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Asbestiform Fibers: Nonoccupational Health Risks (1984)

Chapter: 6 Laboratory Studies of the Effects of Asbestiform Fibers

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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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Suggested Citation:"6 Laboratory Studies of the Effects of Asbestiform Fibers." National Research Council. 1984. Asbestiform Fibers: Nonoccupational Health Risks. Washington, DC: The National Academies Press. doi: 10.17226/509.
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6 Laboratory Studies of the Effects of Asbestiform Fibers .! This chapter describes experimental studies that have elucidated some biological effects of asbestiform fibers and their interactions with cells. STUDIES IN ANIMALS In humans, inhalation of asbestos is associated with increased risks of lung tumors (bronchogenic carcinoma and peripheral adenocarcinoma), pleural and peritoneal mesotnelloma, 1ncers~al pulmonary fibrosis (asbestosis), pleural thickening, and possibly other tumors, including those of the gastrointestinal tract and kidney. Investigators have induced lung tumors, mesothelioma, and fibrosis after administration of asbestos to animals. This section a''mmarizes the results of toxicological studies in an attempt to determine whether (1) certain physicochemical properties of asbestos are important in the induction of disease and whether (2) other asbestiform fibers exhibit pathogenic potential in animals. Lung Cancer ~. ~_ ~ ~ ~ In the lung, malignant tumors arise from the bronchial or alveolar epithelial Celia and are classified according to their histological features (e.g., squamous cell carcinoma or adenocarcinoma; Small or large cell carcinoma). Although small numbers of these types of tumors appear in rats after inhalation (Appendix F. Table F-1) or intratracheal instillation of asbestos and chemical carcinogens (Appendix F. Table F-2), benign (papilloma, adenoma) and malignant (fibrosarcoma) tumors uncommon to humans occur more frequently. In general, results are difficult to evaluate because of different experimental protocols (e.g., Amounts of dusts, exposure regimens, different species or strains). For example, in inhalation studies conducted by Davis et al. (1978, 1982), Wagner et al. (1974, 1982a), and Bozelka et al. (1983) asbestos concentrations of approximately lO mg/m3 of air were used, whereas Reeves and colleagues (1971, 1974, 1976) used concentrations approximately fivefold higher. 165

166 Another shortcoming is the lack.of dose-response information at various concentrations of asbestos. With the exception of Davis et al. (1978) and Lee et al. (1981), all in~restigators have used orgy one concentration of dust. The different size distributions of fibers in these studies also present problems to those attempting to compare the pathogenic potential of different types of asbestos. For example, inhalation studies by DaVi8 et al. (1978) (Appendix £, Table Fit) demonstrated that clouds of ZErysotile contain many more fibers longer than 20 Am than are found in aerosolized amphiboles. This phenomenon could account for the greater tumor incidence observed after exposure to chrysotile in these experiments. However, despite these general limitations in interpretation, data in Appendix F. Tables F-l and F-2 support the for owing conclusions: I. The development of lung cancers in rodents after inhalation of asbestos is specie~-specific. For example, rats and mice develop both benign and malignant neoplasms, whereas hamsters, guinea pigs, and rabbits develop only benign neoplasms (Botham and Holt, 1972a,b; Gardner, 1942; Gross et al., 1967; Reeves et al., 1974; Vorwald et al., 1951; Wagner, 1963~. In the few studies conducted in cats (Vorwald et al., 1951) and monkeys (Wagaer, 1963; Webster, 1970), fibrosis but no tumors developed. Thus, cats and monkeys seem to be inappropriate animal models for studies of asbestos-liDked carcinogenesis. 2. Small numbers of benign and malignant lung tumors have been observed after inhalation of all types of asbestos (Appendix F. Table F-1). The short lifespan of rodents may not allow sufficient time for development of larger numbers of tumors. 3. A striking increase in the number of neoplasm has been observed after rodents were exposed by instillation to a combination of asbestos and chemical carcinogens such as polycyclic aromatic hydrocarbons (PAHs). Thus, asbestos appears to act synergistically with PAN to induce lung tumors (Appendix F. Table F-2~. 4. For unexplained reasons, a synergistic effect has not been observed in rats exposed to both asbestos and cigarette smoke by inhalation (Shabad et al., 1974; Wehner et al., 1975~; however, development of fibrosis in these ~nima\B leads to reduced lifespan. 5. When asbestos is inhaled by rats simultaneously with the intratracheal instillation of sodium hydroxide, a caustic agent affecting mucociliary clearance, an increase in the number of tumors has been observed (Gross et al., 1967~. The sodium hydroslde pres''-=bly leads to a greater retention of asbestos in the respiratory tract. 6. Several types of man-made mineral fibers (MMMFa) have been evaluated in inhalation studies. Among the fibers studied are fibrous glass (Gross 1974; Gross et al., 1970; Lee et al., 1981; Moorman, in press; Schepers and Delahant, 1955; Schepers et al., 1958) and insulation (Morrison et al., 1981~; alumina (Piggott et al., 1981~; glass wool, rock

