R E Bolton

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.

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.

Kinetics of the bronchoalveolar leucocyte response in rats during exposure to equal airborne mass concentrations of quartz, chrysotile asbestos, or titanium dioxide.

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.

Increased release of hydrogen peroxide and superoxide anion from asbestos-primed macrophages

The ability of asbestos-elicited murine peritoneal macrophages to release superoxide anion and hydrogen peroxide, followingin vitro triggering, has been investigated. The asbestos-elicited macrophages produced increased levels of super oxide and hydrogen peroxide compared to control macrophages and similar levels to those produced byCorynebacterium parvum elicited macrophages. The supernatants from asbestos-elicited macrophages which had been triggered invitro were capable of impairing the ability of α1protease inhibitor to inhibit elastase function. The catalase sensitivity of this effect showed it to be due to hydrogen peroxide.

ASSESSMENT OF HUMAN RISKS FROM EXPOSURE TO LOW TOXICITY OCCUPATIONAL DUSTS

Recent animal studies have demonstrated three separate and distinct mechanisms by which low toxicity dusts can cause important chronic pulmonary effects; through overloading of clearance mechanisms, through increased toxicity associated with ultrafine particle size or by increasing the toxicity of known carcinogens in mixed exposures. The problem to be addressed is how the pathogenicity to man of various airborne dusts should be evaluated, when epidemiological evidence is often insufficient, and the reliability of extrapolation of quantitative risks from animals to man is not established. In this paper we examine the feasibility of evaluating the likely human risks of low toxicity dusts by: (1) semi-quantitative comparisons of the ability of various dusts, in animal studies, to cause overload of clearance and resulting inflammation and fibrosis; (2) postulating that these relativities apply quantitatively to human risks; and (3) estimating approximate human risks by comparisons with reference dusts for which adequate animal and human data are available. Such a decision-making framework appears feasible, provided: (1) comparable and quantitative methods are used consistently in animal studies for the measurement of impairment of clearance leading to overload and resulting inflammation and fibrosis; (2) the quantitative relationships between impairment of clearance leading to overload, and resulting inflammation and fibrosis, can be defined adequately in animals for various dusts; (3) the particle size distributions, including those in the ultrafine range, for dusts to which animals and/or humans are exposed, are taken into account (or are comparable); (4) at least two reference dusts with well-documented activities spanning the range of toxicity can be identified; and (5) the reliability of the predictions of human pathogenicity of a sample of other dusts is tested, in toxicological studies and by observation in humans. Some possible candidate reference and test dusts are identified.

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.

Pulmonary clearance of UICC amosite fibres inhaled by rats during chronic exposure at low concentration.

Clearance of UICC amosite asbestos from the lungs during chronic--that is, repeated--exposure was investigated by using the scanning electron microscope to measure lung burdens from rats which had inhaled amosite asbestos at an approximately constant concentration of 0.1 mg/m3 or, equivalently, 20 fibres/ml for seven hours a day, five days a week for up to 18 months. The lung burdens were compared with previous results for higher exposure concentrations of 1 and 10 mg/m3. Those previous lung burdens had been measured using other analytical methods (infrared spectrophotometry) that were not suitable for the new lower lung burdens. Taken together, these results showed lung burdens rising pro rata with exposure concentration and exposure time. This accumulation of lung burden has been described by a kinetic model that takes account of the sequestration of material at locations in the lung from where it cannot be cleared. Unlike some earlier models in which lung burdens eventually reach a plateau with equilibrium between deposition and clearance during chronic exposure, this sequestration model shows lung burdens continuing to rise with exposure time. The latest results reported here support the application of such a model to lower exposure concentrations closer to those of asbestos in workplaces.

Release of Superoxide Anion and Hydrogen Peroxide by Macrophages in Response to Asbestos

The multiple roles of the macrophage in the inflammatory response are well documented (Nathan et al. 1980). In particular the importance of reactive oxygen intermediates (ROI) during inflammatory defence against microbes has been recognized (Klebanoff 1980). However, it has also been suggested that inappropriate ROI release by phagocytes during non microbially induced inflammation may, under some circumstances, prolong and exacerbate tissue damage and inflammation (Fantone and Ward 1982). The possibility that the deposition of pathogenic dusts in tissue could result in just such a build up of potentially toxic ROI has been suggested (Gee and Walker-Smith 1984). Using mouse peritoneal macrophages elicited with asbestos we have found support for this contention in the raised oxidative status of asbestos activated macrophages as measured by chemiluminescence (Donaldson and Cullen 1984). In the present paper we report on the levels of superoxide anion and hydrogen peroxide released by asbestos-activated macrophages. We also describe the effect of hydrogen peroxide and superoxide anion on the functional activity of lymphocytes as an indicator cell population.

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.