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REPORTS NOT SENT ffiCAUSE NOT OWNED BY LIBRARY RLE IS INCOnMPruLEu^ IN USE HOLD PLACCEEDO \ REQUEST AGAIN ^ PUBLICATION NOT YET RECEIVED PLEASE VERIFY YOUR REFERENCE OTHER: SUGGEST YOU REQUEST OF: ESTIMATED COST OF MICROFILM PHOTOPRINT PLEASE PAY IN ADVANCE PLEASE DO NOT PAY IN ADVANCE PLEASE SEND COST ESTIMATE FOR MICROFILM PHOTOPRINT GOAHEAD WITH ORDER IF IT DOES NOT EXCEED: S-----------------------SPECIAL INSTRUCTIONS: ----- FOLD / OLI 1545 |CANCER RESEARCH 31,514-522. May 1*711 LLZ--- Oncogenic Response of Rat Skin, Lungs, and Bones to Vinyl Chloride1 P. L. Viola. A. Bigotti, and A. Caputo Refine Elena Inoiwie for Cancer Rtttarch, Rome. Italy SUMMARY Rats (Ar/IRE Wistar strain) exposed for 12 months to vapors of vinyl chloride developed tumors of the skin, lungs, and bones. The cutaneous tumors, which always appeared in the area in which submaxillary *nd 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 in 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 polyurethans which, as 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 capsule that gradually covers the plastic film. Thus, Brand et al (2--4) have observed premalignant areas made of poorly differentiated fibroblasts which were firmly attached to the plastic film up to the moment of malignant transformation. This investigation demonstrates that 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 1A preliminary report of the results reported here was given at the Tenth International Cancer Congress, 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 die sat. MATERIALS AND METHODS The experiments were performed with vinyl chloride (CH]CHC1, the monohalogenate derivative of ethylene) ofa commercial grade (99% purity) assumed to contain insignificant amounts of various noncarcinogenic 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 wen kept in metal or plastic, air-tight cages in which a constant flow of air, containing 3% v/v (equal to 30,000 ppm) of vinyl chloride, was introduced. Twenty-fire rats of the sane strain were the control group. At the end of the treatment, the surviving animals were killed at 20-day intervals, and the moat important tissues and organs were examined histologically by standard methods. During the period of exposure, the anbnali were slightly soporific; however, the first few months f treatment were well tolerated end no changes in growth or behavior were noticed. After 10 months of treatment, come animals began to show a hard mass in the paraauricular region which became progressively larger until it reached the Mae of a walnut or slightly larger. In most cases, the swelling was unilateral; it was bilateral in only a few animals. After 1 or 2 months, the growing masses became ulcerated and dischwged necrotic debris, while a certain amount of tumorous tianre began to form on their surfaces. In addition, we observed that the masses were an integral part of the paraauricular region and could not be distinguished from the local tissues. Caseous necrotic zones were found. Pleura and pericardium often showed diffure inflammation of a fibrous nature and, in many cases, the lungs were covered with a number of white formations as large as grains of rice or even larger and harder than the lungs themselves. In 2 cases, die lungs were hemorrhagic with milky, thick fluid in the pleural cavities. The liver was sometimes increased in size and rery fragile. The animate were subjected to X-ray analyses at different intervals from the beginning of treatment in order to control the stare of the skeletal bones. All the animate that inhaled vinyl chloride showed a reties of parenchyma] lesions. Among there, most prominent were the disappearance of granular and Purkinje cells, degeneration 516 CANCER RESEARCH VOL. 31 Oncogenic Activity of Vinyl Chloride of the cerebellum, severe chronic hepatitis, interstitial pneumonia, and moderate swelling of the kidney parenchyma, often assuming the pattern of tubulonephrosis. 