167 wool, and glass microfibers (Wagner et al., 1982a); ceramic aluminum silicate glass (CASG) (Davis et al., 1982~; and potassium titanate, i.e., Fybex and pigmentary potassium t~tanate (PKT) (Lee et al., l9Bl). In comparison to the various types of asbestos included as positive controls in many of these studies, these fibers are generally less carcinogenic. Tumors have been reported after exposure to CASG (Davis et al., 1982~. Results with fibrous glass vary (see footnote to Table F-1 in Appendix F.). Lee et al. (1981) produced two malignant lung tumors in hamsters exposed to potassium octatitanate fibers and bronchogenic tumors in rats with use of fine fiber glass. No malignant tumors were produced in hamsters, rats, or guinea pigs inhaling ball-milled fiberglass. Wagner et _ . (1982a) reported Small numbers of lung cancers in rats exposed to glass wool, rock wool, and glass microfibers by inhalation and no cancers in control animals, whereas in comparable studies, McConnell _ al. (1982) did not detect such an increase. 7. In comparison to amosite asbestos, more bronchogenic tumors developed in rats after intratracheal instillation of ferroactinolite--an unusual and impure asbestiform fiber (Coffin et al., 1982; Cook et al., 1982~. Ferroactinolite appears to undergo longitudinal splitting as a result of dissolution in the respiratory tract, thereby producing an increased dose of extremely thin fibers. When the animals were exposed to nonfibrous grunerite, no carcinomas were observed. Mesothelioma Pleural and peritoneal mesotheliomas originate in the serosal cells lining the body cavities occupied by the lungs and digestive organs, respectively. They occur in small numbers after inhalation of asbestifo~m fibers by animals, but in larger numbers after intrapleural and intraperitoneal injection of the fibers. The injection technique has been used most frequently in reported studies because of its reproducibility. However, injection of fibers bypasses normal lung clearance mechanisms and is criticized as a nonphysiological method of exposure. Table F-3 in Appendix F summarizes the results of experiments in animals injected with asbestiform fibers. From these data and the results of inhalation studies (Table F-1 in Appendix F), the following observations can be made: 1. Mesotheliomas have been observed in rats after injection of all types of asbestos and a number of nonasbestos fibers, including fibrous glass, ceramic fibers, attapulgite (palygorskite) from the USSR, brucite, and erionite. Fewer mesotheliomas occurred in rats after inhalation of UICC chrysotile, amosite, and crocidolite, but none appeared after inhalation of respirable fibrous glass, alumina, and ceramic aluminum - silicate glass fibers. 2. The incidence of fiber-induced mesothelioma in hamsters is lower than in rats and in mice, but tumors appear earlier, perhaps because of the shorter lifespan of the hamster.

168 3. Tumor response was related directly to the dosage of fiber administered in experiments by Smith and Hubert (1974) and by Wagner et al. (1973~. Other investigators (e.g., Pott and Friedrichs, 1972; Stanton and Wrench, 1972) have not observed such dose-response relationships. 4. Exposure to either long (>10 ~m) or short (milled) fibers results in the appearance of mesotheliomas. Nonfibrous particles, including cleavage fragments, do not generally cause tumors (see Appendix F.). 5. Alteration of crocidolite by leaching (a process that can cause fragmentation of fibers) reduces its ability to cause mesothelioma (Monchaux et al., 1981; Morgan et al., 1977), but removal of PAR or trace metals toes not affect the deve Shipment of tumors (Wagner et al., 1973~. 6. In studies to evaluate various injected samples, no one type of asbestos has appeared to be more pathogenic than others (see Appendix F). Wagner (1982) has reported erionite from Oregon readily induces mesotheliomas in rats by inhalation or injection. 7. Inhalation of potassium octatitanate fibers (average size 6.7 x 0.2 ~m) produced three mesotheliomas in 59 hamsters surviving 21 months or more. No such tumors were produced in guinea pigs and rats exposed to these fibers, or in smaller numbers of rats, guinea pigs, and hamsters inhaling amosite asbestos or ballimilled fiberglass (Lee et al., 1981~. Fibrosis _ Asbestos-associated fibrosis is an irreversible disease characterized as an excessive deposition of fibrous tissue. Thin cellular response is thought to occur as a reparative or reactive process. All types of asbestos cause pulmonary fibrosis of the lung (also called pulmonary interstitial fibrosis, or PIF) after relatively long periods of administration (Appendix F. Table F-4~. A range of morphological alterations are observed after exposure to other fibers. The important features of these experiments can be summarized as follows: 1. There are species and strain differences in fibrogenic response. After inhalation of chrysotile by rats, guinea pigs, and mice, granulomas (distinctive focal lesions formed as the result of an inflammatory reaction) and focal fibrosis have been observed in the rat and guinea pig but not in the mouse (Reeves et al., 1974~. Moreover, studies by Lee et _. (1981) show a direct relationship between tosage of fibers and development of fibrosis in the rat, whereas less prominent changes occur in the hamster and guinea pig. 2. Studies with fibrous glass in a diversity of size ranges suggest that responses are minimal in comparison to those induces by asbestos of comparable dimensions. Morphological changes in exposed animals include

169 At, . . 3` mild microphage infiltration without fibrosis (Gross et al., 1970; Moorman, in press), minimal peribronchiolar fibrosis (Kuschner and Wright, 1976), alveolitis (inflammation of the alveoli) (Begin et al., 1982), and alveolar proteinosis (alveolar accumulation of granular proteinaceous material) fine et al., 1979~. No abnormal pathology has been observed with use of fibrous glass in inhalation experiments by Lee _ al. (1981), Schepers (1959), and Schepers et al. (1958~. 3. Inhalation and pleural injection studies implicate the increased fibrogenic potential of longer (~10 ~m) fibers of both asbestos and fibrous glass. Hinimal or no change has been observed after exposure of animals to smaller fibers or particles of chrynotile (Gardner, 1942), other types of UICC asbestos (Vorwald et al., 1951), fibrous glass, arid a Pathetic fluoro~amphibole (Kirchner and Wright, 1976~. 4. Other fibers evaluated in rodents by inhalation include alumina, which produced no fibrosis (Piggott et al., 1981~; PKT and Eybes, which caused fibrosis, but to a lesser extent than amosite (Lee et al., 1981~; and ceramic aluminum silicate glass, which induced alveolar proteinosis (Davis _ al., 1982~. These fibers tended to fall within the same size dimensions as asbestos. 5.^ Davis and Coniami (1973) evaluated pleural fibrosis in mice after injection of the material resulting from heating chrysotile to temperatures exceeding 600°C. Compared with control material, fiber length was reduced and the fibrotic changes in animals diminished. 6. Pleural plaques have not been observed in laboratory animals after exposure to asbestos (Craighead and Mosaman, 1982~. A number of investigators have attempted to induce gastrointestinal tumors in animals by administering oral dosen of asbestos (Gross et al., 1974; Smith et al., 1965, 1980a; Workshop on Ingested Asbestos, 1983~. Thus far, these studies have yielded negative results, as have three National Toxicology Program bioassays (McConnell en al., 1983 a,b). Moreover, asbestos was not found to be cocarcinogenic when administered to rats with azoxymethane-~a documented intestinal carcinogen (Ward et al., 1980~. After being ingested by rodents, asbestos has been observed in mucosal cells of the gut (Westlake et al., 1965~. In guinea pigs, epithelial injury has been observed (Jacobs et al., 1978) but erosion and degenerative changes regressed by 24 hours after administration of an oral 500-mg dose (Sasena et al., 1982~. The acid environment of the stomach and the secretion of mucin by gastrointestinal cells could contribute to the modification of fibers as a result of the leaching or adeorbance of mucin. Moreover, the rat gut may be resistent to asbestos. Studies by Reiss et hi. (1980b) show that asbestos is toxic to intestinal and colonic cells in culture. Lea chiming of chrysotile in ~ N hydrochloric acid ameliorates cytotosicity.

