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(CANCER KliSlsARCH 31, 516-522, May 19711 Oncogenic Response of Vizi Skin, Langs, and Bones to Viny? Chcoride1 P. L. Viola, A. Bigotti, and A. Cnputo Regina Elena Institute far Cancer Research, Rome, Italy < I < F i ! SUMMARY Rats (Ar/IRE Wisi3r strain) exposer! for 12 months to vapors of vinyl chloiide developed tumors of the skin, lungs, and bones. The cutaneous tumors, which always appeared in the area in which submaxillary and parotid glands are located, have been histologically recognized as epidermoid carcinomas, papillomas, and mucoepidermoid carcinomas. The morphological characteristics of lung tumors, which occurred in a lower percentage, were mainly of the adenocarcinoma type, with the exception of a single epidermoid tumor originating from the epithelial covering cells. In a minor number of rats, a large proliferation of cartilaginous tissue diagnosed as osteochondroma developed in the metacarpal and metatarsal regions of the four limbs. INTRODUCTION The oncogenic properties of some chemical organic compounds used for the preparation of "plastics" have been widely investigated, and their limits and effectiveness have been well established. Detailed information on this subject may be found i.i the literature (5, 6, 8); however, all information and references are exclusively related to highly polymerized compounds of roughly the same size as used in various industries. Oncogenic polymers produce sarcomatous tumors, with the exception of polyuiethans which, a* reported by Hueper (6), also induce adenocarcinomas. Recently, it has been demonstrated that sarcomatous tumors (1--4, 7) are transplantable, and it has been suggested that they may originate from the tissues of the cap$uie that gradually covers the pi.tsiic film. Thu?.. Rf-mri tt a> (2-4) have observed premnlignanf areas made of poorly differentiated fibroblasts which were firmly attached to the pLsfic film up to the moment of malignant transformation. This investigation demonstrates th2t even the monomer vinyl chloride possesses oncogenic properties when used with an appropriate model different from the models previously reported by several authors (9--12). The carcinogenic response 1 A preliminary report of the results reported here was given at the Tenth International Cancer Con*res.-.. Houston, Texas, May 22-24, 1970. Received July 31, 1970; accepted January 12, 1971. to vinyl chloride follows a singular pattern for the different tissues and organs of the rat. 1 MATERIALS AND METHODS The experiments were performed with vinyl chloride I (CM---CITCI, the monohalcgenate derivative of ethylene) of a S commercial grade (99% purity') assumed to contain insignificant amounts of various noncarcinogemc ; contaminants. ; Three-month-old Wistar (Ar/IRE) male albino rats (about ; 150 g body weight) were exposed to vinyl chloride vapors for > 4 hr a day, 5 days a week, for 12 months. The animals were kept in metal or plastic, air-tight cages in which a constant ; flow of air, containing }% v/v (equal to 30,000 ppm) of vinyi chloride, was introduced. Twenty-five rats of the same strain ; were the control group. At the end of the treatment, the j surviving animals were `mied at 2'J-dzy intervals, and the most ! important tissues and organs were examined histologically by standard methods. During the period of exposure, the animats i were slightly soporific; however, the firs: few months of treatment were well tolerated and no changes in growth or 1 behavior were noticed. After 10 months of treatment, some [ animals began to show a bard mass in the paraauricular region ' which-became progressively larger until it reached the size of a ; walnut or slightly larger. In most cases, the swelling was ' unilateral; it was bilateral in only a few animals. After ! or 2 ! months, the growing masses became ulcerated and discharged i necrotic debris, while a certain amount of tumorous tissue \ began to form on their surfaces. In addition, we observed that i the masses were an integral part of the paraauricular region i and could not be distinguished from the local tissues. Caseous i necrotic zones were iound. Pleura and pericardium often : showed diffuse inflammation of a fibrous nature and, in many j cases, the lungs were covered with a number of white I formations as large as giains of rice or even larger and harder r than the lungs themselves. In 2 cases, the lungs were ! hemorrhagic with milky, thick fluid in the pleural cavities. The i liver was sometimes increased in size and very fragile. The | animals were subjected to X-ray analyses at different intervals j from the beginning of treatment in order to control the state | of the skeletal bones. i All the animals that inhaled vinyl chloride showed a series ! of parenchymal lesions. Among