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* NTEK ). iSLAKCH Jl, SI4-322, May i 9 i i I % _- Oncogenic Response of It at Skin, Lungs, and Bones to Viny) Chloride1 P. L. Viol, A. Bigotti, and A. Caputo Regius Elen* InttHurt for Concer Research, Rome, Iffy SUMMARY to vinyl chloride follows lingular pattern for the different tissues and organs of the rat. Rats (Ar/IRE Wisur strain) exposed for 12 months to vapors of vinyl chloride developed tumors of the skin, lungs, and bones. Tire cutaneous tumors, which always appeared in MATERIALS AND METHODS the area in which submaxillary and parotid glands are located, have been histologically recognized as epidermoid carcinomas, papillomas, and mucoepidermoid carcinomas. Tire 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 tire epithehal 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. The experiments were performed with vinyl chloride ! (CHi'CMCl, the monohMogcnate derivative of ethylene) of a I commercial grade (99% purity1) assumed to contain j insignificant amounts of various noncarcinogernc 1 contaminants. ! Three-month-old WisUr (Ar/!RE) 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 i kept in metal or plastic, air-tight caves in which a constant ; flow of air, containing 3% v/v (equal to 30,000 ppm) of vinyl j INTRODUCTION chloride, was introduced. Twenty-five rats of the same strain | were the control group. At the end of the treatment, the i surviving animals wen i.i'lcd at 20-dcy ir.tcrvJi, and the must ! Tne oncogenic properties of sotu* chemical organic important mutes and organs were examined histologically by i compounds used for the preparation of "plastics*' have been standard methods. During the period of exposure, lire ar.trr.-ls j widely investigated, and their limits and effectiveness have were slightly soporific; however, the firjt few months of " been well estaMrshed. Detailed information on this subject treatment were well tolerated and no chances in growth or i may be found m tire literature (S, 6, 8); however, all behavior were noticed. After 10 months of treatment, some I information and references are exclusively related to highly animals began to show a hard mass in the paraauricular region > polymerized compounds of roughly the same size as used in which became progressively larger until it reached the size of a : various industries. * Oncogenic polymers produce sarcomatous rumors, with the except ion of polyurethans which, as reported by Ilueper (6), Iso induce adenocarcinomas. Recently, it has been demonstrated that sarcomatous tumors (1-4, 7) ate transplantable, and it has been suggested drat they may originate from the tissues of the capsule that gradually coven she plaMic film. Tims, Rond et nl. (2-->) have observed piemalignairt areas made of poorly differentiated fibroblasts which were firmly attached to the photic 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 tire models previously reported by several authors (V-l 2) The carcinogenic response 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 dischr.rjed necrotic debris, while a certain amount of tumorous tissue 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 diffuse 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, the lungs were hemorrhagic with milky, thick fluid in the pleural cavities. The 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 1A preliminary report of the resutis reported here was given at the Tenth tim.manorial Cancer Cot.(lev. Ilwmon, Tevas May 22-24, 1970. of the skeletal bones. All the animals that inhaled vinyl chloride showed e series of parenchyma; lesions. Among these, most prominent were Received July 31,1970; accepted January 12. 1271. the disappearance of granular and Purkinje cells, degeneration CANCER RESEARCH VOL. 31 j ucc 065657 Oncogenic Activity of Vinyi Chloride 'el the cerebellum, tevere chronic hepatitis, imcmiual preuntonia, and moderate (welling of the kidney parenchyma, often assuming the pattern of tubul nephrosis. RESULTS The rates of survival and the main findings are summarized Ip Table 1. Almost all the animals developed tumors of the tbfn and lungs. Very few animals developed bone tumors; 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 ot 707c. Skin Tumors. The tumor that developed most frequently in thtpanaurlcular region was the epidermoid carcinoma, but we also observed papillomas and rarely mucoepidermoid carcinomas. In all cases, the nc<'j :-<sms were of epithelial astute. The 3 patterns noticed cannot be compared to various tstotype* 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 subauriculv growths occurred in some rat*. The histological picture showed papillar epithelial proliferati n, 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, acmthosk, and areas of individual dyskeratosis or peari-Iike homy formations. The epithelial cells of the penetrating columns (Fig. 2) were irregularly arrangrd and were frequently accompanied by an inflammatory infiltration of the dermis. The typical features of the Malpighian layer and of the stratum comcum 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 "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 I Oncofrric effects ofinhaled vinyl chloride at a function oftime The animal* were exposed io vinyl chloride vapor* for 4 hr a day, S day* a week, fee a total of 12 month*, in a conitant now of tit con laming 3% v/v vinyl chloride. Rat 1 2,3 4 S 7 14 16,17 2! 