mesothelioma cancer

In Vivo Models of Mesothelioma, Their Purposes, and
Roles

Knowledge of the factors and mechanisms responsible for cancer induc¬tion rests
largely on the development of animal models. In these models, both benign and
malignant tumors, as well as preneoplastic lesions, can be induced by proper
experimental designs and with appropriate con¬trols, and their analogy to human
pathology can be determined. The animal models can be used to test and identify
agents (chemical, physi¬cal, or biologic) that are capable of carcinogenic
activity, and to investi¬gate their mechanisms. In addition, species and strains
of laboratory animals with specific genetic susceptibility to specific types of
sponta¬neous or induced tumors can be identified. Recently, genetic
manipula¬tion has given rise to transgenic or gene-deleted (knockout) animals,
which can reveal selective molecular pathways to carcinogenesis.

The study of in vivo animal models of carcinogenesis has sometimes followed the
indications of the evidence derived from studies of human epidemiology,
especially for occupational and environmental carcino¬gens. On the other hand,
with the systematic development of animal bioassays, many experimentally
identified active carcinogens have been subsequently shown to be human
carcinogens in epidemiologic studies.


Bioassays of potential carcinogenic agents in animal models, previ¬ously carried
out on a smaller scale, became more systematic in the past 40 years, and they
provided a basis for public health measures and primary cancer prevention. The
choice of animal biossays depends on our knowledge of the underlying mechanisms
of carcinogenesis for dif¬ferent classes of carcinogens. For example, knowledge
of the biochem¬ical pathways of metabolic activation of many organic carcinogens
in human and animal tissues determined the selection of metabolically competent
animal models and corresponding modes of administration. In turn, the animal
models have provided an indispensable tool for the study of pathogenetic
mechanisms of carcinogenesis, and for the inves¬tigation of inhibitory factors
(chemoprevention).

In the case of mesothelioma, its association with human exposure to asbestos was
first suggested by some case reports in the 1940s, was sub-stantiated by
occupational epidemiology studies in the 1960s (1–4), and was confirmed by many
subsequent reports.

The pathology of human mesothelioma is presented in Chapter 31. The microscopic
patterns of mesothelioma are numerous, but they can be grouped into epithelial,
sarcomatous (or fibrosarcomatous), and mixed types (5,6).

The first experimental reports of mesothelioma induction, obtained by
intrapleural administration of asbestos, were published in the 1960s and 1970s
(7–9), and showed that the epithelial, fibrous, and mixed types of mesotheliomas
could be induced experimentally by the same treatments. Numerous experimental
results subsequently provided increasingly extensive evidence of the
carcinogenicity of various asbestos types and other fibrous minerals, as is
discussed below.

Criteria for the evaluation of in vivo models need to be considered.
Traditionally, animal models have been evaluated for their analogy to the
corresponding human tumors in terms of target organs, tumor morphology,
histologic types, and histopathogenesis. Their response to etiologic agents
known to be carcinogenic in the human has also been an important criterion for
their evaluation and acceptance. Mesothe-lioma animal models, therefore, have
been aimed at the induction of tumors by direct exposure of serosal cells, using
intrapleural and intra-peritoneal administrations of potential carcinogenic
agents; in addi¬tion, other methods have been used to explore the induction of
mesothelial cell proliferation and transformation following indirect exposures,
such as inhalation and intratracheal administration, intra-venous (intracardiac)
injection, and even dietary intake.

With the development of carcinogenesis studies in animal models, new criteria of
analogy with the human counterpart evolved on the basis of mechanism studies,
which revealed common pathways of metabolic activation of organic carcinogens
and specific mechanisms of activation of mineral particles and fibers, leading
to reactive oxygen formation on their surface. Recent advances in the methods of
molec¬ular biology now provide a wide range of new opportunities for the
investigation of molecular mechanisms in the induction and develop¬ment of
experimental tumors, and for the identification of pathways of signal
transduction and gene activation.

Spontaneous Mesotheliomas

Ilgren and Wagner (10) and Ilgren (11) reviewed the “background” inci¬dence of
mesotheliomas in humans and animals, and cited references to their sporadic
occurrence in a variety of animal species in non-experimental settings,
including lower vertebrates, fish, birds, rodents (rats of various strains and
wild; hamsters; Mastomys), bovines (adult, neonatal and fetal), domestic and
wild dogs, felines, marsupials, ovines, and pigs. They also reviewed reports of
mesotheliomas in untreated, historical, concurrent, and sham-operated control
cohorts in

rats of seven inbred strains. Untreated rats showed the following inci¬dences at
different sites: pleura 0.3% to 1.5%; peritoneum 0% to 11%; tunica vaginalis of
testes 1.3% to 22%; ovarian serosa 0.1% to 0.2%; unspecified sites 0.1% to 1.7%.
Vehicle controls showed incidences of 2.0% to 7.8% for intraperitoneally treated
rats, and 2% to 2.5% for intrapleurally treated rats.

Mesotheliomas from the tunica vaginalis of the testes or the epidyd-imis in
Buffalo and Fischer rats were described by Morris et al (12). Among spontaneous
neoplasms in F344 rats observed for life span, five peritoneal mesotheliomas
(from omentum, spleen, liver, pancreas, and intestine) were found in 160 males
(3.1%), but none in 192 females (13). In lifetime studies in NEDH rats, no
mesotheliomas were found in 793 controls; in parabiont rat pairs (rats
surgically prepared to share the peritoneal cavities), no pleural mesotheliomas
and only one peritoneal mesothelioma were found in 624 rats (14). Tanigawa et al
(15) reported 17 mesotheliomas in 395 untreated male Fischer 344 rats (4.3%),
only one of which was in the pleura, and all others in the genital serosa or
peritoneum. Pott et al (16) reported one mesothelioma in 204 female Wistar rats
injected intraperitoneally with saline, and six mesothe-liomas in 394 rats
injected with titanium dioxide (anatase) or corun-dum. Minardi and Maltoni (17)
examined untreated Sprague-Dawley rats kept until spontaneous death and found,
among 1179 males, three peritoneal and one pericardial mesotheliomas, and among
1202 females, only one pleural mesothelioma. Two historical control groups of
male Fischer 344 rats, reported by the National Toxicology Program (NTP) (18),
showed mesotheliomas of abdominal and tunica vaginalis origin, respectively, in
16/752 (2.1%) and 12/416 (2.9%). No mesothe-liomas were reported in all the NTP
studies on female Fischer 344 rats, or in any of the tests in mice. The
International Agency for Research in Cancer (IARC) in its reviews on tumors in
animals, reported that, in rats, naturally occurring mesotheliomas of the pleura
were “virtually unknown” (19), but those of testicular origin were cited by
several authors (20–23). In the IARC volume on tumors in hamsters, only induced
mesotheliomas were reported (24), and in the volume on mice (25), mesotheliomas
were not even mentioned.

