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dust was also sampled on a Whatman QM-A filter and analyzed by emission spectroscopy (ICAP61E™, Thermo Jarrell Ash, Franklin, MA, USA).

2.4. Cytological, histological and ultrastructural examinations

At the end of the experiment, mice were eutha-nized and bronchoalveolar liquids were per-formed with a phosphate buffered salinę solution (pH 7.4). Two washes of 500 //I were carried out. The total number of cells was evaluated after Trypan Blue (0.04% w/v) staining and the nu-cleated cells were recorded in triplicate using a Thoma celi. The pulmonary alveo!ar macro-phages (PAM), the polymorphonucleated cells (PMC) and the lymphocytes (L) were counted on a 100 /il aliąuot of the bronchoalveo!ar liquid which was previously spun in a centrifuge (Cytos-pin 2™, Shandon, Pittsburgh, PA, USA) for 5 min at 1000 x g before staining according to the May Grnwald Giemsa technique (Henderson, 1984; Henderson et al., 1992). The results are the mean of three independent measures.

For histologic observations, lung specimens of control and treated animals were fixed in Bouin’s solution or in 4% (v/v) aqueous formalin solution, embedded in paraffin, sectioned, stained with hematoxylin/eosin and evaluated by adapt-ing the Wagner scalę (McConnell et al., 1984; Fisher et al., 1987) with an Ortoplan™ micro-scope (Leitz, Wetzlar, Germany).

For scanning electron microscopy, the bron-choalveolar liquids were spun as above and the cells were fixed in an aqueous solution of 2.5% glutaraldehyde (v/v) for 1 h, rinsed in 200 mM sodium cacodylate bulfer (pH 7.4), then in a 2% (v/v) osmium tetroxide aqueous solution for 1 h. They were further dehydrated in graded ethanol Solutions before a 10 min incubation in l,l,l,3,3,3-hexamethyldisilazan. They were finally coated with a thin gold layer and examined using a Jeol JSM-840A scanning microscope.

To evaluate the fiber burden in the lungs, the frozen left lobe of three control and treated animals was cleaned in acetone to take the fat off, dried to constant weight and reduced to ashes in a Iow temperaturę oven in 2 h. The ashes were homogenized in ultrapure water and filtered over a carbon-covered Nucleopore™ membranę (25 mm diameter, 0.4 /im porę size). The retained particles were recovered with a second layer of carbon. Under the prepared carbon coated filter, 5 electron microscopy grids were deposited, all rested onto a filtering apparatus. Ten ml of chloroform were filtered through the grids in order to dissolve the Nucleopore™ filters while the eventual fibers were retained on the grids and were observed with the transmission electron microscope (Jeol 100CX) under a magnification of 33000. This technique allowed the fiber nu-meration, the volume determination and with the knowledge of the density (d = 3.35 g/cm³) finally the mass was calculated.

2.5. Tumor necrosis factor determination

The TNF-a biological activities and the bron-choalveolar liquid protein contents, determined according to the Bradford Colorimetry Assay, were measured as previously reported (Martinet et al., 1992). The results are the means of triplicate experiments and the values are related to protein concentrations in bronchoalveolar liquid.

3. Results

The whole fiber size distribution of the bulk crocidolite sample is shown in Fig. 2. The dust mean concentration varied between 10.8 and 16.0 mg/m³ (mean: 13.6 ± 2.1 mg/m³ (n = 8)). The mean fiber number concentration considering the fibers with a length greater than 5 /im and a diameter less than 3 //m was 3594 ± 1142 fibers/ ml (n = 8). The geometrie mean lengths of the aerosol and bulk crocidolite samples were 1.73 (S.D. = 2.12) and 2.22 /tm (S.D. = 2.33) respect-ively. The geometrie mean diameters were 0.14 (S.D. = 1.66) and 0.14 (S.D. = 1.70) for the aerosol and the bulk crocidolite sample respectively, showing that the generation process does not introduce changes in fiber distribution (j* = 7.8, NS). The fiber sampling was always taken at the same chamber port sińce during preliminary ex-periments changing port did not change the value obtained.

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