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 turnon; in these cases, the tumors were localized in the metacarpal and metatarsal bones of all 4 extremities. Skin tumors were by far the most frequent, amounting to 65 or 70%. Skin Tumors. The tumor that developed most frequently in the paraauricular region was the epidermoid carcinoma, but we also observed papillomas and rarely mucoepidermoid carcinomas. In all cases, the neoplasms were of epithelial nature. The 3 patterns noticed cannot be compared to various histotypes 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. Warty subauricular growths occurred in some rats. The histological picture showed papOlar epithelial proliferation, with progressive increase in the thickness of the epidermis (Fig. 1). The papillary warts of exophytic type had a fibrous vascular stroma and various degrees of inflammatory lymphocytic infiltration, marked hyperkeratosis, parakeratosis, acanthosis, and areas of individual dyskeratosis or pearl-like homy formations. The epithelial cells of the penetrating columns (Fig. 2) were irregularly arranged and were frequently accompanied by an inflammatoty infiltration of the dermis. The typical features of the Malpighian layer and of the stratum comeum of the prickle cells and of germinal layers were easily recognized (Fig. 3). The homy layer was composed largely of cell nests, which appeared at certain points within the epithelial masses and assumed the well-known appearance of "homy 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 chhfide as a function <4time The animals were exposed to vinyl chloride vapors for 4 hr a day, S days a week, for a total of 12 months, in a constant flow of air containinc 3% v/v vinyl chloride. m _s 5 a NJ * Mm 4 l OC. CL J5tEto 3 Rat 1 2,3 4 5 6 7 8 14 16, 17 21 22 23 24 25 26 Survival rate 300 280-300 310 333 337 347 347 354 359 380 380 380 380 380 380 Tumors Skin Imp Bones Mucoepidermoid tffdnom Epidermoid rarctooma, kentinizmc type Epidermoid carcinoma, kernUniting type Papilloma, kentotk type Epidermoid ardnoms Epidermoid arenam Mucoepidermoid orcmofBa Epidermoid curinoma Epidermoid cvcmoma Epidermoid carcmoma Epidermoid carcmoma Epidermoid carcmoma Epidermoid carcmoma Epidermoid carcinoma Epidermoid carcinoma Adenoacanthoma No tumor Adenocarcinoma No tumor No tumor No tumor No tumor No tumor Adenocarcinoma No tumor Adenocarcinoma No mmor Mucus-producing adenocarcinoma (alveolar odi carcinoma?) No tumor Squamous etl) cartfcsoma Osteochondroma No tumor No tumor Osteochondroma Osteochondroma No mmor Osteochondroma No mmor No tumor No tumor Osteochondroma KoHM No tumor No toM* No mmor MAY 1971 517 OLI 1547 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 attend fibrous tissue. collective type of keratinization (Fig. 4). In dyskeratotic 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 cellukr proliferation, cartilaginous areas, chondroid of 3 types: mucin-producing cells (originating from the duct developmental nests, and calcified bones occurred in the epitheiium of sweat glands or salivary glands); squamous cells; deeper portions of the trabeculae (Fig. 18). and intermediate cells with minor tendencies towards The new formation appeared to be an osteochondroma and differentiation. conssted essentially of a bony protuberance capped by Respiratory Tract. These tumors, although occurring rarely, cartilage and a fibrous layer, which represented the were mainly adenocarcinomatous. 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 aepta 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 (Fig. 8) and supported by poorly developed conditions as the experimental rats and at the appropriate titn^ fibrous stroma (Fig. 9). were subjected to a constant flow of air without viayk In other cases, glandular structures were often imperfectly chloride. None of these animals developed tumors or the types formed and appeared as sheet-like proliferations of of parenchymal lesions developed by the rats that inhdea undifferentiated or pleomorphic character. The cells showed a vinyl chloride. In a very few of the control rats, there was 4 tendency to extend into the pulmonary parenchyma, thus swelling of the liver and kidneys. simulating the microscopic features of the so-called alveolar cell carcmoma (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 cytoplasm that was faintly eosinophilic (Fig. 11); frequently, As in many carcinogenesis studies in which multiple tumors adenopapillaty 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, either 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 decompostion 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 the mucus glandular cells to the tumorous cells, seemed to derive directly from the cortical bone (Fig. growth. Such kinds of histotypes are often related to some 14) . The periosteum also grew and in some areas spread as glandular aggreptes, and, therefore, the histogenesis of human finger-like 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 ured, saturation and wetting of the 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 be the result of the difficulty of a prolongations into the boundary of newly formed tissue. complete cleansing fay the ret. Besides the chondroblastic, 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. 