22 23 24 25 26 Survival rate 300 280-300 310 333 337 347 347 354 359 350 380 380 380 380 380 Tumor* Skin Lung* Bone* UiKfwpi^rmoift carcinoma Epidermoid caiemoiivi, Lemtmizing type Epidermoid carcinoma, kera tinizing type Papilloma, kcralotic type Epidermoid carcinoma Epidermoid carcinoma Mucoepidermoid carcinoma Epidermoid carcinoma Epidermoid carcinoma Epidcimoid carcinoma Epidermoid carcinoma Epidermoid carcinoma Epidermoid carcinoma Epidermoid carcinoma Epidermoid carcinoma Adcnoacanthom* No tumor Adcoooircinoma No tumor No tumor No tumor No tumor No tumor Adenocarcinoma m No tumor Adenocarcinoma No tumor Mucut-rtoducing adenocarcinoma (alveolar cell carcinoma?) No tumor Squamous ctl) carcinoma Osteochondroma No tumor No tumor Osteochondroma Oateochondroma No tumor Osteochondroma No tumor No tumor No tumor Oftcochondtomn No tumor No tumor No tumor No tumor UY 1971 517 ucc 065658 t. L Viok. A. Bigotti, and A. Capita Keratlnteation was irregular and often parakeratotfs waa cartilaginous zone* possessing regressive features, such m bserved in the area* f the turn r in which the crib were fibrosis and hyalinoris. The perichondrium appeared often ass , loorely aggregated and undergoing an individual rather than a compressed and structurally altered fibrous tissue. i collective type of keratinization (Fig. 4). In dytkeratotic area*, In some cases, the tumor growth was related to the stags ofi a few epithelial celb tended to form pearls. The tumor seldom the endochondral ossification occurring below the "epiphytal : showed an undifferentiated growth with cellular plate." Ossification was irregular, ao that osseous trabeculae pleomorphbm and several mitotic figure* (Fig. 5). A few varied greatly in thickness and contours. In other cases, fod of i tumors showed little nests of isolated pale cells (Figs. 6 and 7) active cellular proliferation, cartilaginous areas, chcndroid \ 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). i and intennediate cells with minor tendencies towards The new formation appeared to be an osteochondroma and ' differentiation. consisted 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 ! celb. Sometimes, the tumors were seen in their early the adjacent cortical bone and extended inward t form septa ; development and consisted predominantly of cubic or separating and enclosing lobules of cartilage. C lumnar celb 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 lime fibrous stroma (Fig. 9). were subjected to a constant flow 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 as shcct-like proliferations of of parenchymal lesions developed by the rats that inhaled undifferentiated or pleomorphic character. The cells showed a vinyl chloride. In a very few of the control rats, there was 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 abdndant | 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 weie present. ----- -- in terms of a more prompt positive response to be obtained In some areas, foci of cellular polymorphism with with the ide.il concentration of the carcinogenic compound. . hypcrcliromatic nuclei were noticed; mucin was produced in The cutaneous system is the most susceptible to the ncogcnic vaiying 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 cells occurred, either alone or in small clusters (Fig. 12). The alveolar walls were often quite thick and, at times, showed an inflammatory infiltration. As mentioned before, a single tumor showed squamous structures and appeared to have been formed mainly by Spindle and oval undifferentiated cells (Fig. 12). Bones. In the metacarpal and metatarsal regions of the 4 Our experimental data do not explain why the cutaneous tumors developed in the same site, Le.. the region including : the area in which the submaxillary and parotid glands ate ' located. It is possible that the salivary glands may by involved in the concentration or excretion of vinyl chloride or some of its active decomposition products. This hypothesis is strongly supported by morphological findings showing the specific . tendency of the developed tumor to appear at * ; 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 fiom 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 aggregates, and. therefore, the