viola-response of rats to vc-1971

7/28/2019 Viola-Response of Rats to VC-1971

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[C ANCER R ES EARCH 31, 516-522, M ay 1971)

Oncogenic Response of Rat Skin, Lungs, and Bones toV inyl Chlo rid e1P. L. Viola, A . B igotti, and A. CaputoReg in a E le na I ns tit ute fo r Can ce r R e se ar ch . R ome, I ta ly

SUMMARYRats (Ar/IRE W istar strain) exposed for 12 months tovapors of vinyl chloride developed tum ors of the skin, lungs,and bones. The cutaneous tumors, which always appeared inthe area in w hich subm axillary and parotid glands are located,h av e b ee n h is to lo gic ally re co gn iz ed a s e pid ermoid c arc in om as ,papillomas, and mucoepidermoid carcinomas. Them orp ho lo gical c haracteristics of lu ng tumo rs, w hich occu rredin a lower percentage, were mainly of the adenocarcinoma

type, with the exception of a single epidermoid tumororiginating from the epithelial covering cells. In a minornum ber of rats, a large proliferation of cartilaginous tissued iag nosed as osteo ch ond roma d ev elo ped in th e m etacarpa l a ndm etatarsal reg io ns o f th e fo ur lim bs.INTRODUCTIONThe oncogenic properties of some chemical organiccompounds used for the preparation of "plastics" have been

widely investigated, and their lim its and effectiveness havebeen well established. D etailed inform ation on this subjectmay be found in the literature (5, 6, 8); however, allinform ation and references are exclusively related to highlypolymerized compounds of roughly the same size as used invarious indus tr ies .O ncogenic polym ers produce sarcom atous tum ors, w ith theexception of polyurethans w hich, as reported by Hueper (6),also induce adenocarcinomas. Recently, it has beendemonstrated that sarcomatous tumors (14, 7) aretransplantable, and it has been suggested that they mayoriginate from the tissues of the capsule that gradually coversthe plastic film . Thus, Brand et al. (2-4) have observedp rem alig nan t areas m ad e o f p oo rly differen tiated fibro blastswhich were firm ly attached to the plastic film up to themomen t o f ma lig nant tr an sfo rma tio n.This investigation demonstrates that even the monomerv in yl chlorid e p osse sses on co gen ic pro perties w hen u sed w ithan appropriate m odel different from the m odels previouslyrepo rted b y se veral au th ors (9 -1 2). T he carcin og en ic resp on se1A prelim inary report of the results reported here w as given at theT enth International C ancer C ongress, H ouston, T exas, M ay 22-24,

1970.R ec eiv ed Ju ly 3 1, 19 70 ; ac ce pted J an uary 12 , 1 97 1.

to vinyl chloride follow s a singular pattern for the differenttissues and organs of the rat.MATERIALS AND M ETHODSThe experiments were performed with vinyl chloride(C H2=C HC 1, the m onohalogenate derivative of ethylene) of acommercial grade (99% purity) assumed to containinsignificant amounts of various noncarcinogenic

