Environmental Health Perspectives
Vol. 9, pp. 205-211, 1974
Small Animals in the Study of Pathological Effects of
by Paul F. Holt*
The main pathological effects attributed to asbestos are carcinogenesis and fibrogenesis.
Statistical studies have shown that asbestos workers may expect a higher morbidity not only
from cancer of the lung and mesothelioma but also from cancer at other sites. Carcinomas have
been reported in animals following the injection of asbestos, but the production of carcinomas
by inhaled asbestos is less easy to demonstrate; most examples of experimental carcinogenesis
with asbestos have been produced in rats. Rats and man react differently to asbestos in that rats
do not produce asbestos bodies.
The fibrosis that follows inhalation of asbestos has been frequently described, but studies
with specific pathogen free animals have shown that, like the fibrosis that may follow the in-
halation of silica dust, gross fibrosis involving the production of abnormal amount of collagen
probably requires the intervention of infection as well as asbestos.
Because of the difficulties encountered in the direct investigation of carcinogenesis and
fibrogenesis resulting from the inhalation of asbestos, attention has been directed to the
mechanisms by which the lung is able to protect itself against these fibrous dusts. While non-
fibrous dusts and short fibers can be ingested by macrophages and removed via the bronchus,
the long fibers that may also reach the alveolar regions may not be removed by this mechanism.
The probability that a fiber may reach the alveoli depends largely on the fiber diameter and only
to a small extent on the fiber length, so that, for example, fibers 100 Mm long may reach the
alveoli of a guinea pig. These long fibers may become coated with a ferroprotein derived from
hemoglobin to form an asbestos body and, after morphological changes, the asbestos body may
be broken up, the fragments ingested by macrophages and dissolved. The lung is thus cleared of
asbestos. In the guinea pig lung, consolidated areas from which the asbestos has disappeared
shows signs of return to normal.
This clearance mechanism is inhibited by other factors: quartz dust may almost completely
inhibit asbestos body formation; tobacco smoke has a considerable effect, and even very heavy
loads of carbon may act similarly.
The normal lung appears able to efficiently eliminate small loads of both nonfibrous and
fibrous dust, including the carcinogenic asbestos fibers. The capacity is not unlimited, however,
and when the load is heavy there is a much greater probability that fibers will not be detox-
icated. In addition, other factors such as silica dust and tobacco smoke may remove the protec-
tive mechanism in the lungs.
Inhalation of asbestos necessarily implies in- for example (1). There appear to be no animal
gestion because much of the dust load of the experiments that demonstrate the penetration
lung eventually reaches the gastrointestinal of the intestinal wall by these ingested particles.
tract. The transfer from the lungs has been Animal experiments have shown that inhaled
studied in animals by using radioactive asbestos asbestos fibers may move from the lung or
dusts and following their excretion in the feces, trachea to other tissues. From the bronchiole,
they may move into the muscular coat. Fibers
*University of Reading, Reading, England. have been found in the lymph nodes of guinea
December 1974 205
pigs that inhaled crocidolite (Fig. 1); kidneys, brain, and lymph nodes (4); chrysotile
presumably they had passed along the injected into the stomach of rats was found in
lymphatics. Asbestos bodies appeared in the every tissue examined (5). Relatively large
thyroid of a guinea pig that inhaled amounts of the asbestos dust are administered
anthophyllite asbestos. in injection experiments, and this fact and the
increased pressure in the tissue may be partly
responsible for the results obtained.
It is established, then, that asbestos dust that
has entered the tissues by inhalation or inges-
tion may be found at the site of entry or at other
sites. It is important to know whether these par-
ticles can be pathogenic. The main pathological
effects attributed to asbestos are carcinogenesis
and fibrogenesis. Experimentally there are dif-
ficulties in studying both these conditions.