histogenesis of human fingcr-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 whiclntie^SctiotTof 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 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 tingle, 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 ritown by the extension of the finger-like concentration could I . the result of the difficulty of piokmgations into the boundaiy of newly formed tissue. complete cleansing by the rat. Besides the chondioblastic, 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 uccSIB CANCER RESEARCH VOL. 3) 065659 i Oncogenic Activity of Vinyl GMbrfei* il, tumots wcjc morphologically similai to rho\c described , the tkm. Hie hypothesis that mucus-producing celts may .-a-iabte of retaining vinyl chloride or it* decomposition edaru seems to be relevant once more. Obviously, a f,*;rnt interpretation required for the pathogenesis of the VM. minors and for their simultaneous incidence at the level J14lin*bs. [FfTtENCES t M., and Bites, R. Prevalence t>f Heteropioidy in Plastic t-lm induced Primary Sarcomas. Brit. J. Cincer, 20: 555-563, W 1 i.-jid, G., and Buoen. L. Polymer 1 umorigenesis: Multiple jvnropLisiie Clones in Priority Order with Clonal Inhibition. Proc. Sn: E\ptl. Biol. Med., 12S: 1153-115'- :968. ) G., Buoen, L., and Brand, I. Malignant Transformation and Slaiuiiucn in Non-dividing Cells Hunng Polymer Tumorigcnexis. Ttc. Soc. Fxptl Biol. Med.. 174; *75-678, 1967 4 !'r;nJ. G,, Bouen, L., and Brand, I. Carcinogenesis front Polymer littphnts- New Aspects from Chromosomal and Transplantation Studies during Premalifnincy. J. Natl. Cancer Inst,. 39: 663-679, tCiO. 5. Drvrkrsy, II. t-.perimenieMe beitraegc wsim Mechanimms dm Carrmnyvnen Wiikung. Arcnciinitiet-Porsch., I: 3836-39$, 1991. 6. lineper, W. C. Cancer Induction by tolyweihan and PolyiRlcene rtasuci. J. Natl. Cancer Intt., 33: 1005-1027.1964. 7. Johnson, K,, Buoen, L., Brand. I., and Brand, C. Polymer Tumorigencris Ctonal Determination of ilistopathological Ch"''eristics during Early Preneoplasia; Relationships to Ka< >pc, Mouse Strain, and Sex. J. Natl. Cancer Inst., 44: 785-793, 1970. 8. Kogan, A. Experimental Blastomogenesis Caused by Synthetic Polymers (Plastics). Meditsina, Moscow, I: 215-237,1965. J. Mastromatteo, E.. t idier, a. M., Christs, If., and Danzigw, D. Acute Inhalation Toxicity of Vinyl Chloride to Laboratory Animals, Ant. lid. Ilyg. Awic, i.,21: 394 - 397,1960. 40s Totkcrson, T. R., Oycn. 1'., and Roux. V, K. The Toxicity of Vinyl " Chloride as Determined by Repeated Exposure of Laboratory Animals. Ind. Ilyg. J.. 354-361. October 1961. 11. Van Duren. B. L., Stvak, A., Coldschmidt, B. M., Kits, C,, and Melchtonne, S. Carcinoge.'idty of Halo-F.thcrv J. Natl. Cancer Inst., 43: 481--486. 1969. 12. Viola, P. L. Pathotopy of Vinyl Chloride. 16rfa International Congress of Occupational lleairi. (Communication No. 98), Tokyo. 1969. Photomicrographs of the original article are not being distributed with this copy because of the poor quality of their reproduction. i ifi. 1 to 18. All sections were stained with II & E. Ft* l.Squutncut cell papilloma of the skin, x 25. Ftp 2. Transitional stage from papilloma to infiltrative type of cancer. X 80. I ig. 3. Tinger-like trabeculae branch to form secondary processes showing horny pearl formation (arrows), x 25. fir. 4. Dyskeratutic area. Few epithelial cells with a tendency to form whorls. Little rapacity to develop into horny pearls. X 250. Fig 5. Atypical cell* partly lacking prickles and showing considerable variation in size, shape an^many mitotic figures. X 250. iii,. 6, Mucoepidermoid !;..( oimo'. Occurrence uf hydropic epidermoid cells, bur.. eel!., wClumnr.; and oxyphil:, cell; in vicious 'ti'iw'tionr. X 100, Fif. 7. Mucoepidermoid skin tumor, showing differentiation of masses of squamous epithelium from columnar celts, lining end proliferating into 'mended tubulai spaces. X 100. 18. Micronodnlar 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. Nodulat bronchiolar alveolar mucus-secreting adenocarcinoma. X 100. I if. 11. Detail of Fig. 10, showing an ah-eolr'gcnic pattern. X 250. fig 12 Mucus-producing cells with hyperchromatic and pleomorphic nuclei and occurrence of signet ring cells. X 250. it 13. Squamous cell carcinoma of the lung. X 100. I it 14. Osteochondroma developing is protubetancq ftom the small bones of metacarpus and metatarsus. X 25. fig 15. Magnification of Fig. 14. The protuberance consists of bone trabeerriae, capped by cartilage, and fibrous layer functioning M jmichondrium. X 100. 1 Fig. 16. Osteochondroma. Cartilaginous growth charactcriied by llnger-like proliferation. X 100. i t*. 17. Osteochondroma. Area of endochondrisl ossification, X 100. 1 g 18. Cellular area of an osteochondroma showing the transition ftom cartilage to otteoid. X 100. MY1921 I i9 ucc 065660