Penetration of cells by asbestos fibers.

Studies on the behavior of asbestos fibers within tissues have shown that the only cells that regularly contain asbestos are macrophages and their derivatives. However, these cells actively incorporate the asbestos fibers by the process of phagocytosis, and there is little evidence of direct penetration. Examination of the gut lining after prolonged asbestos ingestion has shown no evidence of dust penetration either through or between the epithelial cells. The structure and arrangement of these cells is discussed, and it is suggested that they are exceptionally well adapted to prevent penetration by any solid material.

Some observations on the in vitro cytotoxicity of chrysotile prepared by the wet dispersion process.

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.

Oxidant production by control and inflammatory bronchoalveolar leukocyte populations treated with mineral dusts in vitro.

Using a rat model we set out to determine whether exposure of bronchoalveolar-derived leukocytes to pathogenic mineral dusts in vitro caused them to undergo an oxidative burst and release potentially harmful oxidants. Three different populations, obtained by bronchoalveolar lavage, were chosen: control cells, cells obtained following instillation of heat-killed Corynebacterium parvum into the lung, and cells obtained following instillation of quartz. None of the populations showed any evidence of superoxide anion or hydrogen peroxide production when treated in vitro with the pathogenic dusts quartz and chrysotile asbestos, or the inert particulate titanium dioxide. Zymosan caused modest release of superoxide anion with all three populations, indicating that a respiratory burst was being provoked, while PMA, a soluble inducer of leukocyte oxidative burst, caused large-scale production of both oxidants. Preopsonization of mineral dust in rat serum did not render them capable of provoking an oxidative burst from lung-derived leukocytes.

Increased release of hydrogen peroxide and superoxide anion from asbestos-primed macrophages. Effect of hydrogen peroxide on the functional activity of alpha 1-protease inhibitor.

The ability of asbestos-elicited murine peritoneal macrophages to release superoxide anion and hydrogen peroxide, following in vitro triggering, has been investigated. The asbestos-elicited macrophages produced increased levels of superoxide and hydrogen peroxide compared to control macrophages and similar levels to those produced by Corynebacterium parvum elicited macrophages. The supernatants from asbestos-elicited macrophages which had been triggered in vitro were capable of impairing the ability of alpha 1-protease inhibitor to inhibit elastase function. The catalase sensitivity of this effect showed it to be due to hydrogen peroxide.

The effect of products from bronchoalveolar-derived neutrophils on oxidant production and phagocytic activity of alveolar macrophages.

Neutrophils and alveolar macrophages are found together in the alveolar region during pulmonary inflammation where neutrophil products could influence important macrophage defensive functions. We set out therefore to investigate the ability of neutrophil products to modulate alveolar macrophage phagocytosis and oxidant production. Neutrophils derived from acutely inflamed rat lung were incubated along with a range of potential triggers of neutrophil secretion and supernatants collected. Using two quantitative assays of rat alveolar macrophage phagocytosis, the supernatants were found to have no effect except for the quartz supernatant, which slightly enhanced phagocytosis via non-specific receptors and the PMA supernatant, which caused reduction in phagocytosis via non-specific and Fc receptors; this reduction could however be mimicked by PMA alone. None of the supernatants affected macrophage production of superoxide anion or hydrogen peroxide except for the PMA supernatant and once again the inhibitory effect of the PMA supernatant could be mimicked with PMA alone. It is concluded that products of inflammatory neutrophils do not affect phagocytosis or oxidative metabolism of alveolar macrophages, although in quartz-exposed lung neutrophils may exert a small enhancing effect on macrophage phagocytosis.

Immunomodulatory effects of mineral dust. I. Effects of intraperitoneal dust inoculation on splenic lymphocyte function and humoral immune responses in vivo.

Syngeneic PVG rats and C57BL6 mice were inoculated with liquid suspensions of UICC chrysotile A asbestos, DQ12 quartz or the inert particulate TiO2. Rats received 10 mg and mice received 2.5 mg dust intraperitoneally on day 0. Injection of the pathogenic dusts asbestos and quartz was associated with a significant reduction in mitogenic response to PHA and Con A detected in splenocytes removed from animals 14 days after inoculation. Injection of TiO2 had no significant effect on splenocyte mitogenesis. Pretreatment of C57BL6 mice with asbestos intraperitoneally also impaired subsequent production of antibody to the protein antigen KLH. These data show that intraperitoneal injection of chrysotile A asbestos and DQ12 quartz had a marked effect on generalized T lymphocyte function. An additional immunomodulatory effect of asbestos injection was shown in C57BL6 mice where in vivo humoral immune responses were suppressed.

In vitro fibrinolytic activity and viability of rat alveolar macrophages treated with inflammation generating mineral dusts.

Rat alvolar macrophages demonstrated plasminogen dependent fibrinolysis in vitro which was inhibited to varying degrees by the addition of zymosan, the non-toxic particulate titanium dioxide, and the toxic dusts quartz and chrysotile asbestos. Assessment of viability suggested that the inhibition produced by zymosan and titanium dioxide could be accounted for by cytotoxic effects but in the case of quartz and chrysotile asbestos there was evidence that stimulation of fibrinolysis preceded cell death. Zymosan, which caused no observeable enhancement of alveolar macrophage fibrinolysis was found to markedly stimulate peritoneal macrophage fibrinolysis. The choice of assays of cell function to assess the action of toxic dusts are discussed.

