Mesotheliomas and Proliferative Lesions of the Testicular Mesothelium by liaoqinmei

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Mesotheliomas and Proliferative Lesions of the Testicular Mesothelium Produced in
 Fischer, Sprague-Dawley and Buffalo Rats by Methyl(acetoxymethyl)nitrosamine
                                   (DMN-OAc)
                                     J. J. Berman and J. M. Rice
                                       Vet Pathol 1979 16: 574
                                 DOI: 10.1177/030098587901600510

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Vet. Pathol. 16: 574-582 (1979)




    Mesotheliomas and Proliferative Lesions of the Testicular
Mesothelium Produced in Fischer, Sprague-Dawley and Buffalo Rats
       by Methyl(acetoxymethy1)nitrosamine (DMN-OAc)

                                     J. J. BERMAN J. M. RICE
                                                and


      Laboratory of Experimental Pathology. National Cancer Institute. Bethesda. Md.



   Abstract. A single intraperitoneal dose of methyl(acetoxymethy1)nitrosamine ( 13 mg/kg
body weight) given to 78 5-week-old male rats induced 25 mesotheliomas; two mesotheliomas
were found in 67 control rats. All mesotheliomas arose from the peritesticular mesothelium
and had a typical microscopic appearance of branching papillary fronds with a collagenous
core covered by one or many layers of plump tumor cells. Cytoplasm of tumor cells contained
material that reacted positively to a colloidal iron stain and was labile to hyaluronidase. In
addition to frank mesotheliomas. 16 lesions. which we called atypical mesothelial prolifera-
tions, were found. These consisted of a single focus of plump mesothelial cells overlying an
area of thick stroma. Often these foci included short. non-branched papillary projections
above the surface of adjacent normal mesothelium. Twelve of the 16 lesions occurred in
methyl(acetoxymethy1)nitrosamine-treated rats.


   Tumors of the genital or peritoneal mesothelium have been reported to occur in
rats of a wide variety of strains 141. Reports of testicular mesotheliomas usually have
been of one or a small number of cases 12, 3, 10, 1 1 , 13, 161. Carcinogens, other than
asbestos, that have induced testicular mesotheliomas in rats include N-nitrosopyrrol-
idine administered in drinking water [5] and N-methyl-N-nitrosourea administered
via the portal vein to partially hepatectomized rats 171. Hyperplasias of mesothelium
have been reported in the hernia sac and on testicular appendages in man [15, 171
and on uterine and testicular serosa in dogs treated with stilbestrol [12], an agent
reported to produce true mesotheliomas in monkeys [8]. In neither case were these
lesions shown to have neoplastic potential. To our knowledge no hyperplasias of
testicular mesothelium have been reported in rats.

                                       Materials and Methods
  Methyl(acetoxymethy1)nitrosamine (mol wt 132) was synthesized as previously described
[ 141: it was distilled twice before use. Chemicals were dissolved in phosphate buffer at pH 7.0
(physiologic sodium ion concentration). All solutions were prepared within 2 hours of
administration.
  Sixty Sprague-Dawley C D males (Charles River Breeding Laboratories. North Wilmington.
Mass.): 60 Buffalo males (Texas Inbred Mouse Co.. Houston. Tex.): and 46 Fischer 344 males
                                                         574




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                           DMN-OAc-Induced Mesotheliomas in Rats                             575

(NIH, Bethesda, Md.) were obtained. All rats were housed three per 13X 15-inch polycarbonate
cage on 0.125-inch corn cob bedding.
   For carcinogenesis studies 30 rats of each strain were lightly anesthetized with fluothane to
reduce activity and insure the intraperitoneal instillation of carcinogen into the peritoneal
cavity. Rats were injected with 13 mg DMN-OAc/kg body weight. Thirty control rats of C D
and Buffalo strains and 16 F344 control rats were injected with phosphate buffer alone.
   Rats were fed Purina Lab Chow@ (Purina, St. Louis, Mo.) and had access to water ad
libitum. They were weighed weekly for the first month and once a month thereafter. Rats were
examined daily, and those that were seriously ill were killed. Surviving rats were killed 83 to
90 weeks after treatment. All rats were necropsied except for 21 that were cannibalized, grossly
autolyzed or died within the first 4 months of life. Rats that died before 4 months were
considered not at risk for carcinogenesis and were excluded from data tabulation. Tissues from
each rat were studied microscopically, including both testes, abnormal organs or tissue masses.
Tissues were fixed in 10% buffered formalin, embedded in parafin, sectioned, and stained
with hematoxylin and eosin (HE) for histologic examination. Testes were fixed whole and
later bisected longitudinally for embedding. Testes slides were examined without knowledge
of rat strain or treatment group. Testes containing mesotheliomas also were stained by Hale’s
modification of the colloidal iron reaction for mucopolysaccharides with or without hyaluron-
idase [9]. In rats in which a testis contained a small proliferative mesothelial lesion, paraffin
blocks of both testes were cut to produce three more sections, each 60 micrometers apart, and
stained by HE.


