Workshop: Laboratory Models for Chronic Toxicity Testing
THE USE OF ANIMAL MODELS FOR STUDIES ON ASBESTOS BIOEFFECTS J. M. G. Davis
Institute oJ Occupational Medicine Edinburgh, Scotland A frequent criticism of animal experimental studies on disease processes is that extrapolation to the human condition is not justified. While this is probably an extreme view, it is obviously important for an animal model for experimental pathologic studies to duplicate as closely as possible the known human pathologic conditions it is intended to study. The consideration of the best animal model for studies on the pathogenic effects of asbestos dust is facilitated by the fact that this dust produces a number of different disease conditions in human beings. Therefore, the number of parameters for comparison with animal effects is increased, and if an animal model is known to produce results similar to the human condition for more than one type of pathology then this model can be used with added confidence to examine other possible disease effects. In human beings asbestos is known to produce, after inhalation, interstitial pulmonary fibrosis or asbestosis, bronchial carcinomas, and pleural and peritoneal mesotheliomas. In deciding which variety of experimental animal to use for experimental studies with asbestos, it must therefore be considered whether this species can be shown to duplicate these conditions adequately. Most experimental studies of asbestos disease have used rats. This species was included with others in studies by Gardner’ and Vorwald ef a1.’ who reported that long fiber chrysotile produced a “fibrous reaction in the lung” but no details of the histologic picture in this species were given. Later inhalation studies using rats have included Holt, Mills and Young3; Gross and De Treville4; Gross et al.’ and Reeves et a1.6All these authors reported lung fibrosis following the inhalation of various types of asbestos dust, but most of the lesions described were early peribronchial foci of granulation tissue and fibrosis and it was left to Wagner et al.’ to demonstrate that in rats, long periods of asbestos inhalation and subsequent long-term survival result in widespread pulmonary interstitial fibrosis. These results were confirmed by Davis et al.’ The early rat inhalation studies did not result in the development of pulmonary tumors but these were reported by Gross et u I . ~and subsequently in the studies by Reeves, Wagner and Davis. Both benign pulmonary adenomata as well as squamous carcinomata and adenocarcinomata have been shown to develop in this species. Similarly, these inhalation studies resulted in the development of occasional pulmonary mesotheliomas and Davis reported a single peritoneal mesothelioma in a rat that had inhaled chrysotile asbestos. Guinea pigs have been used in a number of experimental inhalation studies with asbestos. These studies include work by Gardner’; Vorwald et al.2;Wagner’; Holt, Mills, and Young”; Gross et aL4; Botham and Holt” and Reeves et a1.6 All these authors reported that guinea pigs developed interstitial pulmonary fibrosis in response to asbestos inhalation, but the development of neither bronchial carcinomas nor mesotheliomas was recorded. Rabbits were included in inhalation studies undertaken by Gardner et a / . ’ ; Vorwald et al.’; Wagner’ and Reeves et a1.6 This species was shown to respond to asbestos inhalation by the development of marked pulmonary fibrosis, but once again no pulmonary tumors or mesotheliomas were caused by the dust.