contaminants.T hree-m onth-old W istar (A r/IRE ) m ale albino rats (about150 g body w eight) were exposed to vinyl chloride vapors for4 hr a day, 5 days a week, for 12 months. The animals werekept in metal or plastic, air-tight cages in which a constantflow of air, containing 3% v/v (equal to 30,000 ppm) of vinylchloride, w as introduced. T wenty-five rats of the sam e strainwere the control group. A t the end of the treatment, thesurv iv in g an im als w ere k illed a t 2 0-d ay intervals, an d th e m ostim portant tissues and organs w ere exam ined histologically bystandard m ethods. D uring the period of exposure, the anim alswere slightly soporific; however, the first few months oftreatment were well tolerated and no changes in growth orbehavior were noticed. After 10 months of treatment, someanim als began to show a hard m ass in the paraauricular regionw hich becam e progressively larger until it reached the size of awalnut or slightly larger. In most cases, the swelling wasunilateral; it was bilateral in only a few animals. After 1 or 2m onths, the grow ing m asses becam e ulcerated and dischargednecrotic debris, while a certain amount of tumorous tissuebegan to form on their surfaces. In addition, we observed thatthe masses were an integral part of the paraauricular regionand could not be distinguished from the local tissues. C aseousnecrotic zones were found. Pleura and pericardium oftenshow ed diffuse inflammation of a fibrous nature and, in m anycases, the lungs were covered with a number of whiteform ations as large as grains of rice or even larger and harderthan the lungs themselves. In 2 cases, the lungs werehemo rrh ag ic w ith m ilk y, th ick flu id in th e p leu ral cav ities. T heliver was sometimes increased in size and very fragile. Thean im als w ere su bjected to X -ray an alyses at differen t in terv alsfrom the beginning of treatment in order to control the stateo f th e sk ele ta l b on es.A ll the anim als that inhaled vinyl chloride show ed a seriesof parenchym al lesions. Am ong these, m ost prom inent wereth e d is ap pe ara nc e o f g ra nu la r a nd P urk in je c ells, d eg en era tio n

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of the cerebellum, severe chronic hepatitis, interstitialp neumo nia, a nd m od erate sw elling o f th e k id ney p aren ch ym a,o fte n a ssumin g th e p atte rn o f tu bu lo ne ph ro sis .RESULTS

T he rates of survival and the m ain findings are summarizedin Table 1. A lmost all the animals developed tumors of theskin and lungs. Very few animals developed bone tumors; inthese cases, the tum ors were localized in the m etacarpal andm etatarsal bones of all 4 extrem ities. Skin tum ors were by farthe m ost frequent, am ounting to 65 or 70% .Skin Tum ors. The tum or that developed m ost frequently inth e p araau ricular reg io n w as th e ep id erm oid carcin om a, b ut w ealso observed papillomas and rarely mucoepidermoidcarcinomas. In all cases, the neoplasms were of epithelialnature. The 3 patterns noticed cannot be com pared to varioushistotypes but rather to a transition of one type into the otheror, and which is more likely, to different stages of the samepro li fe ra ti ve t ype .

Onco gen ic A ctivity of V in yl C hlorid eW arty subauricular growths occurred in some rats. Theh isto lo gic al p icture sh ow ed pap ular epith elial pro liferation ,with progressive increase in the thickness of the epidermis(F ig . 1 ).T he papillary w arts of exophytic type had a fibrous vascularstroma and various degrees of inflammatory lymphocyticin filtra tio n, m ark ed h yp erk era to sis, p ara ke ra to sis , a ca nth osis,and areas of individual dyskeratosis or pearl-like horny

formations.T he epithelial cells of the penetrating colum ns (Fig. 2) w ereirregularly arranged and w ere frequently accom panied by aninflammatory infiltration of the derm is. T he typical featuresof the Malpighian layer and of the stratum corneum of thep rick le cells an d o f ge rm inal lay ers w ere easily reco gnized (F ig.3). The horny layer was com posed largely of cell nests, whichappeared at certain points w ithin the epithelial masses andassum ed the w ell-know n appearance of "horny pearls" (Fig. 3,arrow s); they w ere m ade of flattened and com pressed pricklecells without nuclei and w ere located around a central core ofkeratin.

T able 1Onc og en ic e ff ec ts o f in ha le d v in yl c hl or id e a s a f un ct io n o f timeT he anim als w ere exposed to vinyl chloride vapors for 4 hr a day, 5 days a w eek, for a totalo f 1 2 m on ths , in a co nstan t flo w o f a ir c ontain in g 3% v /v v in yl c hlo rid e.