An improved macrophage spreading assay--a simple and effective measure of activation.

The development of a quantitative spreading assay of macrophage activation is described. The assay involved incubation of macrophages on glass coverslips for 1 hour and assessment of cell size using a microscope attached to a microcomputer-assisted digitising system which allowed the diameter of 200 cells to be assessed within 10 minutes. Mouse peritoneal macrophages were used in the development of the assay. Internal consistency of the assay was shown by minimal inter-observer, intra-observer and inter-animal variation. Validation of the assay as a measure of macrophage activation was confirmed by the use of in vivo and in vitro activating agents. Once validated the assay was used to detect activation in alveolar macrophages from rats exposed to airborne asbestos. The macrophage spreading assay described here is quick, reliable, consistent and easy to perform and has a potentially wide application in studies of macrophage function and dysfunction.

Fibrinolysis by rat mesothelial cells in vitro: the effect of mineral dusts at non-toxic doses.

Rat mesothelial cell cultures were shown to have considerable plasminogen dependent and independent fibrinolytic activity in vitro using an 125I fibrin degradation assay. At non-toxic doses of the mineral dusts titanium dioxide, quartz and crocidolite asbestos, as assessed by 51Cr release, the fibrinolytic activity of mesothelial cells was inhibited. Quartz had the greatest inhibitory effect and crocidolite asbestos had the least. These results suggests that inhibition of mesothelial cell fibrinolysis does not, on its own, explain pleural fibrosis due to toxic mineral dusts.

The ability of inflammatory bronchoalveolar leucocyte populations elicited with microbes or mineral dust to injure alveolar epithelial cells and degrade extracellular matrix in vitro.

Inflammatory cells are recruited to the parenchyma of the lung in a range of conditions where they are considered to have the ability to exert damaging effects on elements of the alveolus. The injurious effects of rat bronchoalveolar-derived inflammatory cells on an alveolar Type II epithelial cell line were therefore assessed. Inflammatory populations produced by intratracheal injection of Corynebacterium parvum or quartz caused non-lethal detachment injury to the epithelial cells on co-culture whereas control bronchoalveolar cells had no effect on epithelial cells. The pathogenic mineral dusts quartz and chrysotile asbestos caused increased detachment injury when added to co-cultures of epithelial cells and bronchoalveolar leucocyte populations; neither titanium dioxide, a control mineral dust, nor zymosan were active in this respect. Detachment injury was particularly marked when quartz was added to co-cultures of epithelial cells and inflammatory bronchoalveolar cells from quartz treated lung. On the basis of anti-protease and anti-oxidant studies, the detachment injury was found to be mediated by protease alone in the case of quartz cells and protease plus oxidant in the case of C. parvum cells. The two inflammatory bronchoalveolar cell populations were found to have increased proteolytic activity, compared to control bronchoalveolar cells, as shown by increased ability to degrade fibronectin, laminin and denatured collagen. Inflammatory bronchoalveolar cells therefore have the potential to attack elements of the septal extracellular matrix as well as to compromise the integrity of the alveolar epithelium.

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.

Experimental lesions in rats corresponding to advanced human asbestosis.

Rats inhaling chrysotile asbestos developed a progressive interstitial fibrosis similar in most respects to human asbestosis. The earliest lesions were focal deposits of fibrous tissue in the walls of respiratory bronchioles and alveolar ducts. Later alveolar septa between adjacent bronchioles became progressively thickened to produce lesions with similarities to human honeycombing. The thickened septa between alveoli or "micro-honeycomb" spaces were mainly surfaced with cuboidal epithelial cells although some spaces lined by ciliated columnar epithelium were also found. Transmission electron microscopy of these advanced lesions showed that the cuboidal epithelial cells retained most of the characteristics of type 2 pneumocytes but that they frequently exhibited apical cytoplasmic blebs normally associated with the apocrine secretion of Clara cells. Columnar cells exhibited all stages from fully cilitated to cells with only an occasional cilium among the normal cell surface microvilli. Alveolar or micro-honeycomb spaces frequently contained clusters of pulmonary macrophages with their surface processes interdigitated but with no signs of fusion to giant cells. At more than 18 months after the end of dust inhalation these macrophages contained no chrysotile asbestos. The basement membranes beneath the epithelial layers of thickened septa were irregular and often convoluted as well as being much thicker than normal. Microscopic deposits of calcification were frequently found within the basement membrane material. Some thickened septa were relatively acellular, consisting mainly of masses of collagen fibrils but others were cellular and contained many macrophages, fibroblasts, plasma cells and mast cells. In these advanced lesions extremely little chrysotile asbestos was found and this was present in two sites only. Some chrysotile, always as individual fibrils and usually of short length, was present among collagen fibrils in areas of fibrosis and some was present within the thickened basement membranes.

Effects of electrostatic charge on the pathogenicity of chrysotile asbestos.

