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CS National Bureau of Standards Special Publication 5D6. Proceedings of the Workshop on Asbestos: Definitions and Measurement Methods held at NBS, Gaithersburg, MD, July 18-20, 1977. (Issued November 1978) MEASUREMENT OF ASBESTOS RETENTION IN THE HUMAN RESPIRATORY SYSTEM RELATED TO HEALTH EFFECTS J. Bignon, P. Sebastien, and A. Gaudichet Universit€ Paris-Val de Marne - Institut de Recherche sur 1'Environnement Centre Hospitalier Intercommunal 94010 Creteil, France and Laboratoire des Particules Inhal4es Direction Departmentale de l'Action Sanitaire et Sociale 75013 Paris, France Abstract The retention pattern of asbestos fibers in the human respiratory system is related to four mechanisms: penetration into the respiratory tract deposition on the surface of respiratory epithelium, clearance, and intra-tissular translocation of asbestos fibers. Knowledge of such retention pattern for people exposed to asbestos dusts could provide useful information concerning the role of these mechanisms and the pathogenicity of fibers. So, asbestos fibers content has been assessed by light and electron microscopy in different samples from the respiratory tract: sputum, broncho-alveolar washing fluid, lung parenchyma, parietal pleural, and mediastinat lymph nodes from people diversely exposed to asbestos dusts and affected by various asbestos- related diseases. In each sample, asbestos fibers, identified as chrysotile or amphibole, have been counted and measured (length and diameter). It has been shown that asbestos fibers found in sputum and in broncho-alveolar washing fluid by light and electron microscopy were reliable for the assessment of inhaled asbestos fibers in the workplace or in the environment. Analytical data concerning asbestos burden in respiratory tissues can be summarized as follows: - despite the fact that most of the consumed asbestos is of chrysotile type, amphibole was more frequently found in lung parenchyma than chrysotile, in most cases; - most of the fibers retained in lung tissues were less than 0.20 pm in diameter and shorter than 5 pm. The intra-alveolar fibers were shorter (3.3 pm) than fibers found in lung parenchyma (4.9 pm). Fibers encountered in mediastinal lymph nodes were shorter (2.5 pm) and of amphibole type, whereas fibers encountered In parietal pleura were the shortest (2.3 Nm), and thinnest (0.06 pm in diameter) and mostly of chrysotile type. The signification of these data concerning the topographic variation in the fiber type and size are discussed in relationship with adverse health effects, particularly carcinogenesis. Key Words: Asbestos; carcinogenesis; fibers; pathogenicity; respiratory tract. Preceding page blank 95

Introduction The factors relevant to the assessment of public health risks of exposure to asbestos have been recently reviewed in two documents [1,2]1. It is now well documented that exposure to asbestos dust can lead to the development of lung fibrosis, bronchogenic carcinoma, pleural plaques, pleurisy, mesothelioma, gastro-intestinal tumors, and perhaps other unexpected diseases. The most critical point today is the establishment of dose- response relationship. Regarding cancer, adequate data to establish a threshold limit are not yet available. "The existence of a theoretical no-effect level may even be doubted; however, there may exist ; a rac4cT no-e ect level-6eTow wTicF any excess incidence cannot be adequately establ ished . As far as asbestos is concerned, because of the various possibilities of exposure, it is difficult to define retrospectively sharp conditions of exposure. So, the exposure- effect relationships are not very reliable and greater reliance should be put upon biological monitoring. Asbestos metrology in human samples could provide information about the most important questions arising for the assessment of dose-effect relationships and for the subsequent definition of prevention practices: A. Is there any relationship between one or several body-burden parameters at autopsy and the cause of death, sex, age, and possibilities of exposure? The problem is that the latency period of asbestos-induced diseases can be very long (up to 30 or 40 years). As the accumulation of fibers in man occurs in a