                                                   Results
   Single intraperitoneal administrations of 13 mg/kg body weight (half the intraper-
itoneal LD5@   determined for 5-week-old male CD rats [ti]) induced 25 testicular
mesotheliomas in 78 treated rats. Large growths were seen grossly as gelatinous or
white fibrous coatings partially surrounding the testis. Small lesions were not seen
grossly, and were found by microscopic examination of a random section in 10 of
our 27 cases. Thin, often branching, fronds with a single layer of plump neoplastic
mesothelial cells covering a collagenous core were always seen (fig. I , 2). The
proliferating mesothelium spread diffusely over the surfaces of the testes, rarely
forming a solid tumor. Occasionally tumor cells were flat, and looked identical to
normal mesothelial cells (fig. 1). Although some tumors contained massive amounts
of stroma (fig. I), there were no examples of the sarcomatoid variant of mesothelioma
common in man. Large mesotheliomas often had dense cellular masses of uniform,
plump tumor cells in addition to papillary structures (fig. 3, 4). Nuclei were round to
oval, eccentric, occupied most of the cell, and contained a single prominent nucleolus.
Mitoses were found readily. Cytoplasm of tumor cells contained a uniformly distrib-
uted material that reacted positively with colloidal iron staining for mucopolysaccha-
rides. This activity was abolished by digestion with testicular hyaluronidase. All
mesotheliomas examined consistently stained positively by the colloidal iron method;
this activity always was associated with the mesothelial cells. The often abundant
tumor stroma gave little or no reaction.
   Also seen were small proliferative lesions of atypical mesothelium that lacked
multiple o r branching papillary fronds and were confined to one discreet clump of
cells rather than the diffuse network of papillae. These lesions consisted of one focus



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576                     Berrnan and Rice




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                            DMN-OAc-Induced Mesotheliomas in Rats                               577

of plump mesothelial cells always overlying a section of thick stroma (fig. 5-8).
Occasionally, lesions were found on parts of peritesticular fascia loosely covering the
testis or epididymis. Often a slight thickening of stroma beneath the proliferated
mesothelial cells was apparent. Sixteen atypical proliferative mesothelial lesions were
found. Twelve of these were in carcinogen-treated groups. Subsequent sections from
testes from six of the 16 cases of atypical proliferative mesothelial lesions contained
foci of frank mesothelioma.
   The greatest incidence of mesotheliomas occurred in the DMN-OAc-treated
groups, although one mesothelioma was found in a control C D rat, and another in
a Buffalo rat (table I). In addition, numerous Leydig cell tumors were seen in control
and treated rats, particularly in the F344 strain. There were no strong coincidences
of mesotheliomas and Leydig cell tumors, but in the treated F344 strain there was a
slight coincidence of mesotheliomas and Leydig cell tumors (P(Chi-square)=O. 1).

                                                 Discussion
   Testicular mesotheliomas have been reported in a variety of animals [4, 5, 7, 10,
13, 161 and in man [2, 3, 11, 171. Reports of induced testicular mesotheliomas or
mesothelial proliferations have involved agents that were administered either in the
drinking water [ 5 ] or intravenously [7] so that the chemical agent was distributed
throughout the body. The induction of testicular mesothelial tumors and hyperplasias
to the virtual exclusion of mesotheliomas arising from other mesothelial-lined surfaces
indicates that testicular mesothelium has properties distinct from mesothelium else-
where. In our study, DMN-OAc induced a high incidence of testicular mesotheliomas
when injected intraperitoneally into rats of three strains. In this study, no mesothe-
liomas arose from any peritoneal mesothelium except that of the testis, although we
have seen occasional splenic and pulmonary mesotheliomas in similar experiments.
   The proposed mechanism through which DMN-OAc is metabolized to a reactive
ultimate carcinogen has been discussed [6, 141. Briefly, it is hydrolyzed by ubiquitous
esterases and converted to the procarcinogen methyl (hydroxymethyl) nitrosamine,
which rapidly decomposes to form a reactive methylating species, methyl diazonium
hydroxide and formaldehyde. DMN-OAc, as judged by trials in rats [l], behaves