795 0077-8923/79/0330-0795 $01.75 0 1979. NYAS
796
Annals N e w York Academy of Sciences
Hamsters, gerbils, and mice have all been used in some experimental studies of asbestos inhalation (Gardner’; Vorwald et d.*; Lynch et ~ 1 . ” ;Gross et ~ 7 1 .Reeves ~; et ~ 1and . Wehner ~ et uI.I3).It may be concluded from these studies that hamsters and gerbils respond readily to asbestos inhalation with the development of pulmonary fibrosis. The position with mice is less certain since, while Vorwald and Lynch reported that mice did develop lung fibrosis following the inhalation of chrysotile asbestos, Reeves reported fibrosis in the lungs of mice only after the inhalation of crocidolite or amosite. No pulmonary neoplasms were reported from these studies in any of the three species under consideration. Considering non-rodent species, Vorwald included cats in his inhalation studies, Wagner9 and Webster“ used vervet monkeys, and Webster is at present undertaking an inhalation study using baboons. Pulmonary fibrosis developed in both cats and monkeys but no tumors were found in either species. No reports of the baboon experiments have yet appeared in the literature, but apparently one pleural mesothelioma has been found which developed five years after the start of inhalation (Webster-personal communication). From this combination of results it would appear that only the rat, of those species studied, has been shown to respond to asbestos inhalation in a similar way to humans. Rats develop interstitial pulmonary fibrosis, bronchial carcinomas, and occasional mesotheliomas. It may be that further experimentation would demonstrate that monkeys and especially baboons would give a response to asbestos dust similar to that of human beings, but the high cost of such experiments would preclude use of primates on a large scale. It would appear therefore that the rat represents the best available animal model for asbestos studies and certainly the best model that can be used in all parts of the world in large numbers. In one respect, however, the tissue reaction of the rat to asbestos fibers is dissimilar to that of human beings. In human lungs, asbestos fibers surrounded by macrophages are frequently coated with a mixture of mucopolysaccharide and ferritin or hemosiderin to form asbestos bodies, whereas these bodies occur only very rarely in rats (DavisIs). Guinea pigs, rabbits, hamsters, mice, monkeys, and baboons all produce asbestos bodies and it is uncertain whether or not the inability of the rat to form these structures is a serious disadvantage to its use in studies intended to reproduce human tissue reactions to asbestos. Originally it was suggested that the coating of asbestos bodies protected the surrounding tissues from the damaging effects of asbestos fibers. This may be true for those fibers that do become coated but it is known (Davis and Grosst6)that only a very small proportion of fibers in the appropriate size range ever do form bodies even in human lungs and it seems unlikely that their presence can noticeably affect the course of asbestos-related disease. Having concluded that of all readily available animal species the rat is the best to use for studies on the bioeffects of asbestos fibers, it may be helpful to consider the type of problems which still need to be examined with this type of system. We know that in humans asbestos will cause lung fibrosis and neoplasia but many of the parameters involved are still uncertain. It is essential to determine whether asbestos carcinogenesis exhibits a dose-response effect and whether there is a finite minimum dose of asbestos dust below which tumors will not develop. With mesotheliomas especially it has been suggested that extremely small doses of asbestos may be sufficient to cause these tumors, and some have concluded that the normal use of asbestos products in the urban environment may lead to an epidemic of mesotheliomas in the next few years. Some work has been done using the animal model to explore these possibilities but more is needed. Smith et al.”, Stanton and Wrench,” and Wagner et al.I9 have all shown that following intrapleural injection in rats the number of mesotheliomas produced is progressively reduced with graded doses. However, the lowest dose level used was 0.5 mg and occasional mesotheliomas did develop in this
Davis: Animal Models for Studies on Asbestos Bioeffects
797