dynamic way (related to inhalation and clearance mechanisms), only the residue-burden can be investigated at autopsy. Research is needed to establish eventual relationships between autopsy residue- burden and burden at the time of disease onset. B. What is the most suitable external indicator of body-burden during life? Such a contamination indicator, if it exists and if available for monitoring, could be very helpful for the detection or the survey of exposed people. If relationships could be established with related diseases or with any biological test, this kind of survey should be specifically relevant to biological monitoring. C. What is the biological significance of physical and chemical properties of fibers (length, diameter, elemental composition, associated pollutants...) regarding the induction of diseases (particularly tumors)? Recent experimental data using intrapleural implantation [3] or intraperitoneal injection [4] of fibers of different sizes indicated clearly that the size parameters are the most important for inducing cancer and that the most carcinogenic fibers, whatever the chemical composition, are those with diameters of less than 0.5 or 0.25 pm, and length more than 5 or 8 pm [5]. How can information provided by asbestos measurements in human respiratory tissues be correlated with these recent findings? 0. These studies on body-burden correlated to environmental monitoring could lead to more appropriate standards or quality guides for the future, in relation to the prevention of asbestos-related cancers. General Considerations Related to Asbestos Retention A. What Could Be the Definition of Body-Burden for Asbestos. The actual amount of pollutants in humans at any time is called retention. The retention of particles In humans occurs in a dynamic way and reaches an equilibrium level depending on the relative rate constants of deposition and clearance processes. The model of lung retention, based on the ICRP Task Group report [6], is suitable for describing the general scheme of deposition, clearance, penetration, and translocation of fibers in humans, as shown in figure 1. So far, the penetration and retention of asbestos fibers through the gastro-intestinal tract have not been intensively investigated [7]. 1Figures in brackets indicate the literature references at the end of this paper. 96

CS Dust in inspired and expred ai- Nasopharynx cofnpartment 1' Blood Tracheo - bironchial compartment Alveolar compartment ~' ~. Parenchymal , tissue Sputum Gastro- intestinal tract ~ ~ I Pulmonary lymph ~ ~ vessels and node '~s Pleural tissue 4 : Feces Figure 1. General scheme for deposition, clearance, translocation and retention of fibers, derived from the ICRP lung model [6]. (Heavy arrows : deposition; light dotted arrows : clearance pathway; light arrows : translocation pathways.) ' As asbestos measurement in tissues requires a destructive process, the retention of asbestos fibers cannot be controlled continuously. Measurement of asbestos in organs will provide information on asbestos retention at a very definite time: time of death for autopsic material, or time of surgical intervention for biopsic samples. So far, few attempts have been made for monitoring asbestos retention in alive people either by means of external magnetic procedure involving no sampling [8], or by means of relating body- burden to the amount of asbestos in sputum [9,10], in gastric juice [11], and in feces [12]. B. Deposition. Distinction has to be made between the two pathways for human exposure to asbestos: the pulmonary tract (PT) and the gastrointestinal tract (GIT). Timbrell has reviewed the mechanisms by which particles deposit in the respiratory system and has addressed specifically to the problem of fibers deposition [13]. He identified settling, inertial impaction and Brownian diffusion as deposition mechanisms which operate for both compact particles and fibers. In addition, he listed a fourth mechanism, direct interception, which is of little significance for compact particles but which may be of marked importance for fibers. In this view, a model for deposition of fibers in the human respiratory system has been described [14]. The effectiveness of these deposition mechanisms depends on the anatomy of the respiratory tract, the effective aerodynamic diameter of the particles (size, shape, density), and the breathing pattern. Asbestos fibers can also deposit in the gastrointestinal tract (GIT) either directly (because of the presence of asbestos in water, beverages and food) or indirectly (fibers coming from the respiratory airways and being swallowed). So far, there is little or no direct information regarding the way of fiber deposition at the surface of the human GIT. 97 2063104896