   Fig. 1: Massive papillary mesothelioma, abundant stroma in papillae. Numerous areas of
transition between round and flat neoplastic mesothelial cells on surfaces of large and small
papillae. Buffalo, 16 months old. HE.
   Fig. 2 Mesothelioma. Papillary projections of tumor tissue. Single outer layer of plump
neoplastic mesothelial cells around prominent fibrous stalk surrounds fibrous connective tissue
(center) supporting efferent ductules of testis. Deposits of neoplastic mesothelial cells like those
of papillae on surface of projecting connective tissue. Transition between flat normal meso-
thelium and pseudostratified neoplastic mesothelial cells projecting from surface of tunica
vaginalis. F344, 15 months old. HE.
   Fig. 3: Highly cellular mesothelioma. Dense areas of tumor cells form tumor masses.
Buffalo, 14 months old. HE.
   Fig. 4 High magnification of papillary mesothelioma; nuclear detail including mitoses.
Buffalo, 14 months old. HE.



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   Fig. 5 Small proliferative mesothelial lesion in rat with no evidence of mesothelioma.
Mesothelial cells are plump and rest on short projections of connective tissue extending from
underlying fascia. Buffalo, 19 months old. HE.
   Fig. 6 Localized proliferative lesion. Single papilla. covered with mesothelium and origi-
nating from hyaline stroma, projects from surface of tunica vaginalis. Charles River CD, 19
months old. HE.
   Fig. 7: Multiple papillae project from fibrous, hyaline stroma. Charles River CD. 21 months
old. Buffer control. HE.
   Fig. 8 Focus of plump neoplastic mesothelial cells adjacent to epididymis. Charles River
CD, 19 months old. HE.
                                                       578

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                           DMN-OAc-Induced Mesotheliomas in Rats                                                  519

   Table I. Incidence of testicular lesions in rats treated with DMN-OAc, 0. I mmole/kg

                  Number of           Average age                                                       Interstitial
                                                                 Mesothe-                 mesothelial
                   rats in             at death,                                                          cell tu-
                                        months                    liomas                  prolifera-       mors
                    group                                                                   tions
   F344
    Control           15                     19.0                       0                     0              7
   Treated            25                     14.8                       9                     5             I1
  CD
   Control            27                     20.9                       I                     2              4
   Treated            27                     14.8                       4                     4              1
  Buffalo
    Control           25                     17.2                       I                     2              0
   Treated            26                     16                        12                     3              0


much like a direct-acting carcinogen; that is, one that does not require metabolic
activation that usually is enzyme-mediated through a mechanism found preferentially
in certain cells. Subcutaneous administration of DMN-OAc induces a variety of
tumors of epidermal and dermal (mesenchymal) cell populations, while intravenous
administration causes a high incidence of lung and Zymbal’s gland tumors. Intra-
peritoneal injection causes a high incidence of tumors of intestinal rnucosa, as well
as a variety of soft tissue neoplasms arising from the intestinal mesenchyme [I].
Intraperitoneal injection also causes splenic angiosarcomas, abdominal schwannomas
and testicular mesotheliomas.
   We did not classify the 16 proliferative lesions of testicular mesothelium as
mesotheliomas because of their small size and apparent absence of either multiple or
branching fronds with a connective tissue core covered by a layer of plump meso-
thelial cells or multilayered masses of mesothelial cells. The lesions were character-
ized, however, by plump mesothelial lining cells and focal proliferations of cells that
often formed short, non-branching papillae (fig. 5-7). In every case the stroma
underlying the proliferations of mesothelial cells was thick, and often the stroma,
with overlying mesothelial cells, was raised (fig. 5 ) . The proliferation of mesothelial
cells and protrusion of underlying stroma resemble features of the papillae seen in
frank mesotheliomas. On a morphologic basis the proliferative lesion differs from
the mesothelioma only in extent of involvement, and on morphologic grounds alone
it was not possible to differentiate with assurance between early, independent
preneoplastic lesions (fig. 5 , 6) and minute foci of dissemination from an inapparent
adjacent primary tumor (fig. 7, 8). The fact that 12 of 16 proliferative mesothelial
lesions were seen in rats treated with DMN-OAc strongly suggests that the small
proliferative lesions usually were related to exposure to the carcinogen. Six of the 16
testes, in which atypical proliferative mesothelial lesions were seen, had areas of
frank mesothelioma elsewhere. Since the tumors progress by seeding the tunica
vaginalis, it is possible that these atypical lesions were metastases. In the remaining
 10 testes no mesotheliomas were detected. All frank neoplasms must go through a