About “spontaneous” cancers, the present writer remembers W.C. Hueper
emphatically saying, some 50 years ago, “Spontaneous? Let’s call them
cryptogenetic!”

Induction by Fibrous Minerals

Experimental induction of mesothelioma by mineral fibers has been obtained in
several species, including rats, hamsters, and mice, and by different routes of
administration. The active fibrous materials include the two types of asbestos:
amphiboles (amosite, crocidolite, antho-phyllite, tremolite) and serpentine
(chrysotile); the zeolite mineral erionite; and man-made mineral fibers and
vitreous fibers, including refractory ceramic fibers, glass wool, glass
filaments, rock (stone) wool, slag wool, and other recently developed, less
biopersistent fibers. Their activity varies considerably, in different test
methods, depending on

several characteristics, such as fiber dimensions and durability or
biopersistence. Ilgren and Wagner (10) and Ilgren (11) reviewed mesothelioma
induction by several nonasbestiform fibrous agents, either naturally occurring
or synthetic. Reports of mesotheliomas induced by intrapleural or
intraperitoneal injections of various asbestos samples were reviewed and their
mechanisms discussed (26–29). Com¬parisons of routes of administration are
reported below.

Detailed reports on the characteristics of tested materials and on
carcinogenicity data from human and animal studies, with extensive references,
are given in the IARC Monographs on the Evaluation of Car-cinogenic Risks to
Humans, for asbestos (30), for erionite (31), for man-made mineral fibers (32),
and for man-made vitreous fibers (33). The 2002 volume (33) partly revises the
classifications given in 1988 (32), on the basis of more recent data.

Intrapleural Administration

In 1962 J.C. Wagner reported the induction of a few mesotheliomas in rats by
intrapleural injection of asbestos. In a larger study, Wagner (34) described a
successful technique for intrapleural injection in SPF Wistar rats, through a
needle attached to a two-way tap connected to a capil¬lary manometer, which gave
a negative reading once the needle reached the pleural cavity, at which point a
suspension of the test dust (which had been ultrasonically dispersed) was
injected. Each rat received 0.4mL of saline containing 20mg of one of the test
dust. The reported preliminary results showed mesotheliomas in rats treated with
natural crocidolite (29/50), extracted crocidolite from which organic
contaminants had been extracted by cyclohexane (37/62), amosite (8/26), and
Canadian chrysotile (55/75). No mesotheliomas were found in 30 rats treated with
crystalline silica and in 19 controls that received saline alone. The induced
mesotheliomas showed either a large mass or multiple nodules (in similar
proportions of animals) and their histologic pattern was tubular epithelial, or
spindle-celled, or, most frequently, mixed.

Comparison of SPF and conventional Wistar rats given intrapleural injections of
various asbestos samples showed analogous percentages with mesothelioma in both
types of rats; the large majority of mesothe-liomas were of the mixed type, with
lower numbers of either fibrous or epithelial types (35). Wagner et al (36,37)
induced mesotheliomas in CD Wistar rats with a single intrapleural injection of
20mg of five ref¬erence asbestos samples from the International Union Against
Cancer (UICC); mesotheliomas were induced in 66% of rats with a “superfine”
Canadian chrysotile, 61% with crocidolite, 36% with amosite, 34% with
anthophyllite, 30% with Canadian chrysotile, 19% with Rhodesian chrysotile, and
also with a fine glass fiber (12%), a ceramic fiber (10%), and a glass powder
(3%); none with a coarse glass fiber. Pylev and Shabad (38) induced 37.5%
mesotheliomas in rats with three intrapleural doses of 20mg of a Russian
chrysotile.

Acid leaching of chrysotile rapidly dissolves magnesium (Mg) from the fibers and
alters their structure: in intrapleural tests in Sprague-Dawley rats, oxalic
acid-leached chrysotile lost most of its carcinogenic

activity as the proportion of leached Mg was increased from 10% to 89%;
hydrochloric acid leaching abolished the activity (39).

In Sprague-Dawley rats injected intrapleurally with 25 mg of asbestos of
different types, pleural mesotheliomas were found in males in 65% with
crocidolite, 70% with Canadian chrysotile, and 40% with asbestos cement, and in
females in 25%, 60%, and 30%, respectively, thus indi¬cating a higher
susceptibility in males (17). For control incidences, see Spontaneous
Mesotheliomas, above.

Stanton and Wrench (9) developed a technique for intrapleural im-plantation in
female Osborn-Mendel rats of gelatin-coated fiberglass pledgets, on which a dose
(usually 40 mg) of the test sample was spread and placed in contact with the
visceral pleura; mesotheliomas were obtained in about 58% to 75% of the animals
in each of the groups given amosite, chrysotile, and four samples of
crocidolite. Samples of fibrous glasses of diverse types and dimensions,
implanted in the pleura, induced different incidences of malignant neoplasms,
called “pleural sarcomas,” but described as identical to mesotheliomas,
including cases with acinar or papillary epithelioid configuration; mesothelioma
incidences ranging from 5% to 100% were reported for 15 samples (40), and for 33
other samples (41). Analysis of 72 experiments with fibrous minerals of widely
different structure showed a correlation with the number of fibers <0.25|jm in
diameter and >8|jm in length (42).

Erionite is a fibrous zeolite structured as a framework of alumino-silicate
tetrahedra (Si,Al)O4, in which each oxygen is shared between two tetrahedra; it
is mined in several countries (31). It was found to induce mesotheliomas in
Sprague-Dawley rats of both sexes after intrapleural injection of 25 mg (43).
Wagner et al (44) injected 20mg samples intrapleurally in Fischer 344 rats of
both sexes and obtained pleural mesotheliomas in 40/40 rats injected with Oregon
erionite, and 38/40 with erionite from Karain, Turkey; for comparison,
mesothe¬liomas developed in 19/40 rats treated with chrysotile and in 1/40
saline controls. In non-inbred rats given three intrapleural injections at
1-month intervals, fibrous erionite from Georgia, in the former USSR, induced
mesotheliomas in 39/40 males and 43/48 females with none in controls (45). In a
dose-response study of Oregon erionite by single intrapleural injection in
Porton rats, Hill et al (46) obtained mesothe¬liomas in 5/10 rats at a dose of
0.1 mg erionite, 9/10 at doses of 1 and 10mg, and 8/10 at 20mg. Thus, erionite
appears to have the highest activity in the induction of mesotheliomas in rats
by intrapleural injection.

In hamsters, the first experimental evidence of mesothelioma induc¬tion was
obtained by Smith et al (8) by intrapleural injection of 25 mg of asbestos in
groups of 15 male Syrian golden hamsters, to which the same type of asbestos was
also fed in the diet; two mesotheliomas were obtained with a sample of “harsh”
crysotile, and three mesotheliomas with a sample of amosite. Of these five
mesotheliomas, two were of the epithelial type and three of the fibrous type
(controls and a sample of “soft” crysotile gave no tumors). In a subsequent
experiment on groups

of 50 hamsters treated with a single intrapleural injection of asbestos, Smith
and Hubert (47) obtained mesotheliomas with 10 or 25mg of chrysotile (4/50 and
9/50 mesotheliomas, respectively), with 10mg of amosite (4/50), with 1 or 10mg
of crocidolite (2/50 and 10/50, respec¬tively), and with 10mg anthophyllite
(3/50).