It, is obvious that accurate quantitative information on the deposition of asbestos fibers in humans is difficult to be obtained because of clearance and translocation mechanisms occurring simultaneously during lifetime. What we measure in the human body results from all these associated mechanisms! C. Clearance. Fibers which are deposited on the muco-ciliated blanket of the trachea and bronchi move toward the pharynx. The clearance of inhaled particles by this mechanism is believed to be more than 98 percent effective for most deposited particles [6]. However, the direct toxic effect of asbestos on the ciliated cells, as shwon recently [15], must impair the effectiveness of this clearance mechanism. The fibers deposited at the surface of the alveoli are either taken by alveolar macrophages or entrapped within the alveolar lining film. From there, some of them are cleared towards the ciliated airways while others should penetrate the alveolar membrane. The clearance is different according to the type of asbestos; for chrysotile, the clearance is important, since Wagner et al. [16], Morgan et al. [17] found that a large percentage of chrysotile asbestos entering the lungs of rats may be removed from the lungs within 58 days; but we do not know the mechanisms involved. However, most of the cleared fibers must reach the GIT as demonstrated by the study of Evans et al. [18] using inhaled neutron activated asbestos; up to 73 percent of this asbestos was found in the feces within 30 days. Measurements related to clearance in human have been carried out in several kinds of samples: sputum [10,19,20], gastric juice [11], and feces [12]. Generally, the finding of asbestos in such samples was related to past exposure, pulmonary burden or pathological features. The feasibility of using such samples as indicators of body-burden will be discussed later. D. Penetration and Translocation of Asbestos Fibers in the Human Body. Measurements in tissues using the transmission electron microscope (TEM) have revealed the presence of numerous fibers and fibrils far more than was ever imagined when the fiber population was evaluated by light microscopy alone. These findings, occurring even in case of moderate exposure and long elapsed time from last exposure, suggest a very high penetration and retention rate for TEM size fibers. In humans, asbestos fibers have been found by TEM in lung parenchyma by many authors [21,22,23, 24,25,26,27] and also in bronchial tissue, lymph nodes [28], parietal pleura [25,26], pleural fluid (29,30], peritoneum [31], liver [24], stomach [32,33], bowel walls [34], and colon [353. These findings suggest the penetration of asbestos in the human tissues and their migration throughout the whole body. Experimentally, penetration of fibers across the alveolar epithelium has been described in TEM by Suzuki (36]. The extreme tendency of asbestos fibers to migrate has also been demonstrated experimentally after subcutaneous injection [37], intrapleural or intraperitoneal inoculation [38,39,40], or after ingestion [41]. However, the penetration of ingested fibers through the wall of the gastrointestinal tract is still in discussion. This point is mostly relevant to asbestos-related extra- thoracic cancers, such as peritoneal mesothelioma, ovarian carcinoma, kidney carcinoma, etc. Some authors pointed out that there was no penetration [42]. However, an experiment In progress in our laboratories has shown that ingested chrysotile and crocidolite fibers did cross the intestinal barrier in the rat, being recovered in the lymph of the thoracic duct [43]. 98