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580                                         Berrnan and Rice

stage when they are small, and the presence of small atypical lesions with all the
attributes of frank neoplasms except for size is consistent with an early stage of
neoplastic development. A final possibility is that the small lesions were either
hyperplastic lesions with no tendency to become mesotheliomas or were pre-neo-
plastic lesions with only a certain probability of progession to mesothelioma. Prolif-
erative lesions of testicular mesothelium exclusive of mesothelioma have not been
reported in the rat.
   All the testicular mesotheliomas seen in our study were identical to those described
by others [4] and involved the testicular tunic and epididymis. The appearance of
small lesions many months after a single exposure to a short-lived reactive chemical
is compatible with incipient neoplasia and in the absence of any sign of chronic
inflammation is unlikely to be reactive hyperplasia. These tumors grew by extension
and expansion, first covering the testes with rather hard, smooth deposits of numerous
thin layered fronds and later enlarging as the mesothelial layer lining the fronds
continued to proliferate, forming dense cellular masses. Because these tumors usually
grow as a thin covering over the testes, they are often undetected at autopsy. Because
incubation of sections with hyaluronidase before staining completely abolished
subsequent colloidal iron reaction, hyaluronic acid or chondroitin sulfate or both
were the mucopolysaccharides chiefly responsible for the colloidal iron reaction.
Additionally, PAS staining of the mesothelial cells was quite faint, indicating that
hyaluronic acid or chondroitin sulfate were the chief mucopolysaccharides in these
cells, as both those sugars release low yields of aldehydes upon periodate oxidation
and therefore stain only weakly by PAS. Other mucopolysaccharides produce a
strong PAS reaction. The observation that tumor cells produce hyaluronic acid or
chondroitin sulfate or both with little production of other mucopolysaccharides
indicates that the tumor cells function similarly to normal peritoneal mesothelial cells
which produce an ascitic fluid rich in hyaluronic acid. In our cases of mesothelioma
we noticed no signs of local invasiveness or of distant metastases, but the tendency
of these tumors to grow progressively and to spread further and further along serosal
surfaces, eventually to enter and expand inside the peritoneal cavity, suggests that it
is appropriate to classify this neoplasm as malignant.
   Notable for their absence were mesotheliomas arising from peritoneal surfaces
other than the testicular coverings. In another study with DMN-OAc [ l ] , one
mesothelioma arising from splenic serosa was seen in a female rat treated with a
single injection of DMN-OAc. This was the only peritoneal mesothelioma to arise
from non-testicular mesothelium in any of our studies of rats of either sex treated
with DMN-OAc. In a study of “spontaneous” neoplasms in Fischer F344 rats [ 161,
all grossly detectable mesotheliomas were intraperitoneal; testes were not examined.
In this and other studies in rats the ability of mesothelial cells to respond to chemical
carcinogens is a property almost exclusive to testicular mesothelium. In a previous
study involving the administration of DMN-OAc by different routes, a high fre-
quency of induced mesotheliomas was seen only in rats injected intraperitoneally
[I]. Fluid given by intraperitoneal injection easily reaches the testicular mesothelium




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                          DMN-OAc-Induced Mesotheliornas in Rats                         58 I

because the peritoneal extension that covers the testes is patent in the rat. Presumably,
DMN-OAc produces testicular mesothelial tumors by direct and immediate action
on testicular mesothelium. The absence of extra-testicular mesothelial tumors is
unexplained. Our laboratory is conducting experiments to determine whether testic-
ular mesothelial cells have a higher normal rate of proliferation than do mesothelial
cells at extratesticular sites. The higher rate of induced testicular mesotheliomas may
simply result from a greater percentage of dividing cells available as targets for
carcinogens in testicular mesothelium.
  Our study demonstrated an agent capable of inducing testicular mesotheliomas (a
tumor seen rarely in untreated rats or in rats treated with most other carcinogens). In
addition, the presence of small proliferative lesions of testicular mesothelium whose
biological identity remains to be elucidated was reported. Because these lesions are
small they can be readily missed by casual examination of the testis, either grossly or
microscopically. Atypical foci may be associated with frank mesotheliomas found by
sectioning deeper into the tissue block.

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582                                         Berman and Rice

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