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

Transcription

1 Vet. Pathol. 16: (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 and J. M. RICE 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 proliferations, 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, 11, 13, 161. Carcinogens, other than asbestos, that have induced testicular mesotheliomas in rats include N-nitrosopyrrolidine 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 CD males (Charles River Breeding Laboratories. North Wilmington. Mass.): 60 Buffalo males (Texas Inbred Mouse Co.. Houston. Tex.): and 46 Fischer 344 males 574

2 DMN-OAc-Induced Mesotheliomas in Rats 575 (NIH, Bethesda, Md.) were obtained. All rats were housed three per 13X 15-inch polycarbonate cage on 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 CD and Buffalo strains and 16 F344 control rats were injected with phosphate buffer alone. Rats were fed Purina Lab (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 hyaluronidase [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 intraperitoneal 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 distributed material that reacted positively with colloidal iron staining for mucopolysaccharides. 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 or 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

3 576 Berrnan and Rice

4 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 CD 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 elsewhere. In our study, DMN-OAc induced a high incidence of testicular mesotheliomas when injected intraperitoneally into rats of three strains. In this study, no mesotheliomas 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 mesothelium 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.

5 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 originating 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

6 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 tumonths liomas proliferamors group tions F344 Control Treated I1 CD Control I 2 4 Treated Buffalo Control I 2 0 Treated 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. Intraperitoneal 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 mesothelial cells or multilayered masses of mesothelial cells. The lesions were characterized, 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

7 580 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-neoplastic lesions with only a certain probability of progession to mesothelioma. Proliferative 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 frequency of induced mesotheliomas was seen only in rats injected intraperitoneally [I]. Fluid given by intraperitoneal injection easily reaches the testicular mesothelium

8 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 testicular 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. References I BERMAN, J.J.; RICE, J.M.; WENK, M.L.; ROLLER, P.P.: Dependence on route of administration of tumor spectrum in rats resulting from single or multiple injections of methyl(acetoxymethy1)nitrosamine. J Natl Cancer Inst (in press. 1979) 2 DEKLERK, D.P.; NIME, F.: Adenomatoid tumors (mesothelioma) of testicular and paratesticular tissue. Virology , FLIGIEI., Z.; KANEKO, M.: Malignant mesothelioma of the tunica vaginalis propria testis in a patient with asbestos exposure. A case report. Cancer GOULD, D.H.: Mesotheliomas of the tunica vaginalis propria and peritoneum in Fischer rats. Vet Pathol , GREENBLATT, M.; LIJINSKY, W.: Nitrosamine studies: neoplasms of liver and genital mesothelium in nitrosopyrrolidine treated MRC rats. J Natl Cancer lnst 48: JOSHI, S.R.; RICE, J.M.; WENK, M.L.; ROLLER, P.P.; KEEPER, L.K.: Selective induction of intestinal tumors in rats by methyl(acetoxymethyi)nitrosarnine, an ester of the presumed reactive metabolite of dimethylnitrosamine. J Natl Cancer lnst 58: , KAUFMAN, D.; KAUFMANN, W.; RICE, J.: Unpublished data 8 MCCLURE, H.M.; GRAHAM. C.E.: Malignant uterine mesotheliomas in squirrel monkeys following diethylstilbestrol administration. Lab Anim Sci MCMANUS, J.F.; MOWRY. R.W.: Staining Methods-Histologic and Histochemical. pp ; Hoeber, New York MORRIS, H.P.; WAGNER. B.P.: RAY. F.E.; SNELI.. K.C.; STEWART. H.L.: Comparative study of cancer and other lesions of rats fed N.N-2.7-fluorenylenebisacetamide or N-2-fluorenylacetamide. Natl Cancer Inst Monogr MUKERJEE. M.G.; NORRIS. M.: STRUM, D.P.: MI ri EMEYER. B.T.; BOKSKI, A.A.: Mucinous adenoid tumor of the paratesticular tissue. J Urol , O SHEA, J.D.; JABARA, A.G.: Proliferative lesions of serous membranes in ovariectomised female and entire male dogs after stilbestrol administration. Vet Pathol I 13 PELFRENE, A.: GARCIA, H.: Histology of chemically-induced rnesotheliomas in MRC- Wistar rats. Tumori , ROLLER. P.P.: SHIMP. D.R.; KEEFER. L.K.: Synthesis and solvolysis of methyl(ace-

9 582 Berman and Rice toxymethy1)nitrosamine. Solution chemistry of the presumed carcinogenic metabolite of dimethylnitrosamine. Tetrahedron Let , ROSAI, J.; DEHNER, L.P.: Nodular mesothelial hyperplasia in hernia sacs: a benign reactive condition simulating a neoplastic process. Cancer , SASS, B.: KABSTEIN, L.S.; MADISON. R.; NIMS. R.M.: PETERS, R.L.; KELLOFF, G.J.: Incidence of spontaneous neoplasms in F:u4 rats throughout the natural life-span. J Natl Cancer Inst W , STAVRIDES, A.; HUTCHESON. J.B.: Benign mesothelioma of testicular appendages. a morphologic and histochemical study of 7 cases and review of theories of histogenesis. J Urol , 1960 Request reprints from Jules Berman. National Institutes of Health. Building 37. Room 3A09. Bethesda. MD (USA).