Intraperitoneal Administration

Effective induction of mesotheliomas was obtained by intraperitoneal
administration of various fibrous minerals, usually at fairly high doses. By
intraperitoneal administration in female Wistar and Sprague-Dawley rats, Pott et
al (16) tested a wide variety of samples and obtained extensive evidence of
carcinogenicity by many fibrous min¬erals; the induced tumors were reported as
sarcoma, mesothelioma, or carcinoma in the abdominal cavity, without a separate
histologic clas¬sification. In subsequent reports of intraperitoneal tests in
female Wistar rats, high incidences were reported for tumors in the abdomi¬nal
cavity described as “mesothelioma or sarcoma” (48), and later specifically as
mesotheliomas in a dose-response study of different samples, with mesothelioma
incidences up to 97% (49,50). Histopatho-logic analysis of the mesotheliomas
induced by intraperitoneal injec¬tion of various mineral fibers in Han:WIS
female rats by Pott and coworkers was subsequently reported (51) as follows: 45%
epithelioid, 34% sarcomatoid, 37% mixed, 18% mixed with bone/cartilage, and 5%
sarcomatoid with bone/cartilage.

Several samples of man-made mineral fibers were tested in groups of 18 to 24
male SPF Wistar rats by single intraperitoneal injection of an estimated dose
containing 109 fibers >5mm in length; in comparison with an amosite sample, four
fiber types were more active and four less active in inducing mesotheliomas (one
ceramic fiber type produced by extreme heating induced no mesotheliomas). The
results pointed to a link with the number of fibers >20mm in length and with
biopersistence in rat lungs of fibers >5mm long (52).

In Sprague-Dawley rats injected intraperitoneally with 25mg of asbestos of
different types, peritoneal mesotheliomas were found with crocidolite in 95% of
males and 100% of females, with Canadian chrysotile in 90% of males and 70% of
females, with Rhodesian chrys-otile in 80% of males and 85% of females, with
California chrysotile in 75% of males and 70% of females, with amosite in 90% of
both sexes, with antophyllite in 80% of males and 85% of females, with asbestos
cement in 45% of males and 60% of females, and none in water con¬trols of both
sexes. In the same study, seven samples of modified chrysotiles induced
peritoneal mesotheliomas with various incidences (30–85% in males and 25–80% in
females) and one other sample in only 15% of males and none in females (17).
This study does not report a clear sex difference in susceptibility for
peritoneal mesotheliomas, whereas the groups treated with intrapleural injection
showed a higher susceptibility in males for pleural mesotheliomas (see above;
and for control incidences, see Spontaneous Mesotheliomas, above). The finding
of a higher susceptibility for peritoneal than for pleural

mesotheliomas with asbestos of various types was not confirmed with tests on a
sedimentary erionite, which induced incidences of 50% peri¬toneal and 87.5%
pleural mesotheliomas (data given for both sexes together) (53). The
administration of a single large intracavitary dose, such as 25mg, may not be
appropriate to detect susceptibility differ¬ences in the target mesothelial
cells.

Dose-response relationships in mesothelioma induction by UICC samples of
chrysotile, crocidolite, and amosite and by Oregon erionite, following a single
intraperitoneal injection in SPF rats of the AF/Han strain, were analyzed in
relation to doses (from 0.005 to 25mg), fiber dimensions (length and diameter),
and number of fibers per milligram (54); this study confirmed the importance of
fiber length and pointed out that of the two fibers with highest carcinogenic
activity, erionite included a fraction of relatively thick fibers, whereas
chrysotile fibers separate largely into individual fibrils in tissues (and
therefore have low durability), but include a high number of long fibers. A
previous intraperitoneal injection study showed the higher activity of long
versus short fibers of chrysotile in terms of mesothelioma incidence and mean
induction period (55).

Several samples of tremolite, an amphibole type of asbestos, were tested by
single intraperitoneal injection of 10mg in 2.0mL saline, in rats of the AF/HAN
strain; all induced mesotheliomas, with different incidences, ranging from 100%
to 5.5% (56).

Repeated intraperitoneal injections in rats were used for tests of biosoluble
synthetic fibers, in groups of 51 female Wistar rats (57); the test materials
were administered by intraperitoneal injection to the midabdominal region, with
two, eight, or 20 injections, each in 2.5mL saline, at 1-week intervals,
depending on the desired total dose [0.5, 2, and 5 ¥ 109, respectively, of World
Health Organization (WHO) fibers]. Glass wool samples B, M, P, and V, of similar
density and surface area, but of different solubility, resulted in mesothelioma
incidences varying from 0% to 14%, and a sample of stone wool only in 0 and 1%.
The incidence of mesotheliomas was correlated with intraabdominal masses,
ascites, and chronic peritonitis. Positive controls with crocid-olite,
administered in a single dose (0.5 and 5.0 ¥ 106 WHO fibers, respectively),
resulted in mesothelioma incidences of 27% and 45%, respectively.

In male BALB/c mice, mesotheliomas were induced by single intraperitoneal
injection of UICC amosite (a dose of 20mg induced 26.7% mesotheliomas; 10mg,
23.5%; 2mg, 35.1%); UICC chrysotile (20 mg, 12.5%; 2 mg, 0); Calidria chrysotile
(2mg, 25%); erionite I (10mg, 45.2%); erionite II (10mg, 37.5%; 2mg, 54.5%;
0.5mg, 33.3%); a double injection of 2mg amosite and 2mg UICC chrysotile induced
mesothe-liomas in 60% (58). In this study, a large proportion of mice treated
with high doses developed severe peritoneal fibrosis and intestinal
obstruc¬tion, and many died before 7 months (mesotheliomas were found only after
this latent period); this pathology accounts for the reverse dose-response
observed with amosite and erionite. Overall, the mesothe-liomas included 88% of
the fibrous type, 11% of the mixed type, and only 1% of the epithelial type.
Peritoneal mesotheliomas were found in

all groups of Swiss mice treated by single intraperitoneal injection with graded
doses (5 to 40mg) of erionite from Karain, Turkey; no evidence of a
dose-response was observed in this study (59).

In C57BL/6 mice, the early mesothelial reactions following intra-peritoneal
crocidolite injection were described as involving cell injury and regeneration,
associated with the development of mesotheliomas (60). Following intraperitoneal
injection of crocidolite, 25% of BALB/c mice and 45% of CBA mice developed
mesothelioma, 7 to 25 months after exposure; cell lines were established from
these tumors (61).