these, most prominent were : the disappearance of granular and Purkinje cells, degeneration ! \ * i( * , 516 CANCER RESEARCH VOL. 31 * i dTH 000011739 fi Oncogenic Activity of Vinyl Chloride 0f the cerebellum, severe chronic hepatitis, interstitial pneumonia, and moderate swelling of the kidney parenchyma, often assuming the pattern of tubuloncphrosis. results The rates of survival and the main findings are summarized in Table 1. Almost all the animals developed tumors of the skin and lungs. Very few animals developed bone tumors; in ihese cases, the tumors were localized in the metacarpal and metatarsal bones of all 4 extremities. Skin tumors were by far die most frequent, amounting to 65 or 707*. Skin Tumors. The tumor that developed most frequently in thcparaauricular region was the epidermoid carcinoma, hut we also observed papillomas and rarely mucoepidermoid carcinomas. In all cases, the nenp'.-isms were of epithelial nature. The 3 patterns noticed cannot be compared to vaiious fcistotypes but rather to a transition of one type into the other or. and which is more likely, to different stages of the same proliferative type.I Warty subauriciihr growths occurred in some rats. The histological picture showed papillar epithelial proliferation, with progressive increase in the thickness of the epidermis (Fig- 0The papillary warts of exophytic type had a fibrous vascular stroma and various degrees of inflammatory lymphocytic infiltration, marked hyperketatosis, parakeratosis, acanthosis, and areas of individual dyskeratosis or pearl-like horny formations. The epithelial cells of the penetrating columns (Fig. 2) were irregularly arranged and were frequently accompanied by an inflammatory infiltration of the dermis. The typical features of the Malpighian layer and of the stratum corneum of the prickle cells and of germinal layers were easily recognized (Fig. 3). The horny layer was composed largely of cell nests, which appeared at certain points within the epithelial masses and assumed the well-known appearance of "horny pearls" (Fig. 3, arrows)', they were made of-flattened and compressed prickle cells without nuclei and were located around a central core of keratin. Table 1 Oncogenic effects ofinhaled vinyl chloride as a function of time The animals were exposed lo vinyl chloride vapors for 4 hr a day, 5 days a week, for a total of 12 months, in a constant flow of air containing 3% v/v vinyl chloride. Rat 1 2,3 4 5 6 7 8 14 16,17 21 22 23 24 25 26 Survival rate 300 280-300 310 333 337 347 347 354 359 3S0 38Q 380 380 380 380 Tumors Skin Lungs Bones Mucoepidermoid carcinoma Epidermoid carcinoma, kera tinizing type Epidermoid carcinoma, kera tinizing type Papilloma, keratotic type Epidermoid carcinoma Epidermoid carcinoma Mucoepidermoid carcinoma Epidermoid carcinoma Epidermoid carcinoma Epidermoid carcinoma Epidermoid carcinoma - Epidermoid carcinoma Epidermoid carcinoma Epidermoid carcinoma Epidermoid carcinoma Adenoacanttieme No tumor Adenocarcinoma No tumor No tumor No tumor No tumor No tumor Adenocarcinoma No tumor Adenocarcinoma No tumot Mucus-producing adenocarcinoma (alveolar cell carcinoma?) No tumor Squamous cell carcinoma Osteochondroma No tumor No tumor Osteochondroma Osteochondioma No tumor Osteochondroma No tumor No tumor No tumor Osteochondroma No tumor No tumor No tumor No tumor / i l i r IAY 1971 517 DTH 000011740 P. L. Viola, A. Bigotti, and A. Caputo Keratinization was irregular and often parakeratosis was cartilaginous zones possessing regressive features, such as ' observed in the areas of the tumor in which the cells were fibrosis and hyalinosis. The perichondrium appeared often as a loosely aggregated and undergoing an individual rather than a compressed and structurally altered fibrous tissue. ; collective type of keratinization (Fig. 4). !n dyskcratotic areas, In some cases, the tumor growth was related to the stage of' a few epithelial cells tended to form pearls. The tumor seldom the endochondral ossification occurring below the "epiphysial ; showed an undifferentiated growth with cellular plate." Ossification was irregular, so that osseous trabeculae , pleomorphism and several mitotic figures (Fig. 5). A few varied greatly in thickness and contours. In other cases, foci of tumors showed little nests of isolated pale cells (Figs. 6 and 7) active cellular proliferation, cartilaginous areas, chondroid of 3 types: mucin-producing cells (originating from the duct developmental nests, and calcified bones occurred in the : epithelium of sweat glands or salivary glands); squamous cells; deeper portions of the trabeculae (Fig. 18). 