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JCANT.f.K RLSLAHCi<3l..$l6-522,,May 1971J Oncogenic Response cf Rat Skin, Lungs, and Bones to iny! Chloride* P. L. Viola, A. Eigotti, and A. Cnputo Kttir.3 Urto Institute for Ctnctr ftetccrch, fiorr.t, Ittty SUMMARY Rats (Ar/IRE Wisiar strain) exposed for 12 months to vapors of vinyl chioiide developed tumors of the skin, lungs, and bones. The cutaneous tumors, which always appeared in the area in which submaxillary and parotid glands aic located, have been histologically recognized as epidermoid carcinomas, papillomas, and mucoepidermoid carcinomas. Tire nrorrhr logical characteristics of lung tumors, winch occurred in a lower percentage, were mainly of the adenocarcinoma type, with the exception of a single epidermoid tumor originating from die epithelial covering ceils. In a minor number of rats, a large proliferation of cartilaginous tissue diagnosed as osteochondroma developed in tire metacarpal and metatarsal regions of the four limbs. INTRODUCTION ~^t-e oncogenic properties of some chemical organic C':n:r-'.'"ml* 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 the literature (5. 6, 8); however, all information and references are exclusively related to highly pc'ymrrircd compounds of roughly'the same site as used in various industries. Once-janic polymers produce sarcomatous tumors, with the exceptinn of po'yuiethans which, as reported by IJucper (6), also induce adenocarcinomas, f'.eccn tly, it has been de-nc:i;:raTcd that sarcomatous tumor*, (1-4, 7) are Uintp-antf-ble, and it has been suggested that they may or.girjti from the rissues of the capsuie that gradually covers liie piis'lc film. Thus. Rrend it si (2--1; have observed riemlignanf areas made of poorly differentiated fibroblasts which v.cr: firmly atrached to the pLstic film up to the mar. er* of malignant rr.msfcrniation. This investigation demons:rates that even the monomer vinyl chloride possesses oncogenic properties when ut-ed with an appropriate model different from die models previously repartee by several authors tV--!2). The carcinogenic response 1A p:r!imi:-.iry report of the results reported here wj pi*en at the Tir.il, I' t.c'.anoiUil Cancel Coi.jieu, I!i':i\;ou,"-T<\a*, May 22--24, >9 TO. Jely 31, 1970;accepreJ January 12. 1971. to vinyl chloride follows a singular pattern for the different ' tissues and organs of the rat. MATERIALS AND METHODS The experiments were performed with vinyl chloride (C!I-=C!!C1, the monohalcgcnate derivative of ethylene) fa commercial grade (99ft purity) assumed to contain insignificant amounts of various noncarcinogetric contaminants. Three-month-old Whtar (Ar/IRE) male albmo rats (about 150 g bojy 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 3ft v/v (equal to 30,000 ppm) of vinyl chloride, was introduced. Twenty-five rats of the same strain were the control group. At the end of the treatment, the surviving animals were M'ivd s! 20-day intervals, and the most important tissues and organs wore examined histologically by standard methods. During the period of exposure, the animals were slightly soporific: however, the first few months of treatment were well tolerated and no changes in growth dr behavior were noticed. After 10 months of treatment, some animals began to show a hard mass in the paraauricular region which became progressively larger until it icachcd the site 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 discharged necrotic debris, while a certain amount of tumorous tissue began to form on their surfaces. In addition, we oburved that I the masses were an integral part of the paraauricular region and could not be distinguished from the local tissues. Caseous 1 necrotic zones were iound. 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 giains of rice or even larger and harder than the lungs themselves. In 2 cases, the lungs were hemorrhagic with nifiky, thick fluid in the pleural cavities. The liver was sometimes increased in size and very fragile. The animal- were subjected to X-ray analyses at different intervals front the beginning of treatment in order to control the stats of the skeletal bones. A!1 the animals that inhaled vinyl chloride showed t. series of parenchyma: lesions. Among these, most prominent wire the disappearance of granular and Purkinjc cell*, degeneration 5K- CANCER RESEARCH VOL. 31 UCC 088297 I. . .1 * e qcrcbellum, severe chronic hepatitis, inlcrrii'.ial Warty nihauricuhr growths occurred, in tome rats. The orris, and moderate swelling of the kidney parenchyma, histological picture showed papillar