31 OLI 1548 Oncogenic Activity of Vinyl Chloride of the tumors were morphologically similar to those described for the skin. The hypothesis that mucus-producing cells may be 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. Banerjee, M., and Bates, R. Prevalence Of Heteroploidy in Plastic Film-induced Primary Sarcomas Brit 1. Cancer, 20: 555-563. 1966. 2. Brand, G.. and Buoen, L Polymer Tumorigenesis: Multiple Preneoplastic Clones in Priority Order with Gone) Inhibition. Proc. Soc. Exptl. Biol. Med.. 128 1153-1158, 1968. 3. Brand. G., Buoen. L., and Brand, 1. Malignant Transformation and Maturation in Non-dividing Cells during Polymer Tumongenesii. Proc. Soc. Exptl. Biol. Med., 124. 675-678. 1967. 4. Brand. G., Buoen, L., and Brand, 1. Carcinogenesis from Polymer Implants New Aspects from Chromosomal and Tran*>Uniation Studies during Premalignancy. J. Natl. Cancer Init.,39; 663-679, 1970. 5. Druckrey. H. Experimentelle Beltra^e wum Meehanismus der Carcinogenen Wirfcung. Arzneimittel-Forsch.. 1: 383-395, 1951. 6. Hueper. W. C. Cancer Induction By Polyuicthin and Polysilicone Plastics. 1. Natl. Cancer Inst,,33:1005-1027,1964. 7. Johnson, K.. Buoen, L,, Brand, U and Brand. G. Polymer Tumorigenetis Clonal Determreatioo of Hrstopathological Characteristics during Early Pieneoplasia; Relationdtips to Karyotype, Moure Strain, and Set, 1. Natl. Cancer Inst., 44: 785-793,1970. 8. Kogan, A. Experimental Blastomogenesit Caused by Synthetic Polymers (Plastics). Meditrina, Moscow, I: 215-237.1965. 9. Masttomatteo, E., Fidier, A. M,, Chtisti. H,, and Danzigcr, D. Acute Inhalation Toxicity of Vinyl Chloride to Laboratory Animals. Am. Ittd. Hyg. Assoc. J.,21: 394-397,1960. 10. Torkerson, T. R.. Oyen, F., and Rowe, V. K. The Toxicity of Vinyl Chloride as Determined by Repeated Exposure of Laboratory Animals. Ind. Hyg. J,, 354-361,October 1961. 11. Van Durcn, B. L., Sivak. A., Goldschmidt, B. M,, Katz. C.. and Mekhionne. S. Carcinogenicity of Halo-Ethers. J. Natl. Cancer Inst.. 43. 481-486.1969. 12. Viola. P. L. Pathology of Vinyl Chloride. 16th International Congress of Occupational Health. (Communication No. 38), Tokyo. 1969. Figs. 1 to 18. AD sections were stained with H A 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 homy pearl formation (arrows), x 25. 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 skin tumor. Occurrence of hydropic epidermoid cells, basal cells, columnar cells, and oxyphilic cells in various proportions, x 100. Fig. 7. Mucoepidermoid skin tumor, showing differentiation of masses of squamous epithelium from columnar cells, lining and proliferating into distended tubular spaces, x 100. Fig. 8. Micronodular adenocarcinoma of the lung arising from a segmental bronchus. X 25. Fig. 9. Detail of Fig. 8 showing tubular aggregates made up by cells with hyperchromatic nuclei. Fig. 10. Nodular bronchiolar alveolar mucus-secreting adenocarcinoma, x 100. Fig. 11. Detail of Fig. 10. showing an alveologenic pattern. X 250. Fig. 12. Mucus-producing cells with hyperchromatic and pleomorphic nuclei and occutrence of signet ting cells. X 250. Fig. 13. Squamous ceU carcinoma of the lung. X 100. Fig. 14. Osteochondroma developing as a protuberance from the small bones of metacarpus and metalareua. x 25. Fig. 15. Magnification of Fig. J4. The protuberance constts of bone trabeculae, capped by cartilage, and a fibrous layer functioning as 'cnchondrium. x 100. Fig. 16. Osteochondroma. Cartilaginous growth characterized by finger-like proliferation, x 100. Fig. 17. Osteochondroma. Area of endochondral ossification, x 100. Fig. 18. Cellular area of an osteochondroma showing the transition from cartilage to osteoid. X 100. IAY 1971 519 OLI 1549 s CANCER RESEARCH VOL. 31 OLI 1550 Oncogenic Activity of Vinyl Chloride MAY 1971 521 P. L Viola. A. Bigotti, and A. Capito frr V- - 4 V.?'* ;fjj'.t'V**'*,ji f l `. '>!' * -j - '^^5-> }*'*- -w* - w.: l* ,. : -- - - 14 522 CANCER RESEARCH VOL. 31 OLI 1552