1 and intermediate cells with minor tendencies towards The new formation appeared to be an osteochondroma and I differentiation. consisted essentially of a bony protuberance capped by Respiratory Tract. These tumors, although occurring rarely, cartilage and a fibrous layer, which represented the j were mainly adcnocarcinomatous. Only in 1 rat did we observe perichondrium. | an epidermoid tumor originating from the epithelium-covering The fibrous layer was continuous with the periosteum of ! cells. Sometimes, the tumors were seen in their early the adjacent cortical bone and extended inward to form septa : development and consisted predominantly of cubic or separating and enclosing lobules of cartilage. ! columnar cells arranged as regular or irregular tubular and Control Animals. The control rats were kept under the same papillary elements (Hg. 8) and supported by poorly developed conditions as the experimental rats and at the appropriate time fibrous stroma (Fig. 9). were subjected to a constant llow of air without vinyl In other cases, glandular structures were often imperfectly chloride. None of these animals developed tumors or the types formed and appeared ar sheet-like proliferations of of parenchymal lesions developed by the rats that inhaled undifferentiated or pleomorphic character. The ceils showed a vinyl chloride. In a very few of the control rats, there was a i tendency to extend into the pulmonary parenchyma, thus swelling of the liver and kidneys. simulating the microscopic features of the so-called alveolar cell carcinoma (Fig. 10). Sometimes, this was the prevailing '! pattern. The pulmonary air sacs were limited by 1 or more DISCUSSION layers of cubic, columnar, or polyhedral cells with abundant : I cytoplasm that was faintly eosinophilic (Fig. 1!); frequently, As in many carcinogenesis studies in which multiple tumors adenopapillary excrescences spreading into the alveolar spaces arise in different tissues and organs, the data must be evaluated were present. - . - in terms of a more prompt positive response to be obtained In some areas, foci of cellular polymorphism with with the ideal concentration of the carcinogenic compound. , hyperchromatic nuclei were noticed; mucin was produced in The cutaneous system is the most susceptible to the oncogenic varying amounts and secreted into the lumen of the tubules effects of vinyl chloride. ; and acini. There were small pools of mucin in which signet ring Our experimental data do not explain why the cutaneous cells occurred, cither alone or in small clusters (Fig. 12). The tumors developed in the same site, i.e., the region including alveolar walls were often quite thick and, at times, showed an the area in which the submaxillary and parotid glands are ' inflammatory infiltration. located. It is possible that the salivary glands may by involved As mentioned before, a single tumor showed squamous in the concentration or excretion of vinyl chloride or some of : structures and appeared to have been formed mainly by its active decomposition products. This hypothesis is strongly ` spindle and oval undifferentiated cells (Fig. 12). supported by morphological findings showing the specific Bones, in the metacarpal and metatarsal regions of the 4 tendency of the developed tumor to appear as a : limbs, a large proliferation of cartilaginous tissues arose mucoepidermoid carcinoma, which indicates the active : outward from the periosteum and, from the appearance of its contribution of tire mucus glandular cells to the tumorous cells, seemed to derive directly from the cortical bone (Fig. growth Such kinds of Imtotypcs are often related to some 14) . The periosteum also grew and in some areas spread as glandular aggregates, and, therefore, the histogenesis of human (Inger-likc prolongations into the newly formed cartilage (Fig. mucoepidermoid carcinoma has been restricted to the cells of 15) . In these places, a gradual transition between fibrous intercalated ducts. This hypothetical interpretion must be ' cartilage, periosteum, and bone could be noticed. confirmed by future experiments in which the action of the The newly formed cartilage appeared irregular with atypical vinyl chloride should be restricted to the salivary system. At ! areas; the cells, which had nuclei larger than the normal the concentrations used, saturation and wetting of tire fur chondrocytes, lay in well-formed, capsulated lacunae. The cells might well be expected. Under these circumstances, the occurred singly, in pairs, or in tetrads and, although of natural cleansing habit of the rat might add a significant different size and shape, they usually contained a single, ingestion problem with subsequent concentration of vinyl darkly stained nucleus. The cartilaginous growth was not chloride in the salivary glands. The local extreme homogeneous, as shown by the extension of the finger-like concentration could ' . :he result of the difficulty of 2 prolongations into the boundary of newly formed