study. Of course, 0.5 mg is quite a large dose in comparison with what a human lung could accumulate from the normal urban environment, and it will be necessary to undertake further experiments with lower doses consisting of only a few hundred fibers to determine accurately the real human hazard associated with urban asbestos. Similarly it has been suggested that only asbestos fibers longer than 8-10 p are carcinogenic and a considerable amount of animal experimentation has been undertaken to support this idea (Smith et a/.”; Stanton and Wrench”; and Stanton et a/.’’). It is extremely important to settle this point finally since at present factory dust legislation in most countries is based on counts of the number of fibers over 5 p in length. If only fibers over 8 p are carcinogenic then only these should be included in future dust counting procedures. Recent studies undertaken in Edinburgh have produced further information on both these points (Davis el a[.*).Inhalation studies using rats were undertaken to determine the relative importance of fiber mass and fiber number in the production of pulmonary disease by the U.I.C.C. samples of chrysotile, crocidolite, and amosite. Three dust chambers were used to test clouds of 10 mg/m3 of all three asbestos types, and since the amosite fibers were found to have the greatest volume, the chrysotile and TABLEI Fiber Number Levels of per ml > 5 p Percentage of Interstitial Fibrosis Asbestos Dust Mass/ by Optical fibers >20 p 29 Months after Bronchial m1 of Air Microscope by S.E.M. Start of Inhalation; Carcinomas Type >20% 9.15 8 Chrysotile 10 I950 >20% 3.86 2 Chrysotile 2 390
THE USE OF ANIMAL MODELS FOR STUDIES ON ASBESTOS BIOEFFECTS J. M. G. Davis
Institute oJ Occupational Medicine Edinburgh, Scotland A frequent criticism of animal experimental studies on disease processes is that extrapolation to the human condition is not justified. While this is probably an extreme view, it is obviously important for an animal model for experimental pathologic studies to duplicate as closely as possible the known human pathologic conditions it is intended to study. The consideration of the best animal model for studies on the pathogenic effects of asbestos dust is facilitated by the fact that this dust produces a number of different disease conditions in human beings. Therefore, the number of parameters for comparison with animal effects is increased, and if an animal model is known to produce results similar to the human condition for more than one type of pathology then this model can be used with added confidence to examine other possible disease effects. In human beings asbestos is known to produce, after inhalation, interstitial pulmonary fibrosis or asbestosis, bronchial carcinomas, and pleural and peritoneal mesotheliomas. In deciding which variety of experimental animal to use for experimental studies with asbestos, it must therefore be considered whether this species can be shown to duplicate these conditions adequately. Most experimental studies of asbestos disease have used rats. This species was included with others in studies by Gardner’ and Vorwald ef a1.’ who reported that long fiber chrysotile produced a “fibrous reaction in the lung” but no details of the histologic picture in this species were given. Later inhalation studies using rats have included Holt, Mills and Young3; Gross and De Treville4; Gross et al.’ and Reeves et a1.6All these authors reported lung fibrosis following the inhalation of various types of asbestos dust, but most of the lesions described were early peribronchial foci of granulation tissue and fibrosis and it was left to Wagner et al.’ to demonstrate that in rats, long periods of asbestos inhalation and subsequent long-term survival result in widespread pulmonary interstitial fibrosis. These results were confirmed by Davis et al.’ The early rat inhalation studies did not result in the development of pulmonary tumors but these were reported by Gross et u I . ~and subsequently in the studies by Reeves, Wagner and Davis. Both benign pulmonary adenomata as well as squamous carcinomata and adenocarcinomata have been shown to develop in this species. Similarly, these inhalation studies resulted in the development of occasional pulmonary mesotheliomas and Davis reported a single peritoneal mesothelioma in a rat that had inhaled chrysotile asbestos. Guinea pigs have been used in a number of experimental inhalation studies with asbestos. These studies include work by Gardner’; Vorwald et al.2;Wagner’; Holt, Mills, and Young”; Gross et aL4; Botham and Holt” and Reeves et a1.6 All these authors reported that guinea pigs developed interstitial pulmonary fibrosis in response to asbestos inhalation, but the development of neither bronchial carcinomas nor mesotheliomas was recorded. Rabbits were included in inhalation studies undertaken by Gardner et a / . ’ ; Vorwald et al.’; Wagner’ and Reeves et a1.6 This species was shown to respond to asbestos inhalation by the development of marked pulmonary fibrosis, but once again no pulmonary tumors or mesotheliomas were caused by the dust.