Statistical studies have shown that asbestos
workers may expect a higher morbidity not only
from cancer of the lung and mesothelioma but
also from cancer at other sites. Kogan et al. (6)
for example, found more carcinomas of the
stomach, intestine, and uterus in asbestos
workers than in the general population. Deaths
from all forms of cancer in asbestos workers
aged 20-50 years were about one and a half
times, in males over 50 years five times, and in
females over 50 years 25 times those of the
Carcinomas have been reported in animals
following the injection of asbestos. Amosite,
chrysotile, and crocidolite all produced
mesotheliomas when administered by in-
trapleural injection (7), but the production of
carcinomas in animals by inhaled asbestos is
less easy to demonstrate. Vorwald and his
collaborators (8) found two squamous car-
FIGURE 1. Asbestos fibers and an asbestos body in a cinomas of the lung in guinea pigs that had in-
lymph node of a guinea pig that inhaled crocidolite. haled crocidolite for 1588 hr. In our experience,
Hematoxylin-eosin. 800X . chrysotile (9) and crocidolite (10) inhaled by
guinea pigs for shorter periods produced a
There is evidence of the movement of asbestos marked proliferation of the bronchiolar
in the tissues in man; human beings with , epithelium, a condition that may precede car-
mesotheliomas were found to have asbestos in cinogenesis. Dutra and Carney (11), for exam-
the lung, abdominal nodes, and peritoneum and ple, stated that in man a squamous metaplasia
asbestos bodies in the spleen and small bowel of the bronchiolar wall that occurs in asbestosis
(2). is comparable with the precancerous metaplasia
After asbestos has been injected into animals of the bronchiolar columnar epithelial cells in
it may be found some distance from the site of cigarette smokers, A similar condition has not
injection. Chrystotile injected into the been induced in animals by ingested crocidolite.
peritoneum migrated to the diaphragm and In man, carcinogenesis by asbestos usually
dome of the liver (3); asbestos injected into the takes some 20 years, and it is possible that the
flanks of mice was found in the spleen, liver, life span of small animals is too short for true
206 Environmental Health Perspectives
carcinogenesis by inhaled asbestos to be man, the hematological changes accompanying
demonstrated.t silicosis (elevated serum mucopolysaccharide,
The fibrosis that follows the inhalation of histamine, and hydroxypyroline values) are
asbestos by animals has been frequently much greater if tuberculosis is present (18), and
described. Specific pathogen-free (SPF) rats and in man no correlation between the incidence of
guinea pigs have been used in our later ex- silicosis and the duration of exposure to silica
periments, and we have not found nodular dust could be found because of the intervention
fibrosis to result from the inhalation of any type of infection (19).
of asbestos. In earlier experiments, fibrosis was It seems that massive fibrosis following
induced by inhaled asbestos in animals that had asbestos inhalation, like the massive fibrosis
lung infections (9, 12). It seems probable that following silica inhalation, requires a combina-
gross fibrosis follows the inhalation of asbestos tion of dust and infection, and that meaningful
into an infected lung both in animals and man. animal experiments require SPF animals,
Fibrosis of the lung has been induced in SPF otherwise a variable and uncontrollable factor
animals by asbestos administered by intra- may invalidate results. The addition of a
tracheal injection, but this is probably an arti- monitored infection would allow experiments on
fact. Gross, Harley, and De Treville (13) showed fibrogenesis that parallel human experience. It
that even aluminum powder would cause is equally important that dusts should be ad-
fibrosis when administered to rats by in- ministered by inhalation or ingestion to obviate
tratracheal injection. Fibrous foci were formed the artifacts introduced by the technique of in-
in which the alveolar structure was entirely jection. If fibrogenesis cannot be regarded as the
obliterated and there were coarse randomly effect of asbestos alone, neither can a
arranged collagen bundles; The same powders lipoproteinosis since quartz and even aluminum
did not produce fibrosis when administered by produce this effect.