170 IN VITRO SAVVIES Investigators using suspensions of red blood cells (RBCs), monolayers of cells, and organ cultures have contributed to the understanding of the mechaniams by which cytotoxicity and carcinogenicity are induced by asbestiform fibers. Results of these studies have been discussed in various proceedings and reviews (e.g. , Brown et al. , 1980; Harington et al., 1975; Mossman and Craighead, 1981; Mossman et al., 1983a). The types of studies and experimental information of most importance In elucidating the interactions of fibers with cells are summarized below. Hemolytic Assays The mechanisms of particle-induced cytotoxicity are complex. A critical part of this process appears to be the ability of particles and fibers to bind to ant damage cellular membranes (Chamberlain et al., 1982; Craighead et al., 1980; Harington et al., 1971, 1975; Jaurand et al., 1979a, 1980; Woodworth et al., 1982~ The disruption of the membranes can result in hemolysza, which is the leakage of hemoglobin from the RBCs. Hemolysis can be quantified spectrophotometrically and often is used to define: · mechanisms of membrane damage by particles (Chamberlain et al., 1982; Craighead et al., 1980; Desai et al., 1975; Jaurand et al., 1979a, 1980; Harington et al., 1971, 1975; Light and Wei, 1977a,b; Manyai et al., 1969; Zitting and Skytta, 1979) and · the fibrogenic potential of particles (Hefner and Gehring, 1915~. The degree of hemolysis by particles, however, does not correlate directly with their fibrogenic or carcinogenic effects. The hemolytic activity of fibers relates to physicochemical properties such as size (Schnitzer and Pundsack, 1970), magnesium content (Harington et al., 1971), crystallinity (Palekar et al., 1979; Zitting and skyeta, 1979), and surface charge (Harington et al., 1971, 1975; Light and Wei, 1977a,b?. A large surface area also facilitates interactions with the RBC membrane. For example, hemolysi~ is enhanced as the number of fibers (Desai and Richards, 1978; Schnitzer and Pundeack, 1970) and the degree of their dispersion (Schnitzer and Pundsack, 1970) increase. Particle shape is not critical since certain fibers, such as crocidolite asbestos and fibrous glass, or particles with sharp edges, such as Carborundum, are only marginally hemolyeic (Harington et al., 1975~. Moreover, some nonfibrous particles, such as montmorillonite, are as hemolytic as chrysotite asbestos (Woodworth et _., 1982). The importance of surface charge in hemolysis by chrysotile and crocidolite has been suggested by Light and Wei (1977a,b). Other investigators have reported that chrysotile is both hemoly~cic and

4 I 171 cytotoxic in a variety of cell systems, whereas crocidolite is relatively inactive (Chamberlain and Brown, 1978; Miller and Harington, 1972; Mossman et al., 1980b; Woodworth et al., 1982~. The magnitude of the surface charge on the fibers (e.g., chrysotile, +44.5 mV, and crocidolite, -43. 5 my, in distilled water, as measured by their zeta potential) 1 is directly related to their hemolytic potential. For example, when chrysotile fibers are leached in acid, their zeta potential decreases as does their hemolytic activity. In contrast, the hemolytic ability of crocidolite increases after leaching as the fibers become more negatively charged. Adsorption of components of surfactant or serum to fibers also renders them less hemolytic (Craighead et al., 1980; Desai and Richards, 1978; Jaurand et al., 1979a; Light and Wei, 1977a). Harington et al. (1971, 1975) hypothesized that negatively charged residues of 8 ialic acid from membrane glycoproteins bind to Poe it ively charged sites on minerals. This ionic interaction might result in the aggregation of integral membrane proteins and leakage of hemoglobin. To test this hypothesis, sialic acid was removed enzymatically from RBCs before hemolysis by chrysotile was measured. The treated RBCs were resistant to hemolysis, suggesting the importance of sialic acid in this process. Experiments with tracheobronchial epithelial cells have suggested that chrysotile also interacts with other carbohydrates on the cell surface (Mossman et al., 1983c). Cytotoxic ity Studies Cytotoxicity is the ability of an agent to interfere with cellular function to the extent that the cell is either damaged or killed. Because asbestos and other particles are believed to play a role in the pathogenesis of respiratory tract diseases, the mechanisms whereby these substances induce cytotoxicity have been investigated in cell culture (Chamberlain et al., 1982; Harington et al., 1975; Mossman et al., 1983a). Although some researchers have attempted to correlate the cy~cotoxicity of fibers with their ability to cause pulmonary fibrosis and mesothelioma (Hefner and Gehring, 1975; Kaw et al., 1982; Wade en al., 1980), the validity of this correlation is not accepted in general, and one must conclude that cytotoxicity is not related directly to pathogenici~cy. For example, crocidolite is less damaging to red blood cells and lees cytotoxic than chrysotile in macrophages and in cultures of tracheobronchial epithelia (Doll et al., 1982a,b; Haugen et al., 1982; Landesman and Mossman, 1982; Miller and Harington, 1972; Mossman et al., 1980b; Woodworth et al., 1982~. Reiss and colleagues (1980a,b) and Wade et al. (1979) have suggested that sensitivity to asbestos differs among Zeta potential in a measure of surface charge. A zeta potential of zero indicates no measurable surface charge.

