Tobacco Documents Online

(CS Figure 3. Electron micrographs showing chrysotile type fibers isolated from sputum in people resident inside asbestos sprayed buildings. E. Respiratory Tissues. 1. Lung Parenchyma Lung parenchyma samples from 27 autopsic cases diversely exposed to asbestos and with different malignancies have been studied by TEM. Four blocks of parenchyma were sampled in different sites of the same lung: central upper lobe, peripheral upper lobe, central lower lobe, and peripheral lower lobe, as described elsewhere [25]. The geometric mean of fiber count in the 4 sites has been calculated and then the cases have been classified in groups according to the asbestos lung burden (Table 7). The proportion of cases having more than 106 fibers/cc of lung parenchyma was 8 out of 10 for the asbestosis + respiratory cancer group, 5 out of 11 for the mesothelioma group, 0 out of 2 for the lung cancer (without associated lung fibrosis) group, and 2 out of 4 for the other malignancies group. 105 2063104904

e Table. 7. Asbestos fibers burden in lung parenchyma according to pathological features. Pathological Fiber concentration in the lung, Nb cm 3 features ~ <106 106 - 107 >107 Total Asbestosis t Respiratory 2 5 3 10 Cancer Mesothelioma 6 3 2 11 Lung Cancer 2 0 0 2 Others Malignancies 2 2 0 4 Total 12 10 5 27 The mineralogical type of fibers encountered in lung parenchyma has been assessed by TEM and the results are expressed in Table 8 by the percentage of amphibole/all asbestos fibers. The parenchyma retention of amphibole type fibers has been found important in most cases, the amphibole proportion increasing with fiber concentration in all pathological groups. Moreover, whatever the fiber concentration in lung parenchyma, the highest mean proportion of amphibole type fibers was observed in the mesothelioma group. Table 8. Mineralogical type of fibers in lung parenchyma: ratio amphiboles/(amphiboles + chrysotile) x 100. Pathological Fiber concentration in the lung, Nb an 3 features <106 106 - 107 >107 Average Asbestosis t Respiratory 38 59 69 58 Cancer Mesothelioma 53 70 89 64 Lung Cancer 4 4 Others Maligancies 12 41 26 Several size parameters have been assessed: mean length, mean diameter, and proportion of fibers longer than 8 pa. The results are shown in Tables 9, 10, and 11 respectively. The main figures are: 1) the size of fibers increases when the concentration increases; 2) the mean diameter never exceeds 0.16 Nm; 3) the mean percentage of fibers longer than 8 pm does not exceed 20.8 percent. 106

.T, Table 9. Size of fibers in lung parenchyma: mean length (um). Pathological Fiber concentration in the lung, Nb cm 3 features <106 106 - 107 >107 Average Asbestosis ± Respiratory 3.7 5.4 5.5 5.1 Cancer Mesothelioma 4.8 5.7 4.1 4.9 Lung Cancer 1 1 Others Maiignancies 2.8 2.3 2.6 Table 10. Size of fibers in lung parenchyma: mean diameter (pm). Pathological Fiber concentration in the lung, Nb cm 3 features <106 106 - 107 >107 Average Asbestosis t Respiratory 0.11 0.13 0.16 0.13 Cancer Mesothelioma 0.09 0.13 0.12 0.11 Lung Cancer 0.05 0.05 Others Malignancies 0.09 0.13 0.11 Table 11. Size of fibers in lung parenchyma: than 8 pm (%). proportion of fibers longer Pathological Fiber concentration in the 1ung, Nb cm 3 features Asbestosis t <106 106 - 107 >107 Average Respiratory Cancer 11.6 20.1 20.8 18.6 Mesothelioma 13.1 20.5 11.4 15 Lung Cancer Others 0.7 0.7 Malignancies 1.6 6.3 4.1 107