inhalation, however, although the concentration These considerations indicate the difficulties
was high. that are encountered when an attempt is made
In this context the effect of silica on the lungs to follow the pathological effects of asbestos in
of animals is relevant. More than two decades animals. It is because of these difficulties that
ago it was established that a gross nodular we have pursued an additional line of research,
fibrosis of the lung could be produced in rats studying the protective action of the tissues
and guinea pigs by silica introduced by injection against asbestos and the factors that might
or inhalation. Heppleston, Wright, and Stewart enhance or reduce its effectiveness. In the lung
(14) failed to produce gross fibrosis when SPF alveoli and bronchioles the macrophages
rats inhaled silica but found a severe alveolar phagocytize dust particles. Spherical particles
lipoproteinosis. Our own experiments confirm and very short fibers are phagocytized rapidly.
this. Dale (15) showed that the percentage of The process may take many hours or may never
collagen in the lungs of quartz-injected rabbits be completed when a fiber is ingested.
was no greater than in controls when the lung Macrophages containing only small particles in
showed no infection. Apparently gross fibrosis the cytoplasm are released into the lumen of the
requires a combination of silica and infection. bronchiole and move up into the bronchus. If a
King, Sivalingam, and Trevella (16) had shown macrophage contains a long fiber it may not be
that an added infection increased the degree of disposed of in this way.
fibrosis caused by silica in non-SPF rats and During phagocytosis, the macrophage
hematite dust, which caused little fibrosis when releases a subtance that induces the diapedesis
injected alone into the lungs of guinea pigs, of red cells from a neighboring capillary (20).
produced extensive fibrosis and fatalities when The red cells are ingested by the macrophage
injected together with tubercle bacilli (17). In and small granules, probably ferroproteins
derived from the hemoglobin are formed in the
tAn important paper (22) has just appeared that describes cytoplasm. They appear first to adhere to a long
carcinogenesis following the inhalation by 700 rats of four asbestos fiber, then to form a smooth coating on
types of asbestos for periods up to 24 months. the fiber that stains blue with Perl's reagent.
December 1974 207
Morphological changes follow (21), with the
production of the beaded asbestos body that
eventually breaks up into fragments (Figs. 2 and
3). The fragments are taken up by other
macrophages and dissolved, the released iron in
the cytoplasm being demonstrable by a Prus-
sian Blue reaction (Fig. 4). In this way long
asbestos fibers are removed from the lung. If an
animal inhales a small amount of asbestos,
short fibers are ingested by macrophages which
are removed via the bronchus; all the long
fibers-or at least all the fibers visible by phase
contrast microscopy-are coated within 18
months, and most of them fragment and dis-
appear within that time. With heavier doses of
asbestos, some fibers remain uncoated for long
periods and presumably remain pathogenic. FIGURE 2. Beaded asbestos body. Guinea pig inhaled
anthophyllite. Unstained; phase contrast; oil immersion.
FIGURE 3. Disintegrating asbestos body. Guinea pig inhaled anthophyllite for 50 hr and was killed 15 months later. Weigert
and Van Gieson. 250X.
208 Environmental Health Perspectives
FIGURE 4. Fragments of asbestos body phagocytized and dissolved by macrophages. The resulting iron-containing solution
stains intensely blue with Perls' reagent. Guinea pig inhaled chrysotile for 24 hr and was killed 18 months later. Perls-
Perhaps the study of this protective mechanism therefore be used as a measure of macrophage
may prove to be more rewarding than the study activity.
of the pathological processes. Silica dust is cytotoxic. In exploratory ex-
Since the process of detoxication of the periments, SPF guinea pigs have inhaled silica
asbestos fiber and the formation of the asbestos dust for 200 hr, then these animals, together
body is initiated by the ingestion of the fiber by with controls that received no silica, inhaled
a macrophage, any factor that reduces the anthophyllite asbestos for 10 hr only. All the
availability of macrophages would be expected animals were killed 6 weeks after the inhalation
to reduce the number of asbestos bodies formed. of asbestos and sections were made of the lungs
Assuming a limited availability of which were stained with eosin and Perls'
macrophages, the inhalation of dust particles reagent to pick out the asbestos bodies. The
that are cytotoxic, or even a high dose of an inert asbestos bodies in each section were counted by
dust, should reduce the number of macrophages scanning six equally spaced strips 230,um wide.
