal wall or mesenteric lymph nodes of these people show no evidence of storage of the ingested dust particles. Animals fed asbestos over much of their lifetime and allowed to live to the age of cancer production, failed to provide evidence of a cancerogenic effect.

In order to compare methods of counting asbestos fibers in lung tissue, seven laboratories participated in an interlaboratory trial in which tissue samples from five human lungs were analyzed. In some laboratories, fiber concentrations were assessed with the light microscope and, in others, with either scanning or transmission electron microscopes. Within each laboratory the ranking of the results was similar, but there were marked differences in the absolute values obtained by the different laboratories. It is concluded that the laboratories participating in this trial appear to produce internally consistent results, but there is difficulty in directly comparing results from one laboratory to the next.

In this study, the cytotoxicity of 13 fibrous samples of known fiber number and dimensions has been established in P388D1 cells. The cells were exposed in vitro to dust concentrations of 10 or 50 micrograms/ml and, after incubation for 24 or 48 hours, any changes in cellular viability, lactate dehydrogenase, and glucosaminidase levels were determined. In general, there was a close association between the reduction in cellular viability and the loss of intracellular enzymes induced by each dust, the chrysotile asbestos samples proving more cytotoxic than the amphiboles. The cytotoxicity of the fibrous dusts was shown to be related to the number of fibers greater than 8 micron in length in the samples.

Previous studies have shown that long thin asbestos fibres are more pathogenic in in vivo and more active in in vitro assays than short fibre samples. In the present study a long fibre amosite asbestos sample and a short fibre sample prepared from it were tested for ability to cause inflammation in the peritoneal cavity of the mouse; a UICC sample intermediate in fibre size and an inert compact dust, TiO2, were also tested. The ability of the dust samples to cause inflammation, as judged by macrophage and neutrophil recruitment, was ranked in the order long fibre greater than UICC greater than short fibre greater than TiO2. Ability of amosite samples to cause inflammation was therefore related to the proportion of long fibres. The enhanced ability of long fibres to cause inflammation and cause macrophage activation is probably a key factor in the ability of long fibres to cause pulmonary fibrosis and may also be important in fibre carcinogenesis.

The effects of respirable dust from three commercially produced calcium silicate insulation materials were examined in laboratory rats by long-term inhalation and injection techniques. These calcium silicate products have been used as replacements for asbestos in the insulation of the engine rooms of ships, and the particle size distribution of the dust clouds generated for the experimental study closely matched those found in ships during the installation of this type of material. One year of exposure to a dust cloud of 10 mg/m3 of respirable dust had no discernible effect on the length of survival of treated animals compared to controls. No pulmonary lesions were found that appeared associated with the inhalation of calcium silicate per se, but one sample did contain significant amounts of quartz and this did produce a few small pulmonary nodules. While two small pulmonary neoplasms, one malignant and one benign, were found in dusted animals, neither was the cause of death, and the incidence was not significantly different from the control group where no tumors were found. One peritoneal mesothelioma was found in an animal from one of the inhalation groups, but this was considered to be a spontaneous tumor as none of over 100 animals injected intraperitoneally with 25 mg of calcium silicate developed these tumors. While the white blood cell count of dusted animals, compared to controls, was significantly raised in all treated groups at the end of the dusting period, these figures were within the published normal ranges for laboratory rats. It was concluded that the three tested calcium silicate products were harmless to the rats of this species at the doses tested.