As an effective method for mesothelioma induction, A.B. Kane’s lab¬oratory
adopted weekly intraperitoneal injections of 200mg of UICC crocidolite in
C57Bl/6 mice (62); mesotheliomas were induced after 35 to 66 weeks of treatment.
The tumors had the typical histology of epithelial, fibroblastic, or mixed
types. This model was used for p53+/+, heterozygous p53+/-, and homozygous
p53-/- mice. Mesotheliomas developed in 37% of the wild-type mice (mean latent
period: 67 weeks), and in 76% of the heterozygous p53-deficient mice (mean
latent period: 44 weeks); the homozygous p53-deficient mice developed many other
types of tumors early and only one mesothelioma was found in 10 mice, with a
latency of only 10 weeks. In the heterozygous mice, 50% of the mesotheliomas
showed extensive invasion (63,64).

Inhalation

Following inhalation in SPF Wistar rats of both sexes, for various periods of
time, of different samples of asbestos, at doses closely com¬parable for all
samples (as concentrations of mean respirable dust and as cumulative dose), many
animals developed lung tumors (adenomas, adenocarcinomas, and squamous cell
carcinomas), but only a few developed mesotheliomas. After a 1-day exposure to
amosite, 1 of 45 rats at risk developed mesothelioma; 1/28 and 1/18 after
12-month and 24-month exposures to anthophyllite; 1/43, 1/36, and 2/26 after
crocidolite exposures, respectively, of 1 day, 3 months, and 12 months; 3/23 and
1/21 after 12-month and 24-month exposures to Canadian chrysotile; and none
after exposure to Rhodesian chrysotile and in con¬trols (65). In another
asbestos inhalation study in SPF rats of the Han strain, again, lung tumors were
induced by UICC amosite, chrysotile, and crocidolite, but only one peritoneal
mesothelioma occurred in 42 rats exposed to 2mg/m3 chrysotile and one pleural
mesothelioma in 43 rats exposed to 5 mg/m3 crocidolite (66). In a later study
(67), long-and short-fiber samples of amosite and chrysotile were tested by
inhalation in rats; the long-fiber samples were much more carcinogenic in both
cases (inducing both pulmonary tumors and pleural mesothe-liomas), whereas the
short-fiber samples gave some tumors with chrysotile and none with amosite.
Results of many experiments on the induction of lung tumors and mesotheliomas by
inhalation of chrysotile and of amphiboles in rats were analyzed by Pott (68)
and showed that mesotheliomas were much less frequently induced than lung
tumors. Davis et al (54) pointed out that mesotheliomas were induced in rats
following inhalation of all asbestos types, but never

with an incidence of more than 10% of exposed animals, even at massive doses
(55,65–67).

In contrast, a high incidence of pleural mesotheliomas was observed in Fischer
344 rats exposed to Oregon erionite inhalation: 27/28 rats developed
mesotheliomas, with none in controls; in comparison, only 4/124 rats exposed to
crocidolite developed mesotheliomas (44). Johnson et al (69) reported that
pleural tumors induced in rats by eri-onite inhalation had similar
ultrastructural appearance to mesothe-liomas induced by asbestos injection in
the pleural and peritoneal cavities. The high incidence of mesotheliomas induced
by Oregon erionite is discussed by Davis (70), who draws attention to the fact
that its appearance and fiber size distribution are very similar to those of
UICC crocidolite, which causes only rare mesotheliomas when inhaled by rats.
Johnson (26) observed that the peritoneum seemed more sensitive to crocidolite
than the pleura, whereas the pleura was more sensitive to erionite than the
peritoneum.

In Syrian golden hamsters (125 males per group, 140 controls), nose-only
inhalation tests of amosite showed the induction of inflammation and pulmonary
and pleural fibrosis, followed by mesothelial hyper¬trophy and hyperplasia, and
the following incidences of mesothe-liomas: 4% with 25 fibers/cm3, 26% with 125
fibers/cm3, and 20% with 250 fibers/cm3. In comparison, only one mesothelioma
was found with inhalation of fiberglass sample MMVF33 and none with fiberglass
sample MMVF10a of much lower durability (71).

In a comparative inhalation study of a kaolin-based refractory ceramic fiber
(RCF-1) in Fischer 344 rats and Syrian golden hamsters, significant pulmonary
and pleural inflammation was detected for both species; DNA synthesis by pleural
mesothelial cells was higher in ham¬sters than in rats, and was highest in the
parietal pleura; greater colla¬gen deposition was measured in the visceral
pleura of hamsters, but was not significantly elevated in rats; and the number
of fibers longer than 5mm per cm2 of pleural surface was two to three times
higher in hamsters than in rats (72). A previous inhalation study in Fischer 344
rats and Syrian golden hamsters, exposed simultaneously to RCF-1, resulted in a
high incidence of mesotheliomas in hamsters (43%), but not in rats (1.6%) (73).
Both species developed fibrosis and inflamma¬tion of the visceral pleura, but
hamsters developed greater surface mesothelial cell proliferation and had focal
aggregates of mesothelial cells embedded within regions of visceral pleural
fibrosis (74).

Comparing the response of different animal models, such as rats and hamsters, is
especially interesting, because it may suggest different underlying mechanisms.
In studies with crystalline silica, administered either by inhalation or by
intratracheal instillation, hamsters developed no fibrogenic or carcinogenic
responses, but only macrophagic granu-lomas, whereas rats showed a high level of
pulmonary fibrogenic and carcinogenic responses (75). Silica and asbestos are
both active through the formation of reactive oxygen species that produce DNA
damage, but particulate materials differ from fibrous materials in
physicochem-ical characteristics and consequently in their transport and
localization in target tissues. Both asbestos and silica are carcinogenic in the
lungs

of rats, but no mesotheliomas have been induced by silica, even in rats. Wagner
et al (76) found that intraperitoneal injection of silica in rats induced
malignant histiocytic lymphomas, but no mesotheliomas. For combined exposures to
asbestos and quartz, see below.

Intratracheal Instillation

Intratracheal injection of 2 mg Russian chrysotile, together with 5mg
benzo[a]pyrene (BP), resulted in pleural mesotheliomas in 6/11 rats, whereas
none were induced in rats given BP alone or chrysotile alone at a higher dose
(77).

Mohr et al (78) induced pleural mesotheliomas by 8 weekly intra-tracheal
instillations in male Syrian golden hamsters (each of 1mg dust in 0.15mL saline)
of two types of glass fibers (mesothelioma incidences: 37/136 and 26/138) and of
UICC crocidolite (8/142), with 0/142 in TiO2 controls. These mesotheliomas were
all epithelioid, with papillary areas.