172 the various cell types (e.g., epithelial cells, fibroblasts, anal mac rophage ~ ~ . Cytotoxicity in dependent on both the geometry and the length of asbestos fibers. In macrophagelike cells, chrysotile is toxic, whereas its nonfibrous analog, piety serpentine, is not (Frank et al., 1979~. Long fibers of various minerals are more cytotoxic than comparable amounts of short fibers (Beck et al., 1972; Brown et al., 1978; Chamberlain and Brown, 1978; Kaw et al., 1982~. There is overwhelming evidence that short fibers are phagocytized completely, whereas long fibers are only partially enveloped by cells. Uptake of fibers by cells results in the release of lysosomal enzymes (Beck ee al., 1972; Davies et al., 1974) and oxygen free radicals (Mossman and Landesman, 1983), reactive species that cause peroxidation of membranes and damage to macromolecules (Freeman and Crapo, 1982~. Cell injury in tracheal epithelial cell cultures can be prevented by the addition of superoxide dismutase--a scavenger of superoxide (Mossman and Landesman, 1983~. Cell death occurs rapidly when fibers are added to culture media without serum, hut is inhibited or delayed when serum is present (Harington et al., 1975; Mossman et al., 1980b). Serum proteins adsorb to fibers (Desat and Richards, 1978), and this protective coating is believed to be reproved by lysosomal hydrolases after phagocytosis of the particles (Allison, 1971; Heppleston, 1979~. Alterations in Cells of the It ne System After Exposure to Asbestiform Fibers Aberrations of humoral and cellular immunity have been reported in individuals exposed to asbestos (Kagan et al., 1977; Lange, 1980; - Stansfield and Edge, 1974; Turner-Warwick and Parkes, 1973~. These studies suggest activation indoor loss of normal immunoregulatory mechanisms in asbestos-associated diseases. Because macrophages may play a role as an intermediate in pulmonary defense, In vitro studies have been conducted to examine the responses of these cells to asbestos. After exposure to asbestos, macrophages release potent inflammatory factors (Hamilton et 81., 1976) and synthesize prostaglandins (Sirois et al., 1980), chemotact~c factors for neutrophils (Schoenberger et al., 1982), and substances that increase replication of fibroblasts (B~tterman et al., 1981~. Results of other in vitro experiments indicate that asbestos affects both cell mediated (Barbers et al., 1982; Bozelka et al., in press) and antibody-mediated (Lawrence et al., 1982) immunity. In addition, both amphibole and serpentine types of asbestos depress viral induction of inte rfe roe--a g lycoprote in that con fe rs ant ivira 1 de f ense--the reby resulting in increased multiplication of the virus (Hahon and Eckert, 1976) . Conversely, the mineral wollastonite enhances the induct ion of interferon by influenza virus in cultured cells, but the mineral per se

173 toes not induce interferon (Hahon et al., 1980~. Finally, in addition to obvious modulatory effects on ce lle of the immune system, asbestos can activate complement, a complex in serum that is destructive to certain bacteria and cells that have been sensitized with antibody (Hasselbacher, 1979; Saint-Remy and Cole, 1980; Wilson et al., 1977~. Effects on Fibrobla~ts In Vitro Although the microphage appears to be a key cell in the induction of tissue injury by asbestos, the affected cell in the fibrotic process is the fibroblast--a cell associated with the production of collagen. It is not clear whether fibrosis results from increased production of collagen by individual cells, or from an increase in the proliferation of fibroblasts, or from both. Some investigators have suggested that these synthetic responses are elicited by fibrogenic factors released by macrophages (reviewed in Vigliani, 1968~. When noncytolyt ic amounts of asbestos are added to cultures of fibroblasts, abnormal and accelerated production of collagen and ret iculin are observed (Hext and Richards , 1976 ; Richards and Jacoby, 1976~. Fibroblasts undergo a maturation process leading to rapid cellular aging. Surviving cells phagocytize fibers avidly and undergo morphological and biochemical changes such as alterations in secretion of proteoglyeans (Richard and Morris, 1973), metabolism of RNA, and enhancement in cell mat collagen deposition (Hext and Richards, 1976~. Fibrous glass is lens cytotoxic, producing minimal but similar alterations. Long fibrous glans provides a substrate for the attachment of cultured fibroblasts and acts as a stimulus to promote cell division (Maroudas _ al., 1973~. I INITIATION-PROMOTION MODEL OF CMCINOGENESIS . This brief discussion of the initial ion-promotion model of carcinogenesis is provided as a basis for subsequent sections describing possible carcinogenic properties of asbe~tiform fibers. It is possible that cancers induced by asbestiform fibers result from the same fundamental mechanisms as cancers induces by other physical and chemical agents. Carcinogenesis is a complex, multistep process that has been extensively reviewed by Becker (1981), Foults (1969, 1975), Farber (1982), and Farber and Cameron (1980~. Multiple focal proliferations of cells (hyperplasia) in target organs are common early features of carcinogenesi~ and serve as sites for subsequent premalignant changes (Farber, 1982~. Such focal alterations give cell populations selective growth and invasive properties (Cairns, 1975; Fialkow, 1976; Foulds, l9S4; Nowell, 1976~. The concepts of initiation and promotion have been developed to explain this process in ~many experimental models and organ systems, including skin, liver, ; i