tissue. complete cleansing by rhe rat. Besides the chondrobiastic, chondrocytic, and angiomatous The neoplastic response of the lower respiratory' tract, : areas which indicated a rapid growth, there were also although of lesser magnitude, is of relevant interest, since some 518 CANCER RESEARCH VOL. 3! DTH 000011741 Oncogenic Activity of Vinyl Chloride , jjjg tUniois were morphologically similar to fhose described for the skin. The hypothesis that mucus-producing cells may he capable of retaining vinyl chloride or its decomposition products seems to be relevant once more. Obviously, a different interpretation is required for the pathogenesis of the bone tumors and for their simultaneous incidence at the level of all 4 limbs. references 1 Bancrjee. M., and Bates. R. Prevalence t>f Heteroploidy in Plastic film-induced Primary Sarcomas. Brit. J. Cancer, 20: 555-563. 1966. 2 Brand, G., and Buocn, L. Polymer Yumorigcnesis: Multiple Prcncophstic Cones in Priority Order with Clonal Inhibition. Proc. joe. Exptl. Biol. Med.. J2S: 1153-115 s 968. 3. Brand. G., Buoen, L., and Brand. I. Malignant Transformation and Maturation in Non-dividing Cells during Polymer Tumorigcnesis. Proc. Soc. F.xnti. Biol. Med., 174: 675-678, 1967. ( grand. G., Buoen. L., arid Brand, I. Carcinogenesis from Poiyrner Implants: New Aspects from Chromosomal and Transplantation Studies during Premaiignancy. J. Natl. Cancer Inst., 39: 663-679, 1970. 5. Druckrcy, II. Lxpcrimcmcllc llcitracge wum Mechanismus der Carcinot'encn Wirkung. Arr.noiinittcl-l-orsch., /: 383-395, 1951. 6. Ilueper, W. C. Cancer Induction by Polyurcthan and Polysilicone Plastics. J. Nati. Cancer Inst.. JJ: 1005-1027. 1964. 7. Johnson. K.. Buocn, L., Brand, I., and Brand, G. Polymer Tumorigcncsis Clonal Determination of liistopathological Char "-tcristics during Early Preneoplasia; Relationships to Kai; ype. Mouse Strain, and Sex. J. Natl. Cancer Inst., 44: 785-793, 1970. 8. Kogan, A. Experimental Blastomogcnesis Caused by Synthetic Polymers (Plastics). Meditsina, Moscow, /: 215-237, 1965. 9. Mastromatteo, E., Eislier, A. M,, Christi, H., and Danziger, D. Acute Inhalation Toxicity of Vinyl Chloride to Laboratory Animals. Am. Ind. Hyp. Assoc. J., 21: 394-397, 1960. 10. Torkcrson, T. R., Oycn. 1'., and Rowe, V. K. The Toxicity of Vinyl Chloride as Determined by Repealed Exposure of Laboratory Animals. Ind. ilyg. J.. 354-361, October 1961. 11. Van Duren, B. L., Sivak, A., Goldschmidt, B. M.. Katz, C.. and Melchionnc. S. Carcinogenicity of HaTo-Ethers. 1. Natl. Cancer Inst., 43: 481-486. 1969. 12. Viola, P. L. Pathology of Vinyl Chloride. ICrh International Congress of Occupational Health. (Communication No. 38), Tokyo, 1969. Photomicrographs of the original article are not being distributed with this copy because of the poor quality of their reproduction. Figs. 1 to 18. All sections were stained with H &. E. Fig. 1. Squamous cell papilloma of the skin. X 25. Fig. 2. Transitional stage from papilloma to infiltrative type of cancer. X 80. '( Fig. 3. Finger-like trabeculae branch to form secondary processes showing horny pearl formation (arrows). X 25. ' i Fig. 4. Dyskeratotic area. Few epithelial cells with a tendency to form whorls. Little capacity to develop into horny pearls. X 250. Fig. 5. Atypical cells partly lacking prickles and showing considerable variation in size, shape and many mitotic figures. X 250. :, Fig. 6. Mucoepidermoid ski.i tnmo*-. Occurrence of hydropic epidermoid cells, cel!;, column.-.; cells, and oxyphil:- cells in various proportion?, x 100. **' Fig. 7. Mucoepidermoid skin tumor, showing differentiation of masses of squamous epithelium from columnar cells, lining and proliferating into b distended tubular spaces. X 100. tr Fig. 8. Micronodiilar adenocarcinoma of the lung arising from a segmental bronchus. X 25. I Fi*. 9. Detail of Fig. 8 showing tubular aggregates made up by cells with hypcrcliromatic nuclei. ;1 Fig. 10. Nodular bronchiolar alveolar mucus-secreting adenocarcinoma. X 100. Fig. 11. Detail of Fig. 10, showing an alvcologenic pattern. X 250. Fig. 12. Mucus-producing cells with hyperchromatic and pleomorphic nuclei and occurrence of signet ring cells. X 250. Fig. 13. Squamous cell carcinoma of the lung. X 100. 1 Fig. 14. Osteochondroma developing as a protuberance from the small bones of metacarpus and metatarsus. X 25. Fig 15. Magnification of Fig. 14. The protuberance consists of bone trabeeulae, capped by cartilage, and a fibrous layer functioning as ;l perichondrium, x 100. ) Fig 16. Osteochondroma. Cartilaginous growth characterized by finger-like proliferation. X 108. n: Fig. 17. Osteochondroma. Area of endochond.-iai ossification. X 100. or Fig 18. Cellular area of an osteochondroma showing the transition from cartilage to osteoid. X 10ft. ! .MAY 1971 ,|9 I DTH 000011742