Rat12,3456781416,17212223242526Survivalate300280-30031033333734734735435938038Q380380380380SkinMucoepidermoidcarcinomaEpidermoidcarcin

kera-tinizingtypeEpidermoidcarcinoma,kera-tinizingtypePapilloma,

keratotictypeEpidermoidcarcinomaEpidermoidcarcinomaM ucoepidermoidcarcinomaEpidermoidc

tumorAdenocarcinomaNo

tumorNotumorNotumorNotumorNotumorAdenocarcinomaNo

tumorAdenocarcinomaNo

tumorMucus-producingadenocarcinoma(alveolar

cellcarcinoma?)NotumorSquamouscellcarcinomaBonesOsteochondromaNo

tumorNotumorOsteochondromaOsteochondromaNo

tumorOsteochondromaNo

tumorNotumorNotumorOsteochondromaNo

tumorNotumorNo

tumorNotumor

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P. L. Viola, A. Bigotti, and A. C aputoK eratinization was irregular and often parakeratosis w asobserved in the areas of the tumor in which the cells wereloosely aggregated and undergoing an individual rather than aco llectiv e ty pe of k eratin ization (F ig . 4). In d ysk erato tic areas,a few epithelial cells tended to form pearls. T he tum or seldomshowed an undifferentiated growth with cellularpleomorphism and several m itotic figures (Fig. 5). A fewtum ors show ed little nests of isolated pale cells (F igs. 6 and 7)of 3 types: mucin-producing cells (originating from the ductep ithelium o f sw eat g lan ds o r sa liv ary glan ds); sq uam ou s cells;and intermediate cells with minor tendencies towardsdifferentiation.R esp irato ry T ract. T hese tumors, a lth oug h oc currin g ra rely,w ere m ainly adenocarcinom atous. O nly in 1 rat did w e observea n e pid ermoid tumo r o rig in atin g from th e e pith elium-c ov erin gcells. Sometimes, the tumors were seen in their earlydevelopment and consisted predominantly of cubic orcolumnar cells arranged as regular or irregular tubular andp ap illary elem en ts (F ig . 8 ) an d sup po rted b y p oo rly d ev elo pedfib ro us strom a (F ig . 9).In o th er cases, g lan du lar stru ctu re s w ere o ften im perfectlyformed and appeared as sheet-like proliferations of

u nd ifferen tiated o r ple om orp hic ch aracter. T he cells showed atendency to extend into the pulmonary parenchyma, thussim ulating the m icroscopic features of the so-called alveolarcell carcinom a (Fig. 10). Som etim es, this was the prevailingpattern. The pulmonary air sacs were lim ited by 1 or morelayers of cubic, colum nar, or polyhedral cells w ith abundantc ytop lasm th at w as fain tly eo sin op hilic (F ig . 1 1); freq uen tly ,a de no pa pilla ry e xc re sc en ce s sp re ad in g in to th e a lv eo la r sp ac eswere p re sen t.In some areas, foci of cellular polymorphism withhyperchrom atic nuclei were noticed; m ucin w as produced invarying amounts and secreted into the lumen of the tubulesand acini. T here w ere sm all pools of m ucin in w hich signet ringcells occurred, either alone or in sm all clusters (Fig. 12). Thealveolar walls were often quite thick and, at tim es, showed aninflammatory infi lt ra t ion.As mentioned before, a single tumor showed squamousstructures and appeared to have been formed mainly bysp in dle a nd o va l u nd iffe re ntia te d c ells (F ig . 1 2).Bones. In the metacarpal and metatarsal regions of the 4limbs, a large proliferation of cartilaginous tissues aroseoutward from the periosteum and, from the appearance of itscells, seemed to derive directly from the cortical bone (Fig.14). The periosteum also grew and in some areas spread asfin ger-like p rolon gatio ns in to th e n ew ly form ed cartilag e (F ig .15). In these places, a gradual transition between fibrouscartilag e, p erio steum, and bo ne co uld b e n oticed .T he n ew ly fo rm ed cartilage ap peared irreg ular w ith aty picalareas; the cells, which had nuclei larger than the normalc ho nd ro cy te s, la y in w ell-fo rm ed , c ap su la te d la cu na e. T he c ellsoccurred singly, in pairs, or in tetrads and, although ofdifferent size and shape, they usually contained a single,darkly stained nucleus. The cartilaginous growth was nothomogeneous, as shown by the extension of the finger-likeprolongations into the boundary of newly formed tissue.B esides the chondroblastic, chondrocytic, and angiom atousareas which indicated a rapid growth, there were also