174 mammary gland, colon, urinary bladder, and brain (Berenblum, 1941; Boutwell, 1974; Pitot and Sirica, 1980~. Initiation is a change in ache DNA of a cell induced by exposure to a carcinogen. This heritable alteration can be promoted ultimately to mal ignancy . Promotion is the process whereby an initiated cell develops focal proliferations, one or more of which may act as precursors for subsequent steps in the process of carcinogenesis. Promotion creates a mitogenic environment that differentially affects initiated cells. Some investigators suspect that many tissues or organs create a physiological promoting or selecting environment. In the early stages, promotion can be reversible, but it eventually brings about the phenotypic changes needed to stabilize the characteristics exhibited by cancer cells (Trosko and Chang, 1980~. By stimulating a premalignant or initiated cell to proliferate, the process of promotion also enhances the probability that additional genetic errors will occur (Potter, 1981; Trosko and Cnang, 1980). Promotion appears to occur after removal of cells, e.g., as the result of a surgical procedure or the infliction of a wound (Pound and McGuire, 1978~; after physical irradiation (Argyris and Slaga, 1981~; after cell death (Erei, 1976~; or upon exposure to exogenous noncytotoxic chemicals (Trosko and Chang, 1980), endogenou~ chemicals, e.g., hormones (Yager and Yager, 1980), or solid ob Sects, e.g., small plastic squares (Brand, 1982~. Some current views on promotion and promoters have been shaped by studies in which croton oil and its active agent, 12-0-tetradecanoylphorbol-13-acetate (TPA) were used in a mouse skin test sys tem. Both chemical and phys ical tumor promoters have been shown to induce a constellation of biochemical and cellular responses (Diamond et al., 1918) . Depending on the cell type, chemical tumor promoters can induce or inhibit normal differentiation (Diamond et al., 1978) or alter ache proliferation of cells in a given tissue (Yu~pa et al., 1982~. One of the important biochemical markers of cell division is increased synthesis of polyamides, which is often detected as an increase in the activity of ornithine decarboxylase (ODC)--a rate-limiting enzyme in the biosynthesis of polyamides. The induction of ODC is relates directly to tumor-promoting activity of a number of compounds in mouse skin (O' Brien, 1976) . When applied to the skin of rodents, classical tumor promoters (e.g., phorbol esters) also cause inflammatory changes and infil~cra~ ion of polymorphonuclear leukocytes (PMNs) and macrophages. These cells release oxygen free radicals--reactive by-products of oxygen that cause peroxidation of membranes and other macromolecules (McCord and Wang, 1979).

s 175 Detailed reviews of tumor promotion have been prepared by Hecker et _. (1982) and by Slaga et al. (1978~._ Interaction of Asbestiform Fibers with DNA Mutagenicity is the ability of a chemical or physical agent to induce permanent, transmissible changes in the character of a gene by modifying the DNA. This event is believed by many to be an initiating step in the proce s ~ of care inogene si s . Daniel (in press) has prepared a review of In vitro tests that have been conducted with asbestiform fibers to determine their potential for mutagenicity and other types of interaction with DNA. In bacterial assays, such as the Ames Salmonella micro~ome assay, UICC reference samples of asbestos, superfine Canadian fibers, and fibrous glass have not shown mutagenic activity (Chamberlain and Tarmy, 1977; Light and Wei, 1980~. The investigators believe that these negative results may be attributable to the lack of fiber uptake by bacterial cells. Chromosome aberrations and chromatic breaks have been observed after chrysotile and crocidolite asbestos have been addled to rodent cell lines (Lavappa _ al., 1975), but not after the addition of fibrous or powdered glass (Sincock ant Seabright, 1975). Huang (1979) demonstrated that chrysotile, crocidolite, ant amosite are mutagenic to the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus of Chinese hamster lung fibroblasts. However, the rates of induced mutation were low, and the conclusions drawn by Huang are strongly dependent on the method of statistical analysis used in this study. Moreover, neither chrysotile, crocidolite, nor amosite was mutagenic in rodent liver epithelial cells (Reins et al., 1982~. Although amosite and crocidolite produced small increases in sister chromatic exchange (SCE) in Chinese hamster ovary cells (Livingston et al., 1980), the V79-4 Chinese hamster lung cell line and cultured mesothelial cells did not exhibit altered SCE levels after exposure to crocidolite and chrysotile, respectively (Kaplan et al., 1980; Price-Jones et al., 1980~. Furthermore, in tracheal epithelial cells, chrysotile and crocidolite did not cause breakage of DNA, as measured by alkaline elusion (Mossman et al., 19836~. Results of studies by Sincock (1977) and Lechner and colleagues ( 1983) suggest that human cells are relatively resistant to DNA damage by asbestos. Neither crocidolite, SEA chrysotile, nor glass fibers induces chromosome aberrations in human lymphocytes or fibroblasts (Sincock, 1977), although the number of chromatic and chromosome breaks increases in freshly isolated human lymphocytes exposed to Rhodesian chrysot ile (Valerio _ al., 1980~. In another study, UICC chrysoeile, amosite, and crocidol ite Lid not appear to cause DNA strand breakage in human bronchial organ cultures (Lechner et al., 1983~.