Analytical Data Related to Asbestos Body-Burden in Humans A. Sam les Studied as Indicators of Asbestos Body-Burden. ' So far, most of the samples studied in this laboratory for estimating asbestos body- burden in humans were collected from the respiratory tract. We will only focus on data obtained from measurements in 3 kinds of samples: lung washing fluid (LWF) obtained by broncho-alveolar lavage (BAL), sputum collected on alive people, and respiratory tissues (lung parenchyma (LP), parietal pleura (PP), and mediastinal lymph nodes (LN) sampled at autopsy). According to the model shown in figure 1, it has been assumed that asbestos fibers found in LWF were related, on one hand to the intra-alveolarly deposited fraction of inhaled fibers, and on the other hand to the fraction cleared from the deep lung whereas those found in sputum must be related to the fibers cleared from the deep lung and from the tracheo-bronchial compartment [20]. The fibers detected by destroying lung parenchyma correspond to intra-alveolarly deposited fibers and intra-tissularly retained fibers at the time of autopsy. The point is to know if LWF and sputum can be used as external indicators of asbestos body-burden. In this view, a systematic comparative study of fibers encountered in LWF, in sputum and in lung tissue has been carried out and is still in progress. B. Analytical Procedures. For this study, the patients were classified according to their past asbestos exposure. A meticulous history was obtained by questioning each patient in detail about their successive occupations since leaving school. When a history of asbestos exposure was found, the duration of this exposure and the lapse-time since last exposure was recorded (expressed in years). Thus, the degree of exposure was estimated on one hand in terms of its duration and on the other hand according to the type of work done by the patients. All the biological samples were collected within 10 percent formalin. For autopsic lungs, the formalin was injected intratracheally. Pieces of tissue samples were cut and their volume measured. Typically, 1 cc of tissue was prepared for analysis. Each sample to be analyzed was put in a glass vessel containing sodium hypochlorite. This digestive procedure was performed at room temperature during one or two hours. Then, the mixture was directly filtered through a 0.4 pm pore size Nuclepore membrane filter previously coated with a carbon layer. At this stage the filter was scanned under the light microscope looking for ferruginous bodies. For TEM study, a second carbon layer was deposited upon the filter and the particles, entrapped in a double carbon-film, were transferred to TEM grids. The preparations were scanned at X 30,000 direct magnification, looking for fibers. Each fiber encountered was identified on the basis of its morphological features and its electron diffraction pattern and was called chrysotile, amphiboles, or non-asbestos fiber. The length and diameter of each asbestos fiber was measured using a calibrated mark on the viewing screen. For each grid square scanned, the data (number, mineralogical type, and size of fibers) were recorded directly on a computer. Several grid squares were scanned until the variation around the mean calculated for numerical concentrations was less than 30 percent. Concentrations of fibers were expressed in terms of number per sputum, number per total lung washing fluid recovered, and number per cc of tissue. Identification of associated non-fibrous particles has been assessed by means of electron microprobe analysis [44], but quantitative information concerning numerical or mass concentration of such particles has not been obtained. 99 2063104898

An intercomparison study between two laboratories (The University College of Cardiff - F. D. Pooley and Laboratoire des Particules Inhalees, Paris - P. Sebastien) has yielded very similar results concerning the assessment of asbestos fibers in tissues, using the procedure previously described [45]. C. Lung Washing Flui'd (LWF). The possibility of assessing the asbestos endo-alveolar content by means of broncho- alveolar lavage is now under investigation in diversely exposed people. Such a technique has been used by different workers in order to collect free cells and proteins from the human lung [46,47] and it has been shown in the baboon that pulmonary washing was an efficient procedure for the recovery of particles deposited in the alveolar compartment of the lung [48]. 