I.Y. Adamson and coworkers used intratracheal instillation of crocid-olite in
mice and rats in a series of short-term studies, to investigate early-phase
proliferation of mesothelial cells in vivo. They showed that mesothelial cell
proliferation was induced by long fibers in mice. Since this effect was also
induced by other agents, such as hyperoxia, bleomycin, and silica, they
suggested that it was a response to injury to the lung and probably mediated by
the same cytokines that trigger interstitial fibrosis (79). The early
proliferative response of mesothelial cells was also seen after intratracheal
instillation of crocidolite in rats, and it was found to be blocked by an
antibody to keratinocyte growth factor (KGF) (80). Since mesothelial cells share
properties of fibroblas-tic and epithelial cells and they express both vimentin
and cytokeratin (81), Adamson et al (80) suggested that KGF, secreted by lung
fibro-blasts to promote epithelial repair after asbestos, may diffuse to the
pleural surface and induce the short-term proliferation of mesothelial cells; in
extended exposure periods, this stimulation may be responsi¬ble for mesothelial
cell proliferation. Further studies by intratracheal instillation of crocidolite
in rats showed that during the phase of mesothelial cell proliferation, both KGF
and hepatocyte growth factor (HGF) were significantly increased in both
bronchoalveolar and pleural lavage fluids (82), and both HGF and KGF stimulated
mesothelial cell proliferation (83).

Ingestion

Extensive long-term feeding tests of mineral fibers were reviewed (84). Two
hamster studies showed no toxic effects of feeding amosite or taconite tailings,
although one mesothelioma was reported in 30 amosite-treated males (none in
controls). No mesotheliomas were reported in 10 experimental groups of rats fed
amosite or crocidolite. In seven feeding studies with chrysotile, only one
showed a peritoneal mesothelioma (in 95 male rats). These tumors were not
considered significant. In a large feeding study in rats of chrysotile (alone or
mixed with 25% crocidolite, with untreated or palm-oil controls), seven
mesotheliomas were reported (two in control groups) and they included three in
the thoracic cavity, one peritoneal, one near salivary

glands and two in the testicles (85); again, these mesotheliomas were considered
unrelated to treatment, but their origin remains unclear.

Induction by Other Carcinogens

Reports have been published on mesotheliomas in animal models fol¬lowing
exposure to a variety of chemicals. The evidence of induction by the test
chemicals is often difficult to establish. Ilgren and Wagner (10) and Ilgren
(11) listed a large number of tests from the literature in which mesotheliomas
were reported, but they did not assess the sig¬nificance of the findings, and so
included tests in which, for example, only one or a few mesotheliomas were
reported in a group of animals, compatible with occurrence in control groups. In
other cases, the tumors were not clearly diagnosed as mesotheliomas. This
section includes only references to studies showing a likely induction of
mesotheliomas.

N-nitrosopyrrolidine (fed at 16mg/kg body weight for 67 weeks) resulted in
papillary mesotheliomas of the testis or epididymis in 4/12 (33%) MRC rats, with
0/34 in controls (86). Mesotheliomas and proliferative lesions of the testicular
mesothelium (tunica vaginalis and epididymis) were produced by a single
intraperitoneal injection of 13mg/kg body weight of
methyl(acetoxymethyl)nitrosamine (DMN-OAc) in male Fischer 344 rats (9/25),
Sprague-Dawley rats (4/27), and Buffalo rats (12/27) (87).

N-2-fluorenylacetamide, fed at 0.06% in the diet to male Fischer 344 rats, in
three cycles of 3 weeks each, with 1-week intervals on normal diet, resulted in
testicular mesotheliomas in 9/25 rats (36%), with none in a group with only one
cycle and in controls (88). A previous study of N-2-fluorenylacetamide, fed at
0.025% in the diet to Buffalo strain rats, gave mesotheliomas of the testes or
epididymis in 3/18 rats, but 2/6 controls also had these tumors (12). Ethylene
oxide, tested by inhalation in male Fischer 344 rats, induced significant
incidences of peritoneal mesotheliomas at 33 and 100ppm in one study (89) and at
100 and 300ppm in another study (90).

Mice of different strains, treated with intragastric administrations of
3-methylcholanthrene (MCA), 20mg/kg in olive oil, weekly for 10 weeks, developed
peritoneal mesotheliomas that frequently invaded the diaphragm and other organs;
their incidence varied in different strains: mesotheliomas were induced in 12/31
(39%) mice of the C3H strain, and 9/32 (28%) mice of the BALB/c strain; low
incidences of mesothelioma were obtained in C57BL/6 mice (1/31) and DBA/2 mice
(1/26), and none in Swiss mice (0/30) and AKR mice (0/32). Most of the induced
mesotheliomas were of the mixed type and mainly fibrous. In addition, lesions
consisting of severe mesothelial hyper-plasia associated with tissue necrosis
and inflammation were consid¬ered as possible early stages of mesothelioma
development (91). This study shows marked susceptibility differences among
different mouse strains to mesothelioma induction by a polynuclear aromatic
hydro¬carbon. This model, which has not been used so far for mechanism

studies, could provide clues to the underlying genetic susceptibility factors.

Other organic chemicals were linked to mesothelioma induction, as reviewed by
Peterson et al (92), Ilgren and Wagner (10) and Ilgren (11). Among them,
sterigmatocystin given by repeated intraperitoneal injec¬tions, which induced
mesotheliomas in 50% of Wistar rats (93); possi¬bly 1-nitroso-5,6-dihydrouracyl
given in three intraperitoneal injections to MRC-Wistar rats (2/40 with
mesothelioma) (94); N-methyl-N-nitrosourea given repeatedly intraperitoneally to
guinea pigs (only one mesothelioma) (95); and diethylstilbestrol, which, after
subcutaneous implantation in squirrel monkeys, resulted in uterine malignant
mesotheliomas in 7/10 animals (96).

Ferric saccharate and nitrilotriacetic acid (NTA) were injected
intraperitoneally daily for 3 months in male Wistar rats, separately or
together: 9/19 rats given ferric saccharate (5mg Fe/kg body weight/day, 6
days/week for 3 months) developed mesotheliomas from the serosa of the tunica
vaginalis or spermatic cord; among 19 rats treated with ferric saccharate and
also with NTA (83.5mg/kg body weight, 6 days/week for 5 months), seven developed
mesotheliomas at the same locations and six had widespread peritoneal
mesothe-liomas; the mesotheliomas showed all three histologic types; none were
found in rats given NTA alone or just saline (97). Potassium bromate (KBrO3), an
oxidizing agent, given in drinking water to F344 rats, induced mesotheliomas on
the surfaces of various abdominal organs in males, in 59% at 500ppm and 33% at
250ppm, but in 6% in untreated controls (!), with none in female F344 rats,
female B6C3F1 mice, and male Syrian golden hamsters; different durations of
treatment in male rats gave mesothelioma incidences near 40% for treatments up
to 52 weeks, and 75% for 104 weeks of treatment (98).