176 Tumor Promotion Properties of tumor promoters have been extensively reviewed and discussed (Farber, 1982; Hecker et al., 1982; Slaga et al., 1978~. In studies of cultured tracheal epithelial cells from hamsters, Mossman et al. (1977, 1980a,b, 1983a,b), Moseman and Craighead (1981), and Woodworth et al. (1983a,b) have demonstrated that several different types of asbestiform fibers exhibit the properties of tumor promoters. Both long (>10 ~m) and short ~ <2 um) chrysotile ant crocidolite fibers interact with the membranes of differentiated superficial epithelial cells in organ culture. Short fibers are phagocytized successfully and are observed thereafter in basal epithelial cells (presumably the progenitors of carcinoma); longer asbestos fibers are enveloped by membranes, but appear incapable of being phagocytized (Mossman et al., 1977; Woodworth en al., 1983~. Thin latter phenomenon occurs concomitantly with release of the oxygen free radical, superoxide, into culture medium (llossman and Landesman, 1983~. After exposure to amosite or crocidolite asbestos, there are increases in the incorporation of 3H-.chymidine (an indication of DNA synthesis) and basal cell hyperplasia in tracheal epithelial cells In vitro. The morphological changes were prevented by the addition of vitamin A, which has been associated with the reduction of cancer incidence in a number of studies in rodents (Mosaman et al., 1980a). Enhanced uptake of 3H-thymidine and morphologic changes have also been observed in monolayers of tracheal epithelial cells exposed to either crocidolite or chrysotile (Landeeman and Mossman, 1982~. Some asbestiform fibers have been observed to alter normal epithelial cell function. For example, crocidolite, amosite, and fibrous glass facilitate the progression of basal cell hyperplasia to squamous metaplasia, i.e., the conversion of a differentiated epithelium to a squamous cornified layer resembling skin (Moseman et al., 197B, 1980b; Woodworth et al., 1983a,b). Chry~otile fibers induce significant increases In squamous metaplasia, but high concentrations of long fibers cause permanent destruction of the mucosa. Ground glass, attapulgite, and nonfibrous analogs of asbestos (e.g., riebeckite and antigorite) neither cause squamous metaplasia nor stimulate DNA synthesis (Woodworth _ al., 1983a). Chrysotile, crocidolite, and fibrous glass induce OI)C and stimulate cell division in tracheal epithelial cells in a dosage~dependent fashion (Landeaman and Mossman, 1982), whereas these changes do not occur after exposure to nonfibrous crocidolite (e.g., riebeckite), chrysotile (e.g., aneigorite), glass particles, or hematite (Mossman, personal communication, 1983~. mere experiments suggest that fibrous glass exhibits some promoterlike features In vitro, but experiments exploring this phenomenon in whole animals are lacking.

177 In Vitro Studies with Mesothelial Cells - l~ne interaction of mesothelial cells and asbestos fibers has been studied In vitro to investigate the genesis of mesothelioma (Allison, 1973; Domagala and Koss, 1977; Jaurand et al., 1979c; Ra jan and Evans, 1973; Rajan et al., 1972; reviewed in Whitaker et al., 1982~. Chrysotile asbestos (1-2 Em length) is ingested by cultured mesothelial cells in both organ and monolayer cultures, whereas it is unclear whether larger fibers are phagocytized (Allison, 1973; Jaurand et al., 1979c ~ . After introduction into cultures of pleura, crocidolite causes proliferation of cells in a manner similar to that observed in tracheal organ cultures (Rajan et al., 1972~. Interactions Between Fibers and Polycyclic Aromatic hydrocarbons (PAHs) Because PAHs are incomplete produc to of combustion, they are ubiquitous in the urban environment and are found in association with various types of atmospheric aerosols (Pierce and Katz, 1975~. A number of investigators have explored the possibility that fibers and particles flay act as "carriers" of these HAHN into the cells of the respiratory t rac t . | Equal milligram amounts of crocidolite asbestos, carbon, hematite, and kaolin have been compared for their abil ity to bind and release the radiolabeled PAN, 3~methy~cholanthrene (3MC), into culture medium ? (Mossman and Craighead, 1982~. Asbestos neither adsorbed more 3MC nor eluted greater amounts of the hydrocarbon than did the other materials. However, when tested for release of PAN to artificial membranes or microsomes, asbestos fibers appear to be more effective than the nonfibrous materials tented (Lakowicz and Bevan, 1980 ; Lakowicz et al., 1978a,b) . The association of PAR with the fiber surface before the fibers are added to tracheal epithelial cell culture appears to be critical to f iber-induced ce 1 lular uptake of the hydrocarbon. For example, increased uptake and retention of radiolabeled benzo~a~pyrene (BP) have not been observed with fibrous glass, a poorly adsorpt ive f iber, or when BP is added 1 hour after the addition of asbestos (Eastman et al., 1983; Mos~man _ al., 1983b). CONCLUS IONS Asbestiform Fibers: Initiators and/or Promoters of Lung Tumors? Asbestiform fibers do not seem to damage UNA directly (Fornace, 1982; Hart et al., 1979; Haugen et al., 1982; Moseman et al., 1983b) or to act as mutagens (Chamberlain and Tar - , 1977; Reiss et al., 1982~. mug, the role of asbestos in t'ne initiation of lung tumors is questionable. Some

178 investigators have observed a weak mutagenic reaction (Huang, 1979; Huang et al., 1978) that could also be interpreted as indicating an epigenetic reaction (Isobe et al., 1982~. The term epigenetic is used here to define the alteration of the expression, not the information, of genes. In other words, it is the repression or derepression of genetic information. One possible explanation of asbestos-faciliteeed carcinogenesis is that PAHo (known initiators of carcinogenesis) are more efficiently transferred to the target cells because they adhere to the asbestos fibers (Eastman et al., 1983; Mossman et al., 1983b). Lung tumors generally do not appear after asbestos LS ~ntratracheally instilled into rodents, but they do appear when PAHs are adsorbed to fibers before instillation (Miller et al., 1965; Shabad ee al., 1974; Smith et al., 1968~. However, small numbers of tumors of the respiratory tract have been observed after inhalation of UICC samples of asbestos by rats (Wagner et al., 1974~. The interpretation of these studies is complicated by the finding that these reference standards of asbestos may be contaminated with PAN (Harington, 1962~. The stimulation of cell proliferation by asbestiform fibers may result in the promotion of initiated epithelial cells lining the airways. In support of the hypothesis that asbestos is a tumor promoter, Topping and Nettesheim (1980) have shown that asbestos has a promoting effect in rodent tracheal grafts exposed sequentially to an initiating PAM and then to chrysotile. In these studies, asbestos increased the incidence of tumors obtained with small amounts of the PAH, although the asbestos was not carcinogenic when administered by itself. However, when asbestos was applied to tracheal grafts in tO-fold higher amounts, a low incidence (5~) of squamous cell carcinoma was observed (Topping en al., 1980) . Although this let ter observation might be interpreted as indicating a weak initiating or carcinogenic potential of asbestos, it seems to be a common feature of many promoters (Iversen and Iversen, 1979). Additional evidence that asbestos acts as a promoter is provided by histological observations of hyperplasia and metaplasia in organ cultures of the respiratory tract after exposure to asbestiform fibers (Landesman and Mossman, 1982; Mossman and Craighead, 1974; Grossman et al., 19BOb; Woolworth et al., 1983a,b,c). In addition, the dosage-dependent induction of ODC in tracheal epithelial cells has been seen after addition of chrysotile and crocidolite, but not after exposure to the nonasbestiform particle hematite (Landesman and Mossman, 1982~. The increase in enzyme induction occurs concomitantly with a mitogenic response as measured by uptake of 3H-thymidine. The accumulation of macrophages and inflammatory cells in the air spaces of rodents after inhalation of asbestos appears to be similar to