1. Material and Method Up to date, this type of investigative procedure has been used in 26 cases (Table 1). The cases studied were divided in 4 groups: Table 1. Groups of 26 patients investigated by broncho-alveolar lavage. Nb Cases Asb. Exposure Diseases Definite Asb : 9 Group 1 9 Heavy Pl Pl : 5 Br Ca : 1 Definite P1 P1 : 2 Group 2 5 Moderate Silico-Asb : 1 Sm irr op : 1 Chr bronch : 1 Suspected Fibrosis + Group 3 3 Moderate P1 P1 : 1 P1 P1 : I Chr bronch : 1 None Lar Ca : 1 tuberculosis : 1 Controls 9 fibrosis : 1 histiocyt, x : 1 Chr bronch : 5 Abbreviations: Nb - number; Asb = asbestosis; P1 P1 - pleural plaques; Br Ca : bronchogenic carcinoma; Sm irr op • small irregular x-ray opacities; Chr bronch = chronic bronchitis; Lar Ca = larynx carcinoma. 100

CS Group 1 included 9 cases with definite heavy asbestos exposure (OH), subdivided into 7 insulation workers, 1 asbestos-cement worker, and 1 asbestos-textile worker. Lung asbestosis from 0/1 to 2/2 was diagnosed by x-ray according to the IL0 U/C International classification of radiographs of pneumoconiosis 1971. Asbestosis was associated with bronchial carcinoma in one case and with pleural plaques in 5 cases (Table 1). Group 2 included 5 cases with definite moderate asbestos exposure (DM), confirmed by minutious occupational inquiries. The occupation and associated diseases are indicated in Tables 1 and 2. Table 2. Occupations, associated diseases, and mineralogical results in cases of Group 2. (definite moderate exposure) Results LWF in Group 2(definite moderate exposure) Years Years since Nb coated Nb Cases Occupation occupation asb. exp. Diseases fibers fibers % A MOU... Boiler Fitter 10 19 P1 P1 10 + 0 GAN... Glass Blower 27 0 P1 P1 0 0 - MAR... Asbestos 19 11 Silicosis 0 + 50 Plate Cutting ± Asbest. ESS... Plumber with 18 3 Small Irr 0 0 - Welding, Brazing Opacities BOD... Isolation of 3 24 Chronic 0 0 - Central Heating Bronchitis Abbreviations: P1 P1 = pleural plaques; Years occupation = years of occupational exposure; Nb = number; LWF = lung washing fluid; % A= ratio of amphiboles number/ amphiboles number + chrysotile number. (See Table 1 a1so.) Group 3 included 3 cases with suspected (but not proven) moderate asbestos exposure (SM) according to the past occupational history of the patients. The occupation and associated diseases are indicated in Tables I and 3. Table 3 Results LWF in Group 3 (suspected moderate exposure) Years Nb coated Nb Cases Occupation occupation Diseases fibers fibers % A ABD... Autonabile 10 Chronic 0 + 0 Worker Bronchi ti s MON... Wood 10 Fibrosis 0 + 0 Worker + P1 P1 DEC... Plumber 25 P1 P1 0 0 - 'Abbreviations: See Table 2. 101 2063104900

CPS The 9 control cases included patients without specific dust exposure. The method used for broncho-alveolar lavage (BAL) has been extensively described elsewhere [49]. It was assumed that the volume of the lung washed by this procedure corresponded to about one segment. For mineralogical analysis, a 10 mL sample was taken from the whole lavage before the centrifugation was performed for cells recovery. 2. Results No asbestos fibers have been detected by LM and TEM in the LWF of the 9 control cases. Some other no fibrous mineral particles have been encountered in 50 percent of these cases, identified as chlorite, calcite, quartz, aragonite, phlogopite, magnetite, and Al metal. In the group I of heavily exposed patients (Table 4), the mean number of fibers was 12.1x106 per lavage. The mean number of alveolar macrophases (AM) was simultaneously estimated to be 12.6x108 per lavage. However, there was no correlation between the number of fibers and the number of AM. Asbestos fibers were mainly of the amphibole type in insulation or asbestos cement workers. The highest fiber count (50x10 ), only of the amphibole type, was observed in the patient working in an asbestos-cement plant. By contrast, in the case of having worked in an asbestos-textile plant, all the fibers were of the chrysotile type. The percentage of coated fibers was low, less than 1 percent in 7 out of 9 cases. The mean length and diameter were 3.3 and 0.13 pm respectively. Table 4. Mineralogical studies of lung washing fluid (LWF). Results LWF in Group 1(definite heavy exposure) Nb Nb % Mean Mean Exp. Yrs Yrs