The reports of the National Toxicology Program (NTP) (18), National Institute of
Environmental Health Sciences, were reviewed for tests resulting in
mesotheliomas. The following long-term tests in male F344 rats yielded high
incidences of mesotheliomas originating in the peritoneum/tunica vaginalis
testis: (a) by inhalation: 1,2-dibromoethane (up to 25/50 rats with
mesotheliomas); (b) by feeding: o-nitrotoluene (up to 44/60 and to 54/60 in a
stop-exposure test); 2,2-bis(bromomethyl)-1,3-propanediol (up to 26/60 in a
stop-exposure test); (c) by gavage: glycidol (up to 39/50); (d) by
intraperitoneal injec¬tion: cytembena (a cytostatic agent) (up to 37/50). Lower
incidences of mesotheliomas were induced by the following compounds: (e) by
inhalation: dichloromethane (up to 5/50); (f) by feeding: pen-tachlorophenol (up
to 9/50); ethyl tellurac (up to 8/50); nitrofurazone (up to 7/50); o-toluidine
(no incidence given); 3,3¢-dimethoxybenzidine dihydrochloride (up to 6/60);
3,3¢-dimethylbenzidine dihydrochloride (up to 4/60); pentachlorophenol (up to
9/50 in a stop-exposure study); (g) by gavage: methyleugenol (up to 12/50);
trichloroethylene (up to 5/50); (h) by dermal application:
2,3-dibromo-l-propanol (up to 4/50). In addition, a test of acronycine by
intraperitoneal injection in Sprague-Dawley rats induced mesotheliomas (in the
abdomen or tunica

vaginalis) in both male and female rats, but the incidence was not clear because
of high mortality. The historical control incidence of mesothe-liomas in male
F344 rats was reported from two laboratories as 2.9% and 2.1%. No mesotheliomas
were reported in all the studies on female F344 rats, or in any of the tests in
mice.

Radioactivity was shown to induce mesotheliomas; in rats exposed to 239PuO2 by
intraperitoneal injection, 27% developed epithelial mesotheliomas and 38%
sarcomatous mesotheliomas; the tumor inci¬dence was dose-dependent and a greater
dose was required to induce epithelial than sarcomatous mesotheliomas (99). When
chrysotile or 3,4-benzo[a]pyrene was injected with the 239PuO2, the resultant
tumor incidence was additive [Sanders, 1973, quoted by Hahn et al (100)].
239PuO2 also induced pleural mesotheliomas by inhalation or intrapleural
injection (101). Four life-span inhalation studies of radionuclides were
reviewed by Hahn et al (100) for the induction of mesotheliomas; the rats were
exposed briefly (10–40min) per nasum once or repeatedly (seven times over 1
year); in a total of 3076 rats (approximately equally divided by sex), exposed
by inhalation to aerosols of 239PuO2, mixed uranium-plutonium oxide, or 144CeO2,
a total of 28 pleural mesotheliomas were induced (21 epithelial-papillary
diffuse, two epithelial-papillary focal, two sarcomatous, and three mixed); four
mesotheliomas were found in 1641 controls (0.24%). These studies showed that
mesotheliomas can be induced with either alpha-or (less effectively) with
beta-emitting radionuclides.

Combined Exposures

Warren et al (14) reported a large study in NEDH rats of both sexes treated with
UICC chrysotile by the intratracheal, intrapleural, and intraperitoneal routes,
alone or combined with x-radiation or 3-methylcholanthrene administration. The
results (limited by incomplete reporting) are indicative of a marked synergism
of asbestos with radi¬ation and with methylcholanthrene for peritoneal, but not
pleural, mesotheliomas. Combined effects in mesothelioma induction were also
reported for intrapleural chrysotile combined with radon 222 inhala¬tion in rats
(102).

In a study of the combined effects of asbestos inhalation coupled with the
inhalation of a particulate material, namely, quartz or rutile (a titanium
dioxide polymorph), unexpected results were obtained (103). Rats of the AF/Han
strain (sex not specified) were exposed to inhala¬tion (5 hours/day at 10mg/m3)
of UICC chrysotile or to a sample of long-fiber amosite. Separate groups were
exposed to the same asbestos inhalation schedule followed by a 2 hours/day
inhalation of either rutile (at 10mg/m3) or quartz (at 2.2mg/m3). The inhalation
exposures were continued for 1 year, with a 2-year follow-up. Early lung lesions
from quartz and asbestos were more diffuse than those from asbestos alone. After
6 months, the asbestos + quartz groups showed more extensive fibrosis and
alveolar epithelial hyperplasia, while titanium dioxide did not change the
degree of fibrosis seen with asbestos alone. The incidence of pleural
mesotheliomas was 6/38 (16%) with chrysotile

+ quartz, 0/37 with chrysotile alone and 2/41 with chrysotile + rutile; and it
was 8/39 (21%) with amosite + quartz, 2/40 with amosite alone and 2/40 with
amosite + rutile. Peritoneal mesotheliomas were 1/40 with amosite alone, 2/40
with amosite + rutile, 1/39 with amosite + quartz, and none with chrysotile. The
induced mesotheliomas were his-tologically biphasic. The remarkable finding that
the induction of pleural mesotheliomas by asbestos inhalation is significantly
increased by quartz was paralleled by the increase in pulmonary fibrosis. Quartz
also increased the incidence of pulmonary adenocarcinomas in com¬parison with
the two groups treated with asbestos alone. The role of quartz, an ubiquitous
dust and a known pulmonary carcinogen, in the potentiation of pleural
mesothelial carcinogenesis remains to be further investigated. Quartz shares
several molecular mechanisms with asbestos (104), and may also have affected the
transport of inhaled fibers to the pleura.

Induction by SV40

High incidences of mesotheliomas were induced in 21-day-old male Syrian golden
hamsters following intracardiac, intraperitoneal, and intrapleural injections of
wild-type (wt) simian virus 40 (SV40) (see also Chapter 3). Specifically, wt
SV40 830 resulted in the induction of 13/21 (62%) mesotheliomas by intracardiac
injection, and wt SV40 776 resulted in 2/5 (40%) mesotheliomas by intracardiac
and 4/6 (67%) mesotheliomas by intraperitoneal injection. When injected
intrapleu-rally, both wt strains resulted in a 100% incidence of mesotheliomas
(6/6 and 5/5, respectively) with no mesotheliomas in controls (105). The tumors
developed between 3 and 6 months after injection. After intraperitoneal or
intracardiac injection, the tumors formed a continu¬ous layer over pleural and
pericardial surfaces, obliterating the cavi¬ties; and those induced by
intraperitoneal injection spread widely over the serosal surfaces. No distant
metastases were observed. The histo-logic appearance of the tumors was mostly of
the mixed type, with spindle cell and epithelioid areas in the same tumor, but
some tumors showed only one type of differentiation. The diagnosis of
mesothe-lioma was confirmed by histochemical staining, showing production of
hyaluronic acid, and by electron microscopic features. Tumor cell lines were
established from these mesotheliomas (105).