available for the detoxication of asbestos fibers. The strips were traced on an enlarged
The -formation of asbestos bodies could photograph of the whole section and the length
December 1974 209
of tissue traversed was measured. From these Table 1. Asbestos bodies formed from asbesios fibers
measurements the number of asbestos bodies in in lungs containing silica and carbon dust compared
a square millimeter of tissue section was with control counts on lungs containing only asbestos.
calculated. Asbestos bodies,
When asbestos fibers were inhaled into a lung count/mm2 lung
in which the number of active macrophages had Animal Lung Survival time,
section x 10
been reduced by silica, the formation of asbestos no. days Carbon Quartz Control
bodies was almost completely inhibited,
although many uncoated asbestos fibers were 1 Right 28 4
visible in the sections (Fig. 5). This fact is 1 Left 28 5
emphasized by the counts given in Table 1. Long 2 Right 28 3
2 Left 28 1
asbestos fibers will not then be removed from 3 Right 28 37
the lung by the usual protective mechanism, and 3 Left 28 23
they will presumably remain pathogenic. 4 Right 28 37
In another experiment, SPF guinea pigs in- 4 Left 28 17
haled carbon dust in high concentrations for 400 5 Right 41 0.3
5 Left 41 0
hr then anthophyllite asbestos for 10 hr. In sec- 6 Right 41 8
7 Left 41 16
8 Right 57 0.7
8 Right 57 0
9 Right 57 47
10 Right 57 27
a Survival time is the time elapsing between inhalation of
asbestos and death.
tions cut from the lungs of animals killed 4
weeks after inhaling the asbestos, the carbon
particles were seen to be almost entirely in-
tracellular, packed into the scavenging
macrophages. Some asbestos fibers were also in
macrophages but others were extracellular. In
sections of the control lungs from the animals
that had only received asbestos, many asbestos
bodies were visible. Very few asbestos bodies
were found in the lungs that also contained car-
bon (Table 1). This observation is relevant to
studies on progressive massive fibrosis, the
pneumoconiosis of coal miners.
Apparently, the number of long asbestos
fibers that are detoxicated by coating, conVerted
into asbestos bodies with subsequent fragmen-
tation, is largely reduced by a dust that destr9oys
macrophages or even by a dust that is regarded
as "safe" if the latter is inhaled in large quanti-
ty. Presumably, infection would act in a similar
manner to reduce the number of macrophages
FIGURE 5. Unaffected asbestos fibers lying in the lung available for removing asbestos particles, and a
of a guinea pig that first inhaled silica dust. None of similar role may be envisaged for the very high
these fibers is coated, whereas many asbestos bodies
were found in control animals that inhaled asbestos concentration of particles found in tobacco
from the same atmosphere for the same time. Eosin; smoke. An attempt is being made to
phase contrast. 216X. demonstrate this.
210 Environmental Health Perspectives
It has already been shown (10) that the rate at 5. Pontefract, R. D., and Cunningham, H. M. Penetration
which fibers are coated to form asbestos bodies of asbestos through the digestive tract of rats. Nature,
243: 352 (1973).
depends on the composition of the fiber; for ex- 6. Kogan, F. M., Gusel'nikova, N. A., and Gulevskaya,
ample, chrysotile asbestos and glass fiber are M. R. The cancer mortality rate among workers in the
coated and fragmented more rapidly than asbestos industry in the Urals. Gig. Sanit., No. 7:
crocidolite asbestos. This implies that the rate (1972).
could be affected by coating the fiber and that 7. Wagner, J. C., and Berry G. Mesotheliomas in rats
following inoculation with asbestos. Brit. J. Cancer,
perhaps some substances might accelerate the 23: 567 (;1969).
detoxication process. Among the substances 8. Reeves, A. L., et al. Experimental asbestos carcino-
used in a preliminary survey, poly(2- genesis. Environ. Res. 4: 496 (1971).