epithelial proliferation, -rung the pattern of lubuionephrosis. with progressive increase In the thickness of the epidermis (Fig. I). The papillary warts of exophytic type had a fibrous vascular stroma and various degrees of inflammatory lymphocytic rates of survival and the main findings are summarized le 1. Almost all the animals developed tumors of the id lungs. Very few animals developed bone tumors*, In ascs, the tumors were localized in the metacarpal and rial bones of all 4 extremities. Skin tumors were by far s', fiequcnt, amounting to 65 or 70X. Tumors. The tumor that developed most frequently In auricular region was the epidermoid carcinoma, but we observed papillomas and rarely mucoepidermoid mas. In aU cases, the ncykiMns wcic of epithelial Tne 3 patterns noticed cannot be compared to various pcs but rather t a transition of one type into the other I which is more likely, to different stages of the same rative type. infiltration, marked hypcikciatosis, 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). Tiie 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 ceils without nuclei and were located around a central core of keratin. Tabic 1 . Oncogenic effects ofMinted rinyt chloride at a function of time The animals were exposed to vinyl rhloriJa vapors for 4 hr a day, 5 days a week, for a total of 12 months, in a constant fidw of air containing 3 X v/v vinyl chloride. Rat 1 2.3 4 5 6 7 8 14 16.17 21 22 23 24 25 26 Survival nte 300 280-300 310 333 337 347 347 354 359 3*0 380 380 380 380 380 Tumors Skin Lung* Bones Mucr<cpidcrinoid carcinoma Epidermoid carcinoma. kera tinizing type Epidermoid carcinoma, kera tinizing type hpillonu, keiatotic type Epidermoid carcinoma Epidermoid carcinoma Mucoepidermoid carcinoma Epidermoid carcinoma Epidermoid carcinoma Epideimoid carcinoma* Epidermoid carcinoma Epidermoid carcinoma Epidermoid carcinoma Epidermoid carcinoma Epidermoid carcinoma Adcnoacanthom? No tumor Adenocarcinoma No tumor No tumor No tmnor No tumor No tumor Adenocarcinoma No tumor Adenocarcinoma No tumoi Mucus-producing adenocarcinoma (alveolar cell carcinoma?) No tumor Squamous cell carcinoma Osteochondroma No tumor No tumor Osteochondroma Osteochondroma No tumor Osteochondroma No tumor No tumor No tumor Osteochondroma _ No tumor No tumor No tumor No tumor -- * 1971 517 UCC 088298 P. L. Viola, A. Eigotti, and /t. Cap.no Kuatlnizatlon w?s Irregutar end 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 asi loosely aggregated and undergoing an individual rather than a compressed and structurally altered fibrous tissue. collective type of kcratinization (Fig. 4). In dyskeratotic areas, In some eases, the tumor growth was related to (he 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, to that osseous trabeculae plcomorphism 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 ccliular proliferation, cartilaginous areas, chondroid of 3 types: mucin-producing cells (originating from the duct developmental nests, and calcified bones occurred In the epithelium or sweat glands or salivary glands); squamous cells; deeper portions of the trahecutae (Fig. 18). and Intermediate evils 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 adcnocaieinomatous. Only in I rat did \vc 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 (Fig. 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 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 sheet-like proliferations of of parenchymal lesions developed by the rats that inhaled undifferentiated or pleomorphic character. The cells showed a vinyl chloride. In a arcry few of the control rats, there was a 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 cytoplasm that was faintly eosinophilic (Fig. 1 ), frequently, adenopapfllary excrescences spreading into the alveolar spaces weie present. .... In some areas, focj of cellular polymorphism with hyperchromatic nuclei were noticed; mucin was produced in As in many carcinogenesis studies In which multiple turnon arise in different tissues and organs, the data must be evaluated in tenns of a more prompt positive response to be obtained with the ideal concentration of the carcinogenic compound. 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, ie., 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 iljay by involved As mentioned before, a single tumor showed squamous in the concentration or excretion of vinyl chloride or sonicbf 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 limbs, a large proliferation of cartilaginous tkiiies 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 K). 