Mesotheliomas in hamsters induced by asbestos or by SV40 were compared (106).
Intrapleural injection of crocidolite asbestos induced mesotheliomas in hamsters
in a dose-dependent manner; the tumors appeared late (>18 months) and were small
and well differentiated, with adjacent pleural fibrosis, and rarely caused local
invasion and death. In contrast, SV40-induced hamster mesotheliomas showed no
adjacent pleural fibrosis, and they were large and multicentric, with both
epithelioid and sarcomatoid areas; histologically they were anaplastic and
showed invasion of adjacent tissues; and they were uniformly fatal within 3 to 6
months. Such different patterns in the histopathology and clinical course of two
animal models of mesothe-lioma obtained by different etiologic agents should
prove valuable for

investigating the respective molecular pathways involved in their pathogenesis.

Induction by Other Viruses

Proliferative lesions of the pleura, pericardium, and peritoneum were reported
in Swiss mice inoculated in utero with polyoma virus, but none of the lesions
progressed to invasion or metastasis; the lesions showed hypertrophied
mesothelial cells over a thickened hyalinized matrix, or stalks attached to the
visceral surface and covered by one to five layers of cuboidal mesothelial
cells, sometimes forming polypoid masses (107). In a group of 16 Syrian golden
hamsters injected intra-tracheally with polyoma virus (which induced lung
carcinomas and liver angiomas), one hamster developed a bilateral pleural
mesothe¬lioma (108); given the absence of spontaneous mesotheliomas in hamsters
(see above), this is probably a polyoma-induced tumor.

The MC29 avian leukosis virus (an RNA virus) induced mesothe¬liomas in 35% of
chickens injected in the peritoneal, pericardial, and air sac cavities (109).
Diffuse mesotheliomas were induced in chickens of the inbred SC line, by
inoculation of preparations of v-src DNA (the oncogene of Rous sarcoma virus);
pRLV-src resulted in mesothelioma incidences of 9/10 by intravenous, and 22/37
by intraperitoneal administration; and intraperitoneal pJDD 11 construct gave
10/13 mesotheliomas. The mesotheliomas in all these experiments were of the
three morphologic types: epithelial, fibrous, and mixed (110).

Mechanisms and Pathways

Mechanisms Specific to Fibrous Minerals: The roles of fiber type, dimen¬sions,
durability, surface properties, deposition, translocation, and dis¬solution have
been shown to be of critical importance in many studies (42,54,68,70,111-114).
The critical factors are fiber dimensions (>8um in length and <0.25 um in
diameter), the number of fibers, their chemical composition, and the durability,
or biopersistence, of the fiber type. Suzuki (115) remarked on the need for a
long latent period for mesothe¬lioma induction, dependent on the longevity of
the species: >7 months in mice, >1 year in rats, >6 years in baboons, and >20
years or longer in humans.

In considering the dose of fibrous minerals that was used in most animal
experiments, especially in single injection tests, one may become concerned that
the induction of mesotheliomas is obtained by high doses, as compared with the
amount of fiber inhaled in human expo¬sures over a long time. The animal models
are often primarily chosen to obtain the desired end point, i.e., the
development of tumors compara¬ble with those of human pathology. Concerning the
dose, one has to remember that particulate and fibrous minerals act by
mechanisms involving the reactivity of their surfaces, not their bulk, and that
there¬fore their total weight is not representative of the active surface
molec¬ular layer, a fundamental difference from the mode of action of soluble
carcinogens. It is also important to consider the probability of tumor

occurrence in relation to the number of individuals exposed and the duration of
their survival, which are obviously very different between groups of
experimental animals and human populations. The fact that the carcinogenic
activity of fibrous minerals has been observed under many different test
conditions, species, and routes of administration, and its correspondence with
human occupational epidemiology, leave no doubt about the carcinogenicity of
these materials. More difficult is the evaluation of negative results obtained
in certain tests, in terms of risk estimates for human exposure. Here,
dose-response relationships are important in the experimental models as well as
in corresponding human exposures, and mechanistic considerations can contribute
to the understanding of the results. An example is given by a recent report on
biosoluble synthetic mineral fibers tested by intraperitoneal injection in rats
(57). Much remains to be investigated about the mechanisms by which fibrous
minerals induce tumors, especially mesotheliomas. For example, asbestos fibers
(chrysotile) were found to be effective in trans-fecting exogenous plasmid DNA
in monkey cells COS-7, suggesting another possible role for the fibers in the
mechanism of neoplastic trans¬formation (116). Well-defined biologic models can
provide new insights into the mechanisms of fiber carcinogenesis.

An extensive critical discussion was provided by the consensus report of a group
of experts convened by the International Agency for Research on Cancer (IARC)
(117), who considered fiber characteriza¬tion, biopersistence, genotoxicity,
cell proliferation, animal models in relation to species, routes of exposure and
doses, and the relevance of in vivo and in vitro assays and of mechanistic data.
The report recog¬nized that fibers can activate macrophages and epithelial cells
to release inflammatory mediators, cytokines, and growth factors, which may
alter epithelial and mesothelial cell proliferation; that fibers can bind to the
plasma membrane and activate cells; and that asbestos fibers can activate
multiple intracellular signaling pathways and transcription factors, including
oxidative stress-related pathways via redox-sensitive transcription factors such
as nuclear factor NFkB and the activator protein AP-1. The report expressed
concern about the interpretation of experiments using intratracheal and
intracavity injections, because high-dose exposures may result in uneven
deposition. It recommended that a multidose chronic inhalation study should be
undertaken in rats and hamsters, using a well-characterized amphibole sample,
and including relevant short-term end points or biomarkers, to be evalu¬ated in
future mechanistic studies.

A workshop report on chronic inhalation testing, sponsored by the U.S.
Environmental Protection Agency (118), reviewed test methods and conditions.
Concerning mesotheliomas, it noted that the rat inhala¬tion model may not be
sensitive enough to the induction of mesothe-liomas (except for erionite), and
that the hamster appeared more sensitive than the rat with respect to
fiber-induced mesothelioma, but less sensitive to the induction of lung tumors
and fibrosis. Comment¬ing on histopathologic evaluations, it recommended that a
dissecting microscope should be used to examine for mesotheliomas (a practice
that has not been widely used).

Muhle and Pott (119) expressed their concern about the adequacy of inhalation
models for risk evaluation of asbestos fibers, considering that rat lungs
weighing about 1g at the start of an experiment, with survival of little more
than 2 years, are compared with human lungs of 1000g, surviving several decades,
and that the number of cells at risk and the number of cell generations is much
higher in humans. They stated, “All German institutions that were involved with
the assess¬ment of the cancer risk from fibers concluded that the inhalation
model with rats is not sensitive enough to predict cancer risk due to the much
longer lifespan of humans.” The complexities and pitfalls of human risk
evaluations based on tests on different animal species were reviewed by Maxim
and McConnell (120). With advances in tracing the molecular pathways of
mesothelioma carcinogenesis, one hopes that the biologic significance of
different animal models will become better understood.