2. Asbestos Fiber Parameters A~_ccordin to ~Samp~lin_q Sites in Respiratory Tissues: ar~ enhymaar et~TP leura, ~iastinaT Lymp~i Nes. Besides lung parenchyma samples „ parietal pleura samples were available in 13 cases and mediastinal lymph node.samples in 4 of these cases. The comparison of fiber concentration in lung parenchyma and parietal pleura is indicated on figure 4. The absenceoF-correlation between asbestos fiber content in parenchymal and pleural tissue is emphasized. It is noteworthy that in some mesothelioma cases, even with high concentration inside lung parenchyma, the fiber concentration in the parietal pleura was very low. By contrast, a correlation seemed to appear between the fiber concentration in parietal pleura and in lymph nodes (figure 5). 107 108 10 106 10 f LUNG PARENCHYMA M A AM M M A A 105 104 M M M M M FIBERS IN LUNG AND PLEURA (Nb.cw3) M. Masetheliom. A. Asbsstesis ± Luny Cancer Figure 4. Correlation between asbestos fiber concentration in lung and in parietal pleura (see text for comments). 108

CS A A y W PARIETAL PLEURA 105 106 10 7 A I I I FIBERS IN PLEURA AND LYMPH NODES (Nb.cm3) Figure 5. Correlation between asbestos fiber concentration in parietal pleura and mediastinal lymph nodes. The comparison of mineralogical types has been carried out in the same way. The most striking features were: a) Most of TEM fibers encountered in parietal pleura were of chrysotile type even when the proportion of amphibole/amphibole + chrysotile type fibers was higher than 0.5 in the lung parenchyma (figure 6). b) By contrast, so far in the few cases studied, most of the fibers encountered in lymph nodes were of amphibole type (figure 6). The fiber size has been compared in the different sampling sites (Table 12). The longest ftT ers were found in the lung and the thinnest in the parietal pleura. Mean fiber length was of 4.9 pm for lung parenchyma, 2.3 pm for parietal pleura, and 2.5 pm for lymph nodes. N ~ 109 W f+ ~ ~ 00

COS Ratio (AmPhiielas/Amp hib olms + ChrYsotilo) x 100 100 E-50 A 50 1g0 1~ _ AA A • LUNG PARENCHYMA 100 Dp 0 F-5• 50 100 PARIETAL PLEURA LYMPH NODES Figure 6. Ratio of amphiboles count/total asbestos fibers count in lung parenchyma coopared to the ratio in parietal pleura (top) and to the ratio in lyvph nodes (bottom). 110

CS Table 12. Fiber size in lung parenchyma, parietal pleura, and lymph nodes. Lung parenchyma Parietal pleura Lymph nodes Mean Length um 4.9 2.3 2.5 Mean Diameter um Proportion of Fibers 0.13 0.06 0.16 Longer than 8 um percent 15 2 3 Discussion { The contribution of this metrologic study of asbestos dusts in the human PT is relevant to three major points relating to the pathophysiology of fibrous particles: - It allowed a check of the reliability of monitoring asbestos in sputum and lung washing fluid for the assessment of asbestos exposure. - It provided a better understanding of the partition of fibers in the different compartments of the respiratory system, which allows hypothesis about the translocation of fibers in the PT. - It yielded quantitative data concerning the actual fiber dimensions in humans in different diseases, including pleural mesotheliomata, which have to be discussed in view of recent experiments concerning the mesothelial response in relation to fiber dimension. A. External Indicators of Asbestos Lung Burden. The present work demonstrated that the study of sputum and LWF by LM and TEM was very reliable for the assessment of asbestos exposure in heavily exposed people. The advantage of LWF over sputum is that it yields a greater amount of fibers which are most representative of the alveolarly deposited fraction. This technique, which requires that the patient accept a fiberoptic bronchoscopy, might help to diagnose asbestos-related diseases. However, this possibility has some limitation. Indeed, the information provided by BAL carried out in moderately exposed people was much less reliable than the study of lung parenchyma. This can be easily understood since we will discuss later on that the percentage of intraalveolar fibers is very low compared to the fibers retained in lung parenchyma. However, it seems that LM and TEM study of sputum is an excellent tool for detecting and following exposed people [9,11]. A cytological control of the sputum looking for, AM is needed to be sure that it represents the mineral content of the deep lung. It is possible that the measurement of asbestos fibers in other biological samples could be better indicators of asbestos body-burden, as discussed elsewhere [50]. Thus the search for asbestos fibers in feces appeared to be a very sensitive method, allowing detection of low intake of asbestos fibers [12]. B. Translocation of Asbestos Fibers in the Respiratory System. The figure 7 summarizes all the mean data concerning number, length, and diameter of fibers in four sites of the respiratory system: alveoli, LP, PP and LN. Moreover, the figure 8 gives the distribution of length fibers in these four sites. 111 2063104910