After the intrapleural injection of asbestos dust in experimental animals, granulomas were produced and large areas of the pleural surface became thickened with fibrous tissue. In guinea-pigs injected with chrysotile dust, alveolar epithelial cells often migrated through the lung surface and were found lining spaces in the areas of pleural fibrosis. Initially these cells exhibited the exact structure of the surfactin-secreting type of alveolar cell, but later many of the cells lost the surfactin granules from their cytoplasm and became ciliated. In some animals cystic formation occurred in the lung tissue and groups of cysts grew out from the lung to invade the areas of pleural fibrosis. These cysts were also lined with surfactin-secreting alveolar cells. The possible association of these epithelial outgrowths and the well-known difficulties of diagnosing pleural mesotheliomas are discussed. Excroissances Epitheliales dans le Tissu Pulmonaire Aprks Injection Intrapleurale de Poussikre D'Amiante A des Animaux D'Experience Apres l'injection intrapleurale de poussiere d'amiante a des animaux d'experience, des granulomes sont apparus et de vastes zones de la surface pleurale ont ete epaissies par des tissus fibreux. Chez les cobayes ayant recu une injection de poussiere de chrysotile, les cellules epitheliales alveolaires emigrent souvent a la surface du poumon et vont se grouper en certains points des zones de fibrose pleurale. Ces cellules presentent d'abord la structure exacte du type de cellule alveolaire qui secrete la surfactine, mais par la suite un grand nombre d'entre elles perdent les granules de surfactine de leur cytoplasme et deviennent ciliees. Chez certains animaux, des formations kystiques sont apparues dans le tissu pulmonaire et des groupes de kystes se sont formes sur le poumon et ont envahi les zones de fibrose pleurale. Ces kystes etaient egalement recouverts de cellules alveolaires secretant de la surfactine. Les auteurs etudient la possibilite d'une association entre ces excroissances epitheliales et les mesotheliomes pleuraux, dont les difficultes du diagnostic sont bien connues.

Intraperitoneal asbestos injection in mice has previously been reported to elicit an activated macrophage population. In the present study supernatants from such macrophages were tested for their effect on thymocyte mitogenesis in response to concanavalin A; control supernatants were obtained from saline- and latex-elicited macrophages. Supernatants from asbestos-elicited macrophages were significantly inhibitory to thymocyte mitogenesis while saline- and latex-elicited macrophages did not release significant amounts of such activity. Asbestos-activated macrophage supernatants were inhibitory in a dose-dependent way and the activity was not secreted by macrophages from mice which had received asbestos in the long term. The inhibitory activity was partially dialysable. Supernatants prepared by treating macrophages in vitro with a lethal dose of asbestos were not inhibitory suggesting that the inhibitory activity in the supernatants of asbestos-activated macrophages did not leak from dead or dying cells. The asbestos macrophage supernatant was also significantly inhibitory to mature T-cell-enriched spleen cells but had no effect on fibroblasts, suggesting that the inhibitory effect could be lymphoid cell specific.

Samples of commercially used asbestos, especially chrysotile, are frequently contaminated by small amounts of other fibrous minerals. Among these are tremolite and brucite although pure tremolite is also produced commercially in relatively small quantities. In order to determine how harmful commercially exploited tremolite might be in comparison with other asbestos types and to explore the possibility that small amounts of tremolite and brucite as contaminants could significantly affect the pathogenicity of industrially used chrysotile, long-term animal inhalation and injection studies using rats were undertaken with what were considered to be mineralogically pure samples of these minerals. Rats treated with tremolite developed very high levels of pulmonary fibrosis as well as 16 carcinomas and two mesotheliomas in a group of 39 animals. Tremolite thus proved to be the most dangerous mineral that we have studied. Animals treated with 'brucite' developed moderate levels of pulmonary fibrosis and two carcinomas. Both tremolite and brucite produced mesotheliomas in greater than 90% of animals following i.p. injection. However, it was found that the supposedly pure brucite in fact contained 10% chrysotile, a level of contamination that could well have been responsible for the pathological changes found in both inhalation and intraperitoneal injection studies. The greatest care should be exercised by industry in handling tremolite or materials contaminated with it.

The kinetics of the bronchoalveolar response was assessed in rats exposed, at equal airborne mass concentration (10 mg/m3), to titanium dioxide--a non-pathogenic dust--and the two pathogenic mineral dusts quartz and chrysotile asbestos. Rats were killed at intervals over a 75 day exposure period and groups of rats exposed for 32 and 75 days after recovery for two months. Bronchoalveolar lavage was carried out and the lavage fluid characterised for cellular content, macrophage activation, and concentrations of free total protein, lactate dehydrogenase, and N-acetyl-beta-D-glucosaminidase. Inhalation exposure to the two pathogenic dusts resulted in an increased number of leucocytes, macrophage activation, and increased levels of free enzymes and total protein. The pattern and magnitude of the responses to quartz and chrysotile differed. Chrysotile caused less inflammation than quartz, and the main cellular response peaked around the middle of the period of dust exposure whereas the highest levels of enzymes occurred towards the end. The difference in timing suggests that macrophages were not available for lavage towards the end of the exposure, owing to their playing a part possibly in deposition of granulation tissue. Quartz caused a greater cellular and enzyme response than chrysotile, particularly towards the end of the dust exposure phase. There was a noticeable progression of inflammation in the quartz exposed groups left to recover for two months, but not in the chrysotile recovery groups.