Molecular Mechanisms in Mesothelioma Pathogenesis, as Studied In Vivo in Animal
Models: As reviewed in this chapter, mesotheliomas can be induced in animal
models by a variety of agents, not only by fibrous minerals, but also by several
organic carcinogens, by radioactive mate¬rials, and by viruses. In addition,
co-carcinogenic effects have been reported, for example, by inhalation of quartz
particles combined with asbestos fibers.

Considerable advances have been made in the past decade in iden¬tifying
molecular pathways that characterize mesothelioma cells. Most of these studies
have used human or animal mesothelial cells or mesothelioma cells in culture and
were recently reviewed (121). The study of messenger RNA (mRNA) expression
patterns at different stages of asbestos-induced carcinogenesis in rats showed
that several genes (c-myc, fra-1, and egfr) were upregulated in pretumorous
tissue, in asbestos-induced tumors and in cells treated with asbestos in vitro;
and proteins associated with cell adhesion were also upregulated (122).

The role of the tumor suppressor genes was studied mostly in human mesotheliomas
and derived cell lines, as recently reviewed (123). Homozygous loss of the p16
INK4 locus was found in the tumor cells, and alteration of p16INK4a appears to
play a critical role in mesothelial cell tumorigenesis. Alterations of the
p16(INK4) locus were found in about one third of human malignant mesothelioma
specimens (124). It was concluded that the available data suggest that
alteration of either product of the CDKN2A locus, i.e., p14ARF or p16INK4a,
contributes to the pathogenesis of mesothelioma (123).

Wild-type p53 was detected in human mesothelioma-derived cell lines at levels
about four times higher than in human fibroblasts (125). In rat mesotheliomas
induced by erionite or asbestos and in cell lines derived from spontaneous or
induced rat mesotheliomas, p53 was found to be rarely mutated (126). The role of
p53 was discussed above in relation to mesothelioma induction in transgenic
p53+/- mice.

The rat neurofibromatosis 2 (NF2) tumor suppressor gene was found to be mutated
in 40% of human mesotheliomas; in contrast, rat

mesotheliomas and derived cell lines showed no DNA sequence alter¬ations,
suggesting a different role of this gene between human and rat asbestos-induced
mesotheliomas (127).

Cells derived from the appropriate animal models, such as mesothe-liomas induced
by intraperitoneal injection of asbestos, were used to identify the
transcription factor AP-1 as a major target of asbestos-induced signaling
pathways, and the induction of c-fos and c-jun mRNAs in rat pleural mesothelial
cells early after exposure to carcino¬genic fibers (128). The AP-1–dependent
member of the fos family of proto-oncogenes, fra-1, was recently identified as a
factor required for asbestos-induced transformation of mesothelial cells through
the extra¬cellular signal-regulated kinase – mitogen-activated protein kinase
(ERK-MAPK) cascade (129). The multifunctional cytokine interleukin-6 (IL-6) was
shown to be an autocrine growth factor for normal human pleural mesothelial
cells and to be expressed together with mRNA for the transmembrane components of
the IL-6 receptor complex, i.e., the IL-6 binding molecule, gp80, and the
signaling molecule, gpl30 (130). The interconnections of the pathways of the
IL-6 complex, the AP-1 and other transcription factors, and the redox-sensitive
pathways were pointed out (131). The role of several growth factors, including
platelet-derived growth factor (PDGF), has been considered (132). In murine
peritoneal mesotheliomas, a profound downregulation of lymphocyte surface
markers was found in tumor-infiltrating lymphocytes; signifi¬cant amounts of
transforming growth factor (TGF)-b, IL-6, IL-1, and of tumor necrosis factor
(TNF)-a were produced by the mesothelioma cells (133,134).

Two recent studies evaluated the expression of transmembrane adhe¬sion molecules
in human mesotheliomas and in other tumor types. Comparing epithelial-type
cadherin (E-cadherin), E-selectin and vas¬cular cell adhesion molecule (VCAM) in
mesotheliomas and peripheral pulmonary adenocarcinomas, it was found that only
E-cadherin showed a significant difference of expression: highly positive in the
adenocarcinomas, but negative or weak in the mesotheliomas (135). In a study on
20 human diffuse mesotheliomas (16 epithelioid and four sarcomatous), compared
with other tumors, E-cadherin was found positive in 4/20 (20%) and neural-type
cadherin in 14/20 (70%), and immunoreactivity was observed mainly in
epithelioid-shaped tumor cells (136). Whether this marker for differential
diagnosis is appli¬cable to the various animal models of mesothelioma remains to
be determined.

Several of these signaling and transcription pathways are related to conditions
involving chronic inflammation and fibrosis, and have been studied also for
silica-induced pathology (104). Their specificity in rela¬tion to mesothelioma
induction remains to be further defined.

The concept of “pathway pathology” was recently proposed to refer to different
pathways of signaling molecules and transcription factors linked to specific
morphologic patterns in the corresponding tumor pathology (137). Such criteria
of molecular pathway identification could be applied to the study of tumors of
different etiologies; they

would become pointers to the causative agents and mechanisms of different groups
of tumors, and could be termed “pathway etio-pathogenesis” (104). The critical
differences in the pathology of mesotheliomas induced in hamsters by
intrapleural injection of either asbestos or SV40, cited above (106), could be a
fruitful case study for both of these approaches to molecular pathways.

Conclusion

This review of in vivo animal models of mesothelioma shows that in the 40 years
since their first development they have provided solid evi¬dence for the
experimental induction of mesothelioma by a variety of carcinogenic agents, most
prominently fibrous minerals, and by some viruses, especially SV40, which was
first identified by animal tests as a specific etiologic agent of mesothelioma.
The close similarity of histopathology in the animal models and in human
mesotheliomas has strengthened their usefulness for mechanism studies.
Variations in sus¬ceptibility to mesothelioma induction by species, strain, sex,
route of exposure, and site of origin offer a choice of animal models for
mech¬anism studies. The high susceptibility of hamsters versus rats, male versus
female rats, and of peritoneal and especially testicular mesothe-lium in rats,
demonstrated in a number of studies with different agents, could be used to
investigate the underlying mechanisms.

The recent expansion of our knowledge of molecular pathways involved in
carcinogenesis opens up a remarkable opportunity for a better definition of the
relationships of specific etiologic factors and cofactors and induced tumor
types. The development of in vitro cellu¬lar models, derived from established in
vivo models, both human and animal, allows further comparative investigations.
One wishes to see a greater development of animal models addressed to specific
mecha¬nisms and pathways. The recent study of mesothelioma in p53 transgenic
mice (64) provides a stimulating example. Transgenic and gene-deleted (knockout)
animal models offer further opportunities to explore molecular mechanisms and
pathways, and to evaluate their relevance for conditions of genetic
susceptibility, tumor biology, and potential tumor therapy.