since A.M. fibers coated length diam Cases type exp. last exp. Diseases 106 106 fibers % A µm um CHA... I 16 2 A 7.6 21 5 100 3.9 0.15 KRE... 1 10 4 A 24.6 5 0.3 100 4.04 0.12 FRA... 1 11 3 A 26.1 6 0.5 100 3.02 0.14 CHE... I 10 11 A + B, CA - 2.4 0.15 100 2.9 0.10 BEN... I 15 4 A - 3.8 0.9 99 3.2 0.15 LAI... I 11 0 A 9.7 11.4 2 90 3.05 0.12 MAA... I 14 3 A+ p1 pl 7.3 7 0.8 100 2.07 0.15 MAR... AC 19 0 A 10.7 50 0.001 100 2.07 0.15 FAL... AT 4 1 A 2.4 3 0.02 0 5.6 - Average 12.2 3.1 12.6 12.1 1 3.3 0.13 ±4 ±3.1 ±8.4 ±14.4 t1.5 ±1 ±0.1 Abbreviations: Exp type = type of exposure; I• insulator; AC = asbestos-cement plant worker; AT a asbestos-textile plant worker; NB A.M. - number of alveolar macrophages per tavage; Nb fibers = number of asbestos fibers per lavage; % A• see Table 2; diam = diameter. 102

CS In the group 1, two parameters, duration of exposure in years and lapse-time since the last exposure, have been assessed and correlated with the fiber count in the LWF. The two curves show that the number deposited within the alveolus increases with duration of exposure, whereas this number decreases when the time since the last exposure increases (figure 2). t + Yrs of exposure • Yrs since the last exposure 5 10 15 20 Yrs I I I I Figure 2. Relationship between fiber count in lung washing fluid and exposure patterns for cases of group 1(definite heavily exposed people). The fiber count increases with the duration (years) of exposure; it decreases when the delay since the last exposure increases. In this group, the fiber yield obtained by BAL and by collecting one sputum has been compared (Table 5). The numbers of coated and uncoated fibers were one or two orders of magnitude higher in LWF than in sputum. Moreover, the fibers were shorter in LWF (mean length 3 pm) than in sputum (5 Nm). Elsewhere, the proportion of amphibole type fibers was less in sputum. - By contrast, in groups 2 and 3, with moderate exposure, the asbestos fiber count in LWF yielded less significant results (Tables 2 and 3). In some cases, both LM and TEM analysis were negative. In others, only a few fibers were found, but at a level not allowing a significant count to be expressed. 103 2063104902

COS Table S. Comparison of asbestos fibers in sputum and lung washing fluid (LWF) from cases of Group 1 (9 cases). Coated 4ibers Uncoated fibers % amphibole type fibers Mean length um Mean diameter um Sputum (one sample) 7.102 1.105 65 5 0.16 LWF (whole lavage) 3.104 5.106 88 3 0.13 In groups 2 and 3, the comparison of asbestos fibers found in sputum and LWF yielded the following results: in many cases, the numerical concentration was low or null; in other cases, one or the other sample showed some fibers. The asbestos content either in sputum or in LWF was similar, within the ranges: 0 to 10 for coated fibers and from not detectable to 5x10s for TEM size fibers, mostly of chrysotile type. D. S utum. It has been demonstrated in this laboratory [9,11] and by others [10] that the amount of coated fibers or ferruginous bodies (FB) in the sputum was significantly related to the asbestos exposure and to the amount of FB in lung parenchyma further measured at the autopsy time [11]. This test is very simple and can be used as a retrospective proof of asbestos exposure, even in the case of long lapse time after the end of exposure. Another advantage is that the coating around the fibers is the evidence that the fibers have stayed in the lung. The study of sputum can also be good in the case of light exposure if the TEM is used. As an example, in this laboratory the sputum has been studied from 45 people working inside buildings insulated with sprayed asbestos containing material. The TEM examination has shown the presence of TEM size asbestos fibers, only of the chrysotile type, in 13 cases (29 percent) (Table 6). The influence of duration of exposure on the presence or not of fibers in sputum has not been demonstrated. Chrysotile fibers were mostly short microfibrils (0.5 to 2 pm long) and forming clumps, probably entrapped in mucus (figure 3). Table 6. Sputum monitoring for asbestos in 45 people working in asbestos-sprayed buildings. TEM study Nb Percent Mean duration of exposure (yrs) Presence of Fibers 13 29 8.3 Absence of Fibers 32 71 8.1 ~ w ~ g 104 8 w