179 effects observed in the skin after application of phorbol esters (Gee, 1980; Hamilton, 1980~. Isolated macrophages ant polymorphonuclear leukocytes emit oxygen free radicals into medium and are chemiluminescent after exposure to asbestos In vitro (Gaumer et al., 1979~. One might assume that these reactive species are injurious to mucosal airways. Again, the length of the fiber appears to be critical to the cellular response in that addition of superoxide dismutase (an enzyme converting the superoxide radical to H2O2 and O2) to tracheal epithelial cells prevents the membrane damage caused by long (>10 ~m), but not short (c2 ~m), chrysotile fibers (Mossman and Landesman, 1983~. Retiny1 methyl ether, a synthetic vitamin A that blocks the action of at least some promoters (Venma et al., 1982), inhibits asbestos-induced epithelial changes in organ culture of hamster trachea (Mossman et al., 1980a). Taken together, these results are consistent with the hypothesis that certain asbestiform fibers may act in lung cancer in a manner similar to other known chemical and physical agents that have the properties of tumor promoters. Some experiments have suggested that promoters may exhibit a threshold concentration below which they do not exert their j tumor-promoting effects (Peraino ee al., 1980; Venua ant Boutwell, i 1980~. However, there is no experimental evidence of a threshold for carcinogenic effects of asbestos. In Chapter 7, a linear nonthreshold model is used for risk assessment. Initiated cells in the lung could be stimulated to proliferate of ter exposure to asbest if arm f ibers either by a membrane-triggered response or by a cytotoxic response . Fibers lodged in ce 1 18 might act as continuous promoting stimuli. Asbestiform Fibers: Initiators and/or Promoters of Mal ignant Hesothelioma? The pathogene~is and etiology of mesothelioma, a tumor arising from the membranes enclosing the body cavities, differ from those of lung cancer. There is no positive association between smoking and the development of mesothel~oma in asbestos workers (Craighead and Mossman, 1982; Hammond et al., 1979), and prior extraction of PAN from asbestos does not appear to diminish tumor incidence after injection of fibers into the body cavities of rodent n (Wagner et al., 1973~. Studies by Brand and colleagues (Brand, 1982 ; Brand et al ., 1975a , b) and by Davis (1971, 1974a,b) provide some insight into the development of this lesion. After injection into the pleural or peritoneal cavity of rodents, longer fibers cause an immediate but chronic foreign body response, presumably because of their inability to be phagocytized by resident macrophages. Abnormal mesothelial cells with an increased mitotic index have been observed within a thickened fibrotic pleura (Jagatic et al., 1967~. Studies by Davis (1974b) suggest that tumors arise from

180 undifferentiated mesenchymal cell to just below the me~othe1ium. These cells retain their normal pleiomorphic appearance or differentiate into either epithelial-like (mesothelial) cells or spindle-shaped cells. Lois hypothesized origin could explain the histological variability of tumors in humans and animals, since mesotheliomas commonly contain cells of both epithelial and mesenchymal tissue origin. Other investigators have suggested that tumors arise from multipotential mesothelial cells that can express all the variable structural patterns observed in these neoplasms (Maximov, 1927; Stout and Murray, 1942, Whitaker et al., 1982~. Asbestiform Fibers: Possible Mechanisms of Fibrosis , During development of fibrosis (i.e., asbestosis), the normal architecture of the terminal airways and air spaces is altered by excessive deposition of fibrous tissue. Fibrosis can also occur in the pleura. The sequence of ce 1 lular events that appear to trigger the onset of fibrosis has been hypothesized on the basis of observations in animals after inhalation or intratracheal instillation of asbestos. Deposition of fibers in the terminal bronchioles and alveolar ducts--the sites where fibrosis first appears in humans (Craighead and Mossman, 1982--occurs with a rapid infiltration of macrophages and an acute inflammatory response (Gee, 1980) . There observations suggest that asbestos and possibly other s~bestiform fibers disrupt the noneal proliferation and differentiation of lung fibroblasts either by direct interaction of the fibers wish fibroblas~cs or via effec ts on an intermediary cell type, the macrophage (or by both mechanisms). Again, the observations summarized above are similar to those reported for the mouse skin after exposure to chemical tumor promoters: infiltration of macrophages is observed, and hyperplasia and/or abnormal differentiation occurs in some cell types (Yuspa _ al., 1982~. SUGARY Elucidation of the pathogenicity and mechanisms of asbestiform fiber- induced disease is complicated by the complexity, diversity of sizes, and variety of these materials. The experimental results described above served as a basis for Table 6-1, which summarizes the relationships between properties of fibers, their effects on cells, and diseases assoc fated with asbestos . Fibers greater than approximate ly 3 Am in diameter are not respirable; they do not gain access to the respiratory tract but may be ingested.