vinylpyridine 4-oxide) was found to be ineffec- 9. Holt, P. F., Mills, J., and Young, D. K. Experimental
tive, but the results obtained with an asbestosis in the guinea-pog. J. Pathol. Bacteriol. 92:
alkylpyridine 1-oxide are certainly worth pur- 10. Botham, S. K., and Holt, P. F. Development of asbestos
suing. bodies on amosite chrysotile and crocidolote fibres in
We have concluded that, while it is important guinea-pig lungs. J. Pathol. 105: 159 (1971).
to study by animal experimentation the 11. Dutra, F. R., and Carney, J. D. Asbestos and pul-
pathology, particularly carcinogenesis, that monary carcinoma. Arch. Environ. Health 10: 416
results from the presence of asbestos in tissues, 12. Holt, P. F., Mills, J., and Young, D. K. The early effects
some means must be devised for expressing the of chrysotile asbestos dust on the rat lung. J. Pathol.
severity of early stages in the tissue changes Bacteriol. 87: 15 (1964).
that is better than the subjective assessment so 13. Gross, P., Harley, R. A., Jr., and DeTreville, R. T. P.
far employed. Possible quantitative methods are Pulmonary reaction to metallic aluminum powders.
An experimental study. Arch. Environ. Health 26: 227
being investigated. But, in parallel with this (1973).
work, a study of the protective role of the 14. Heppleston, A. G., Wright, N. A. and Stewart, J. A.
macrophage, which can be assessed objectively, Experimental alveolar lipo-proteinosis following the
appears to offer another line of attack that has inhalation of silica. J. Pathol. 101: 293 (1970).
given encouraging results. 15. Dale, K. A method for inducing unilateral silicosis in
rabbits by an injection technique with some observa-
tions on lung clearance and quantitative evaluation
Acknowledgement of experimental silicosis. Scand. J. Resp. Dis., 54:
Technical assistance was given by Mrs. M. 157 (1973).
16. King, E. J., Sivalingam, S., and Trevella, W. Infective
Horne and Mr. R. A. Jerrome. Financial pneumoconiosis. Arch. Environ. Health 7: 33 (1963).
assistance was provided by the Medical 17. Byers, P. D., and King, E. J. Experimental infective
Research Council. pneumoconiosis with mycobacterium tuberculosis (Var.
Muris) and hematite by inhalation and injection.
J. Pathol. Bacteriol. 81: 123 (1961).
18. Nddudvary, G., and B6hm, B. Biochemical features
of pulmonary fibrosis in uncomplicated experimental
REFERENCES silicosis and when accompanied by tuberculosis and
collagen formation. Int. Arch. Gewerbepath. Gewerbe-
1. Evans, J. C., et al. Studies on the deposition of inhaled hyg. 24: 169 (1964).
fibrous material in the respiratory tract of the rat and 19. Norviit, L. Primary tuberculosis, other lung infections
its subsequent clearance using radioactive tracer tech- and BCG as important factors in developing silicosis.
niques. Environ. Res., 6: 180 (1973). Nordisk Hyg. Tidskr. 45: 17 (1964).
2. Godwin, M. C., and Jagatic, J. Asbestos and mesotheli- 20. Beck, E. G., Holt, P. F., and Nasrallah, E. T. Effects
omas. Environ. Res., 3: 391 (1970). of chrysotile and acid-treated chrysotile on macrophage
3. Morgan, A., Holmes, A., and Gold, C. Studies of the cultures. Brit. J. Ind. Med. 28: 179 (1971).
solubility of chrysotile asbestos in vivo using radio- 21. Botham, S., and Holt, P. F. The mechanism of forma-
active tracer techniques. Environ. Res., 4: 558 (1971). tion of asbestos bodies. J. Pathol. Bacteriol. 96: 443
4. Kanazawa, K., et al. Migration of asbestos fibres from (1968).
subcutaneous injection sites in mice. Brit. J. Cancer, 22. Wagner, J. C., et al. The effects of the inhalation of
24: 96 (1970). asbestos in rats. Brit. J. Cancer 29: 252 (1974).
December 1974 211