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 IS). 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 he 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 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 1 . the result of the difficulty of a piolongatic-ns into the boundaiy of newly formed tissue. complete cleansing by the rat. Besides the chondrohlasttc, chonJrneytie, 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 UCC 088299 mc~;to!opkjtly limilai It* rh*>ic dcserihej j .r.. Ii:e hypothesis that nuciis-proiiuciiis cells may :i t of re:*-inj vinyl chloride or its ocenmposition to be relevant once more. Obviously, a n is required for the pathogenesis of tire jrii for their simultaneous incidence at the level \r..ks. i.\`C5S c,'c. ft., sad Rates, R. Prevalence tf lletcroploidy in Plastic Primary Sarcomas. Brit J. Cancer, 20: 555-563. ./ ra. G.. end Ruocn, L, , Polymer Yumoripenesis: Multiple Clonet in Priority Order uirli Clonal Inhibition. I'roe. Fvr l. 3k!. Med.. J2i: 1153-11V. 'fOti: t, G.. iuoen. L. and Bund. I. Malignant Transformation and r.;ii:an ir. Non-dividing Cellt doimp Polyn.ei Tumoripenesis. r sf J. Biol. Med.. 174: 675-678.1967. *J. j.. C<.uen. U. end Brand. I. Carcinogenesis from Poiynier New ASfectf from Chromosomal and Tiansphnlatinn if, t-nu: Premi'if-nar.cy. J. Natl. Cancer lust.. 29: 663-679. r,. 5. Druekrey. II. I xpeiimcniclle llaitracge wum Mtchanismut dcr Careinorrnen Wiikung. Ar/.nci.nltlcllorsch., /: 383-395, 1931. 6. Iliirpvi. W. C. Cainr Induction by Polyurctlian and Polyrlticone Plastics. 1. Natl Cancer Intt., JJ: 1005-1027, 1964. 7. Johnson, K.. Ituocn, L., Brand, I., and Brand, C. Polymer Tumoiigcucsis Clonal Determination of llislopalhologlcal Cb" trtistie* rlurinp Early rteneoplatia; Relationship! to Ka > pc, Moure Strain, and Sex. J. Natl. Cancer Intt., 44: 783-793. 1970. 8. Kogan. A. Experimental ni.tslomogonesSs Caused by Synthetic Polymer* (Plastics). Mcdirsina. Moscow, I: 215-237, 1965. 9. Maitromatteo, E., Fisher, A. M., Chrisfi, It., and Damiger. D. Acute Inhal.ition Toxicity of Vinyl Chloride to Laboratory Animals. Am. Jnd. Hyp. Aruvr. J..21: 394-397, I960. 10. Toikcison, T. R., Oj'cn. r., and Roux. V. K, The Toxicity of Vinyl Chloride at Determined by Repeated Exposure of Laboratory Animals. Ind. llyp. J.,354-361.October 1961. 11. Van Durcn, B. L., Sivak, A.. Goldschmidt, U. M.. Kata, C.. and Melchionne, S. Careinogr.`icily of Haro-F.thers. J. Natl. Cancer Intt., 43: 481-486. 1969. 12. Viola, P. L. Pathology of Vinyl Chloride. Jfirh International Congics* of Occupational Ilcalt'i. (Communication No. 38), Tokyo. 1969. Pr.otonicrographs of the original article are not being distributed wizh ihis copy because of the poor quality of their reproduction. s. 1 :->! S. Ail lections were ttained with II & E. . l.S^.r.i' js evil pcpiiioma of the skin. X 25. \ . jr.;l stags from papiComa b> infdiralivc type of cancer. X 80. . 3. rirr::-ikc trabeculae branch to form secondary processes showing horny pearl formation (arrows). X 25. . 4. Dj r:-,c;atf.:ic area. Few epithelial cell* with a tendency to form whorls. Little c.ipjeily to develop into horny pearls. X 250, . 5. At) p`.r,I cells parity lacking p.irklct and showing considerable variation in sire, shape and many mitotic figures. X 250. . 1. *1 i.-.rpder.v.r.id rhi.i r,,no'. Oivurrcucs of hydropic epidermoid cells, c;!N. ,o!umnn: ccl!i. and cxyphili. cell; in '.a.1ou* / jr*3. -- . * '!j;"::lrn::oid skin tumor, showing differentiation of masses of squamous epithelium from columnar cells, lining ar.d proliferating int :'*.i-li spaces. X 100, . S. Micc.-.ndular adenocarcinoma of the lunp arising from a segmental bronchus. X 25. . 9. Detail c-f Fig. 8 allowing tubular aggregates made up by crtls with hypcrchromatic nuclei. . 10. NcJa'-ar Lronchiolar alveolar mucus-secreting adenocarrinoma.X 100. . II. Dstail nf Fig. 10, showing, an alveol'-pcnie pattern. X 250. ; 12. 'r-rur-prodacirc cells with hyperchrorsatie and pleomorphic nuclei and occurrence of signet ring cells. X 250. . I3.S'j;.---ms cell carcinoma of the lunp. X 100. . l-t. C.'-eorhcndrona developing as a protuberance from the small bones of metacarpus and metatarsus. X 25. . !S. Metrication of Fig. 14. The protuberance consists of bone trabeculae, capped by cartilage, and a fibrous layer functioning as -ir.an. J IO0. If :h''nJ:oma. Cartilaginous growth characterized by (ingei-like proliferation, x 100. :`.:ond;oma. Area of endochorid.-ial ossification. X 100. ,r area of an osteochondroma showing the transition from cartilage to osteoid. X 100. I1S71 UCC 088300 519