Figure 7. Diagram comparing the mean number (Nb), mean length (L) and mean diameter (d) of asbestos fibers in 4 sites of the respiratory system. Numbers have been estimated for the whole lung for parenchyma (Par) and alveoli (Alv), while they are given per cc of tissue for pleura and lymph nodes (LN). 112

percent 30 20 t0 30 20 10 30 20 10 30 20 10 LUNG PARENCHYMA 1 2 4 6 8 16 32 µm PARIETAL PLEURA LYMPH NODES ALVEOLUS Figure 8. Distribution of fibers length in parenchyma, parietal pleura, lymph nodes and alveoli. Note that long fibers, more than 4{rm in length, are less frequent in pleura, lymph nodes and alveoli than in parenchyma. 113 1

,-- For LP and alveoli, the fiber counts have been integrated for the whole lung, distinguishing intra-alveolar fibers assessed by the BAL and intra-parenchymal fibers assessed by destroying LP. Thus, the fraction corresponding to LP totalizes fibers entrapped in the pulmonary interstitial tissue (plus fibers inside blood vessels?) and fibers within the alveolar compartment. For that estimation, the volume of total lung has been assumed to be 5000 mL and the fraction of alveolar spaces washed by the BAL to be 1/20 of the whole lung volume. Thus, the figure 7 shows that the intra-alveolar fraction of all intra-parenchymal fibers would only represent about 1 percent of all the fibers retained in lung tissue, when assumed that BAL recovered all intra-alveolarly deposited fibers. The mineralogical type of alveolar and interstitial asbestos dusts did not differ significantly, as indicated on one hand by the electron diffraction pattern and on the other hand by the measurement of fiber diameters, identical in both sites (0.13 pm in mean diameter). Elsewhere, it is noteworthy that the intra-alveolar fibers were significantly shorter (3.3 pm in mean length) than the interstitial fibers (4.9 pm in mean length for LP fibers). This difference must even be more important, because the mean length of interstitial fibers is probably reduced by adding the 1 percent of short alveolar fibers to the interstitial fibers when LP is studied; on the other hand, it is possible that the mean length of intra-alveolar fibers is increased by the addition of longer fibers deposited at the surface of the peripheral airways and washed out during the BAL. Indeed, the mean length of fibers in sputum was found to be 5 pm (Table 5). These results clearly indicate a shorter length of fibers inside alveoli compared to pulmonary interstitial tissue. This can be related to two mechanisms, more or less associated (figure 9); either long fibers might penetrate more easily across the alveolar membrane or small fibers are more easily cleared from the interstitial tissue toward the alveolar spaces? As will be discussed, sizing of fibers in pleura and in lymph nodes brings a clue in the favor of the last hypothesis. Indeed, in these two sides (PP and LN), the asbestos fibers were significantly shorter than in lung parenchyma (2.3 pm in PP; 2.5 pm in LN compared to 4.9 pm in LP). These findings are additional clues to the greatest translocation effectiveness of short fibers. The migration of fibers was found even more selective in this study, since mostly chrysotile fibers were found inside the PP, with a mean diameter of 0.06 pm, whereas mostly amphibole type fibers with a mean diameter of 0.16 pm were found in mediatinal LN. This selective migration of fibers might be mostly related to their dimension, as if only short and very thin fibers could be entrapped in the PP tissue (figure 9). C. Fibers Dimension Related to Carcinogenicity. The aforementioned recent animal experiments after implantation of fibers in the pleura [3,5] reinforced the idea that the carcinogenicity of fibers depends only on dimension of fibers, whatever the chemical composition is, in such a way that the probability to induce pleural cancer reaches 100 percent when all the fibers are less than 0.25 Nm in diameter and more than 8 Nm in length (see Stanton et al. , this meeting). In humans, as demonstrated by this work and by others [24,26,57], all the asbestos fibers encountered in different sites of the respiratory system were found to have a diameter less than 0.25 pt. By contrast, the present study has clearly demonstrated that the mean length of fibers was always less than 8 pm in all sites (figure 7). However, a certain percentage of fibers was longer than 8 pm, especially in lung parenchyma (15 percent) (see Table 12 and figure 8). The point is to understand how such few fibers, distant from the parietal pleura, might induce the carcinogenetic transformation of mesothelial cells, or if other mechanisms specific to humans are to be considered. 114