795 0077-8923/79/0330-0795 $01.75 0 1979. NYAS
796
Annals N e w York Academy of Sciences
Hamsters, gerbils, and mice have all been used in some experimental studies of asbestos inhalation (Gardner’; Vorwald et d.*; Lynch et ~ 1 . ” ;Gross et ~ 7 1 .Reeves ~; et ~ 1and . Wehner ~ et uI.I3).It may be concluded from these studies that hamsters and gerbils respond readily to asbestos inhalation with the development of pulmonary fibrosis. The position with mice is less certain since, while Vorwald and Lynch reported that mice did develop lung fibrosis following the inhalation of chrysotile asbestos, Reeves reported fibrosis in the lungs of mice only after the inhalation of crocidolite or amosite. No pulmonary neoplasms were reported from these studies in any of the three species under consideration. Considering non-rodent species, Vorwald included cats in his inhalation studies, Wagner9 and Webster“ used vervet monkeys, and Webster is at present undertaking an inhalation study using baboons. Pulmonary fibrosis developed in both cats and monkeys but no tumors were found in either species. No reports of the baboon experiments have yet appeared in the literature, but apparently one pleural mesothelioma has been found which developed five years after the start of inhalation (Webster-personal communication). From this combination of results it would appear that only the rat, of those species studied, has been shown to respond to asbestos inhalation in a similar way to humans. Rats develop interstitial pulmonary fibrosis, bronchial carcinomas, and occasional mesotheliomas. It may be that further experimentation would demonstrate that monkeys and especially baboons would give a response to asbestos dust similar to that of human beings, but the high cost of such experiments would preclude use of primates on a large scale. It would appear therefore that the rat represents the best available animal model for asbestos studies and certainly the best model that can be used in all parts of the world in large numbers. In one respect, however, the tissue reaction of the rat to asbestos fibers is dissimilar to that of human beings. In human lungs, asbestos fibers surrounded by macrophages are frequently coated with a mixture of mucopolysaccharide and ferritin or hemosiderin to form asbestos bodies, whereas these bodies occur only very rarely in rats (DavisIs). Guinea pigs, rabbits, hamsters, mice, monkeys, and baboons all produce asbestos bodies and it is uncertain whether or not the inability of the rat to form these structures is a serious disadvantage to its use in studies intended to reproduce human tissue reactions to asbestos. Originally it was suggested that the coating of asbestos bodies protected the surrounding tissues from the damaging effects of asbestos fibers. This may be true for those fibers that do become coated but it is known (Davis and Grosst6)that only a very small proportion of fibers in the appropriate size range ever do form bodies even in human lungs and it seems unlikely that their presence can noticeably affect the course of asbestos-related disease. Having concluded that of all readily available animal species the rat is the best to use for studies on the bioeffects of asbestos fibers, it may be helpful to consider the type of problems which still need to be examined with this type of system. We know that in humans asbestos will cause lung fibrosis and neoplasia but many of the parameters involved are still uncertain. It is essential to determine whether asbestos carcinogenesis exhibits a dose-response effect and whether there is a finite minimum dose of asbestos dust below which tumors will not develop. With mesotheliomas especially it has been suggested that extremely small doses of asbestos may be sufficient to cause these tumors, and some have concluded that the normal use of asbestos products in the urban environment may lead to an epidemic of mesotheliomas in the next few years. Some work has been done using the animal model to explore these possibilities but more is needed. Smith et al.”, Stanton and Wrench,” and Wagner et al.I9 have all shown that following intrapleural injection in rats the number of mesotheliomas produced is progressively reduced with graded doses. However, the lowest dose level used was 0.5 mg and occasional mesotheliomas did develop in this
Davis: Animal Models for Studies on Asbestos Bioeffects
797
study. Of course, 0.5 mg is quite a large dose in comparison with what a human lung could accumulate from the normal urban environment, and it will be necessary to undertake further experiments with lower doses consisting of only a few hundred fibers to determine accurately the real human hazard associated with urban asbestos. Similarly it has been suggested that only asbestos fibers longer than 8-10 p are carcinogenic and a considerable amount of animal experimentation has been undertaken to support this idea (Smith et a/.”; Stanton and Wrench”; and Stanton et a/.’’). It is extremely important to settle this point finally since at present factory dust legislation in most countries is based on counts of the number of fibers over 5 p in length. If only fibers over 8 p are carcinogenic then only these should be included in future dust counting procedures. Recent studies undertaken in Edinburgh have produced further information on both these points (Davis el a[.*).Inhalation studies using rats were undertaken to determine the relative importance of fiber mass and fiber number in the production of pulmonary disease by the U.I.C.C. samples of chrysotile, crocidolite, and amosite. Three dust chambers were used to test clouds of 10 mg/m3 of all three asbestos types, and since the amosite fibers were found to have the greatest volume, the chrysotile and TABLEI Fiber Number Levels of per ml > 5 p Percentage of Interstitial Fibrosis Asbestos Dust Mass/ by Optical fibers >20 p 29 Months after Bronchial m1 of Air Microscope by S.E.M. Start of Inhalation; Carcinomas Type >20% 9.15 8 Chrysotile 10 I950 >20% 3.86 2 Chrysotile 2 390