Two groups of 48 rats of the AF/HAN strain were exposed for one year to respirable dust clouds of UICC chrysotile asbestos at a dose level of 10 mg/m3. One group was treated with dust carrying the normal electrostatic charge produced during dust generation, whereas the other was exposed to dust discharged by exposure to ionising radiation from a thallium-204 source. After dusting most animals were retained for their full life span. At the end of the dusting period those animals treated with normally charged dust had significantly more chrysotile retained in their lungs than animals exposed to discharged dust. Subsequently, animals treated with normally charged dust developed more pulmonary fibrosis and more pulmonary tumours. These findings suggest that the charge carried by airborne fibres should be taken into account when considering the health risks from exposure to chrysotile. Highly charged fibres are more likely to be deposited in lung tissue and thus constitute a greater hazard.

Much has been learned concerning the harmful effects of mineral dusts and other toxic materials by both in vivo and in vitro techniques. Both approaches have disadvantages, however. With in vivo studies exposure can be conducted under physiologically normal conditions but it is more difficult to unravel the complex sub-cellular and molecular events. In vitro, target cells can be examined in much greater detail but dose levels may have to be excessive to produce effects in an acceptable time scale and the absence of the whole of the body’s defensive systems and factors such as recruitment of new populations limit this approach. An approach now being adopted more frequently where target cells are readily available by simple techniques such as pulmonary lavage is to undertake exposure to harmful substances in vivo followed by examination of target cells and their products in vitro. This paper reports studies in which rats were exposed to three coalmine dusts, two varieties of asbestos, quartz and titanium dioxide administered by inhalation. Subsequently, their lungs were lavaged and a series of studies undertaken with the cell populations obtained.

Mineral fibers in the lungs of 13 Pittsburghers and 10 Cbarlestonians as well as of 7 workers exposed to asbestos dust have been quantitated, and the methodology has been described in some detail. Although wide variations in fiber concentration were found in people of the same sex and of the same community, the average concentration of optically visible pulmonary mineral fibers of Pittsburghers was about seven times as high as that of Charlestonians. The average pulmonary concentration of optically visible fibers in people not occupationally exposed to asbestos was about 0.7% of the average concentration of optically visible fibers in asbestotic lungs. From 54 to 71% of the EM-size fibers were less than 5 μm long. Of the EM-size mineral fibers in the lungs of people not occupationally exposed to asbestos, an average of 6.8% were identifiable as chrysotile.

The ability of mineral fibers to cause disease in man is well established and the work of Stanton et al. (1977), Stanton and Layard (1978) and Pott (1978) has indicated that the ability of a dust to induce intrapleural tumors depends on the number of fibers in the sample that are greater than 8 pm in length and less than 1.5 μm in diameter.

To the Editor:— This letter outlines the history of the asbestos body from its discovery through a period of little clinical significance to the present day when it holds the position of being possibly one of the greatest industrial medical problems of the age. Asbestos:— Asbestos is a general term that covers a number of minerals of which four are of commercial importance: crocidolite from the Republic of South Africa, amosite also mined chiefly in the Republic of South Africa, chrysolite which occurs in many countries of the world, and anthophyllite which is mined mainly in Europe. Asbestosis:— This is a type of lung fibrosis in workers exposed to large amounts of asbestos dust. It may or may not be associated with recognizable clinical disease during life and is produced by all types of asbestos. Asbestosis was recognized as an industrial disease at the turn of the century, but at

To the Editor:— Regarding our "Asbestos Bodies and Their Bioeffects" ( 203 :1142, 1968) in which we said that types of asbestos other than crocidolite have been almost com-that crocidolite was almost completely ineffective as a cause of mesothelioma, further information qualifying the relative abilities of crocidolite and other forms of asbestos to produce mesotheliomas has come to hand. According to J. F. Knox (personal communication, March 1968) the Ministry of Labour in Great Britain uses a phrase which is considered preferable to our statement and is offered herewith: [while]... the Advisory Panel considers that the evidence to date on balance indicates a particular significance must be given to crocidolite as a cause of mesotheliomas... it cannot be concluded that only this type of fibre (crocidolite) is concerned with these tumours. In this regard also, Sluis-Cremer reports a high incidence of mesothelioma in one crocidolite mining area of South Africa (Cape