cartilaginous zones possessing regressive features, such asfib ro sis an d hy alino sis. T he p erich on drium ap peared often a s ac ompre sse d a nd stru ctu ra lly a lte re d fib ro us tiss ue .In some cases, the tumor growth was related to the stage ofth e e nd oc ho nd ra l o ssific atio n o cc urrin g b elow th e " ep ip hy sia lplate." O ssification w as irregular, so that osseous trabeculaevaried greatly in thickness and contours. In other cases, foci ofa ctiv e cellu lar pro liferation , cartilag in ou s areas, ch on dro iddevelopmental nests, and calcified bones occurred in thedeeper portions of the trabeculae (F ig. 18).The new form ation appeared to be an osteochondrom a andconsisted essentially of a bony protuberance capped bycartilage and a fibrous layer, which represented theperichondrium.The fibrous layer was continuous with the periosteum ofthe adjacent cortical bone and extended inward to form septas ep ar atin g and enc lo sing l obule s o f c ar tila ge .C ontrol A nim als. T he control rats w ere kept under the sam econditions as the experim ental rats and at the appropriate tim ewere subjected to a constant flow of air without vinylchloride. N one of these anim als developed tum ors or the typesof parenchyma! lesions developed by the rats that inhaledvinyl chloride. In a very few of the control rats, there was asw ellin g o f th e liv er a nd k id ne ys.DISCUSSIONA s in m an y carcino gen esis stu dies in w hich m ultip le tumo rsarise in d ifferen t tissu es an d org an s, th e da ta m ust b e ev alu atedin terms of a more prompt positive response to be obtainedwith the ideal concentration of the carcinogenic com pound.T he cutaneous system is the m ost susceptible to the oncogenice ff ec ts o f v inyl c hl or id e.Our experimental data do not explain why the cutaneoustum ors developed in the sam e site, i.e., the region including

the area in which the submaxillary and parotid glands arelocated. It is possible that the salivary glands m ay by involvedin the concentration or excretion of vinyl chloride or som e ofits activ e d ecomp osition pro du cts. T his h yp oth esis is stro nglysupported by morphological findings showing the specifictendency of the developed tumor to appear as amucoepidermoid carcinoma, which indicates the activecontribution of the mucus glandular cells to the tumorousgrowth. Such kinds of histotypes are often related to someg la nd ula r a gg re ga te s, a nd , th ere fo re , th e h isto ge ne sis o f h um anm ucoepiderm oid carcinom a has been restricted to the cells ofintercalated ducts. This hypothetical interpretion must beconfirmed by future experiments in which the action of thevinyl chloride should be restricted to the salivary system . Atthe concentrations used, saturation and wetting of the furmight well be expected. Under these circumstances, thenatural cleansing habit of the rat m ight add a significantingestion problem with subsequent concentration of vinylchloride in the salivary glands. The local extremeconcentration could be the result of the difficulty of ac om plete c lean sin g b y th e rat.The neoplastic response of the lower respiratory tract,a lth ou gh o f le sser m ag nitu de, is o f relev an t in terest, sin ce som e

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P. L. V iola, A. Bigotti, and A. Caputo

V v ' ^*"r>^%' '* V" '.>* ' .- N*y^ (' *; ^ -% . v- ' ^ '.-'M* V..;.::>:;>-/* ' '

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Oncogene A ctiv ity o f V in yl Chlo rid e

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MAY 1971 52 1


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viola-response of rats to vc-1971

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