181 IABLE 6-~. Possible Mechanisms of Disease Induction by Fibrous Materials at the Cellular Level Re levant Diseases Fiber Propertyb A 1, 2, 6 A, C, D 1-7 A, C 1, 2, 5 A, B. (is n 1, 2, 3 A, B. C, 1) 1, 2, 3 A, B. C, D l ~ 3, 7 ! bThe numbers ! properties: 1 Effect on Target Cells and/or Mac rophage 8 Adsorbance and transfer of polycyclic aromas ic hydrocarbons (PAHs ~ to ce 11 membrane 8 Disruption of cell membranes (i.e., lysis and hemolysis) and release of cell enzymes Release of oxygen free radicals Induction of proliferative alterations (e . g ., in DNA, RNA, or prose in synthe ~ i s B. C 1, 2, 5, 7 Al te rat ions in ce l 1 d i f ferent fat ion Interaction with DNA (e.g., chromosomal changes or alteration in normal DNA repair) Effects on immune system (e.g., activation of complement or chemotactic factors) aThe letters in this column represent diseases associated with exposure to f ibrou~ materials: A = Lung cancer B = Mesothe l ioma C= D = Fibrotic lung disease Gastrointe~tional tumors in this column represent the biologically relevant fiber 1 = Respirability (<3 Am diameter) 2 = Size and aspect ratio 3 ~ Durability 4 5 Flexibility and tensile strength 5 a Chemical composit ion 6 a Surface area 7 = Surface charge

182 Although data from the ma jority of investigators show an increased risk of mesothelioma with long, thin fibers in comparison to short, thick fibers, there does not appear to be a critical length below which fibers have no carcinogenic potential. For example, studies by Kolev (1982) and by Pott and colleagues (1972, 1976) suggest that amorphous asbestos and fibers shorter than 5 Em are capable of inducing mesothelioma in rodents. The results of inhalation experiments to determine the importance of aspect ratio in inducing lung cancer are difficult to interpret because the fibers in the aerosols are heterogeneous with regard to size. Irene specific effects of long versus short asbestos fibers of one type have not been evaluated in comparative experiments. Although studies by Gardner (1942), Vorwaid et al. (1951), and Kuschner and Wright (1976) suggest that asbestiform fibers longer than approximately 10 Am are more active than shorter ones in inducing pulmonary fibrosis, not all long fibers (e.g., Saffil, fibrous glass) are fibrogenic. Moreover, some nonfibrous minerals (kaolin, silica) are fibrogenic in humans (Heppleston, 1979~. Asbestos fibers may fragment longitudinally during processing or within the lung and thus increase in both number and surface area. This property may enable more interaction of fibers with cells. Since direct cell contact appears essential to asbestiform fiber-induced diseases, the greater the surface area, the greater the likely pathogenic potential of a fiber. Furthermore, if polycyclic aromatic hydrocarbons (PAHa) adsorb to fibers as a function of surface area, fibers comprised of many fibrils (assuming all were accessible to the PAH) would present a greater surface for adsorption than would a single fiber. Insider these circumstances, their (co~carcinogenic ability might be increased. Longer fibers ~ >ca 10 um), which tend to be more pathogenic, cannot be removed effectively by phagocytic macrophages. Thus, their time of residence in the respiratory tract might be greater than that of shorter fibers. Moreover, lodger fibers appear to be more cytolytic than the shorter fibers, which can be phagocytized completely. Durability is another factor that could account for prolonged retention of asbestos within the lung and other tissues in comparison with a variety of other asbestiform materials. Although leaching may alter the composition of the fiber, asbestos does not tend to dissolve as does glass. It is unclear whether the chemistry of asbestos plays a direct role in pathogenicity. However, since chemistry determines both durability and surface charge, the latter a feature directly related to cytotoxicity, chemistry may play at least an indirect role. Both inhalation and _ vitro studies indicate that asbestos is more pathogenic than a number of man-made mineral fibers (fibrous glass, glass wool, rock wool). However, they fail to identify one type of asbestos as more potent than others. Moreover, there have been few

183 experiments to identify precise dose-response relationships. One obvious conclusion is that there is interapecies variability in response to asbestos. Thus, certain rodents, such as rats and mice, appear to be appropriate models for the study of carcinogenesis and fibrosis, whereas others (e.g., guinea plge) have developed no obvious pathological effect from exposures to asbestos. Different cultured cell types (e.g., epithelial mesothelial, fibroblastic cells) also differ in their susceptibility to the toxic effects of asbestos. Unfortunately, no in vitro model studied to date has been predictive of the fibrogenic or carcinogenic potential of fibers. However, in vitro systems have been helpful in elucidating possible mechanisms of action of asbestos. The evidence that asbestos may act as a gene or chromosomal mutagen is weak and inconclusive, but its ability to function as a tumor promoter at noncyto~ytic amounts and as a cytologic agent at higher levels is well documented. RECOMMENDATIONS To increase our understanding of the health hazards of asbestlform fibers, a necessary first step is to Study the common physical properties of these fibers in relation to their pathogenicity in animals and ability to injure cells. A number of experiments are needed to relate the physicochemical features to biological effects. · In vitro and inhalation studies should be conducted to test whether the biological effects of asbestiform fibers are related to their size and shape. These studies should include as controls appropriate nonfibrous analogs of similar or identical chemical composition. "Positive" responses with nonfibrous analogs would be evidence to support the view that the chemical composition of asbestos is important in the development of disease. All preparations should contain fibers of comparable and Despicable size. · Investigators comparing the pathogenic potential of various fibers and particles should define completely the characteristics chemical constitution, surface charge, crystallization habit, crystallography, and geometry) of their source materials. Different preparations of fibers should be sized to obtain comparable size distributions, thus controlling for this important variable. · Where possible, concentrations of particles should be documented in all experimental systems. There are also gaps in knowledge about such important subjects as the basic molecular mechanisms by which asbestiform fibers induce cell killing, alter differentiation, or cause gene and chromosomal mutations in various cells. The following experiments would be advantageous:

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Much of the more than 30 million tons of asbestos used in the United States since 1900 is still present as insulation in offices and schools, as vinyl-asbestos flooring in homes, and in other common products. This volume presents a comprehensive evaluation of the relation of these fibers to specific diseases and the extent of nonoccupational risks associated with them. It covers sources of asbestiform fibers, properties of the fibers, and carcinogenic and fibrogenic risks they pose.

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