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.

Inflammation generating potential of long and short fibre amosite asbestos 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.

Mesothelioma dose response following intraperitoneal injection of mineral fibres.

The relationship between injected dose and the development of peritoneal mesotheliomas has been examined in rats using the UTCC standard reference samples of chrysotile, crocidolite and amosite as well as a sample of fibrous erionite from Oregon. Doses injected into the peritoneal cavity ranged from 0.005 to 25 mg and with each dust a clear dose response was found. The proportion of animals developing tumours increased with the amount of dust injected while the tumour induction period was reduced. When times to death from mesothelioma were analysed using standard hazard models, erionite was the most carcinogenic dust by mass followed by chrysotile, amosite and crocidolite. The hazard slopes for erionite, chrysotile and crocidolite, over the range of doses examined, were parallel while the slope for amosite was shallower. The relative hazards for the various dust types were also examined with dose expressed as the number of injected fibres in a range of sizes as measured by SEM. No combination of fibre dimensions was found at which the hazard for the four dust types was equal although when dose was expressed as the number of long fibres injected (> 8 microns in length) the hazard slopes for chrysotile, crocidolite and amosite were relatively close. The hazard level of erionite remained well above the other dust types regardless of how the dose was expressed.

An overload hypothesis for pulmonary clearance of UICC amosite fibres inhaled by rats.

Two types of experiments were carried out to examine the effects of deposition and clearance on the accumulation in the lungs of rats of inhaled fibres of UICC amosite. In the first experiment the mass lung burdens of the dust in question were measured as a function of the time at which animals were killed after the cessation of the six week exposure period, and in the second the masses were measured for rats removed from exposure and killed at intervals during the exposure period itself. The experimental conditions were chosen to complement those of earlier work. Taken together with the results of that earlier work, the new results provide the basis for a simple mathematical model of the kinetics of deposition and clearance which appears to account for the major observed trends. Most significantly, there is strong evidence for an overload of clearance at high lung burdens (exceeding about 1500 micrograms/rat), in which a breakdown occurs of the intermediate rate clearance mechanisms (time constants of the order of 12 days). This hypothesis is supported for inhaled asbestos dust, quartz dust, and diesel fume by results obtained elsewhere. Biological explanations for the clearance overload hypothesis are at present speculative, involving discussion of the role of the macrophage in pulmonary clearance. It is believed that the clearance overload hypothesis could have possible consequences for people occupationally exposed to airborne dusts.

Kinetics of deposition and clearance of inhaled mineral dusts during chronic exposure.

New inhalation studies have been carried out with rats exposed to UICC (Union International Contre le Cancer) amosite asbestos, with the main aim of further elucidating the factors the influence the accumulation of dust in the lung during prolonged chronic exposure. The results show that, for exposure times beyond a few weeks, the lung burden rises linearly and does not level off as predicted by simple models based on ideas taken from the 1966 report of the Task Group on Lung Dynamics. Furthermore, the lung burden is found to scale directly in proportion to the exposure concentration in a way that seems to contradict the overload hypothesis stated earlier. Nevertheless, the general pattern exhibited by our results for asbestos is markedly similar to that found elsewhere for rats inhaling diesel fume, leading to the suggestion that it is general (and not specific to fibrous dust); and the hypothesis that, whereas overload of clearance can take place at high lung burdens after exposure has ceased, it is cancelled by the sustained stimulus to clearance mechanisms provided by the continuous challenge of chronic exposure. The linearity of the increase in lung burden is explained in terms of a kinetic model involving sequestration of some inhaled material to parts of the lung where it is difficult to clear. The particular sequestration model favoured is one where, the longer a particle remains in the lung without being cleared, the more likely it will be sequestrated (and therefore less likely cleared). It is believed that such ideas may eventually be useful in forming exposure-dose relations for epidemiology.

The effects of intermittent high asbestos exposure (peak dose levels) on the lungs of rats.

Four groups of rats were treated by inhalation with the UICC preparations of amosite or chrysotile in order to explore the effects of intermittent high dust concentrations (peak dosing). For each of the 2 asbestos types one group of rats was treated for 5 days each week, 7 h a day, for 1 year. Two other groups were treated with amosite or chrysotile at 5 times the previous dose for 1 day each week for 1 year. Results showed that the lung dust levels of both chrysotile or amosite in the lungs of rats after the 12-month inhalation period were similar regardless of whether "peak" or "even" dosing had been used. During the following 6 months, asbestos was cleared from the "peak" chrysotile group more slowly than the "even" chrysotile group but clearance from the "peak" amosite group was faster than that found after "even" dosing with amosite. Levels of early peribronchial fibrosis were generally lower for the "peak" dosing groups than for "even" dosing although levels of interstitial fibrosis were slightly higher following "peak" dosing. The incidence of pulmonary neoplasms did not differ between the "peak"-dosing and "even"-dosing experiments. These findings therefore give no indication that short periods of high dust exposure in an asbestos factory would result in a significantly greater hazard than would be indicated by the raised overall dust counts for the day in question.

Studies on the morphological patterns of asbestos induced mesotheliomas in vivo and in vitro.

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.