Document VjxJ4r7rMdv3ozjZM4QLrjXEo

MLE COFi KE0 2 2 1974 H. Tv: and Ucs, |t Riboflavin *jt|j 1*J Aniiiniau, 0- I'QT re. I * w. ft. M. Hv.nnntTT. U. \[ rk: C'otuniliia |'l(; \ Mvlh,*l for !>,, Arid, li ilumurlr,, 1 TisMioa, J. Ui0| 11. Intracellular 'r ^tudira on tl iver Homogenate, dtorvtic and Ultn. `rotcins of Various .liincthyliuninoato. 1951. icoac-6-phoephataac NeopUatic Tiaetm. Further Studies of Polymers as Carcinogenic Agents in Animals* B. S. T. I.Oppeniieimer, Enid Oppenheimeu, Danishefsky, Arthur Purdy Stout, and Frederick R. Eirich With hi technical assistance or Margaret Wiujiite (Inititute of Cancer Reward!, and tAe Department/ Biockemiitry, ColUyo of Phynciane and Surgeon*, Columbia Unirerrity, Now York, N.Y.) The inception of this investigation is an exam ple of serendipity, a word recently popularised to designate the faculty of accidentally making ob servations or discoveries which were not originally ..ought. In tile present instance, a safe antihyper tensive compound was being sought among the quinoncs to reduce the high blood pressure pro duced by wrapping one or both kidneys of rats with cellophane. After a couple of years, in seven of these fntSliialignant sarcomas hail developed jt the site of the wrapping. In several instances, j these sarcomas had extended into the jieritoncal cavity and had also metastasized. In view of this { unexpected finding, a new series of cx])erimcnts was begun to investigate this phenomenon in vari ous directions. It was found that sarcomas could I* induced not only by wrapping the kidney in cellophane, hut alternatively by imltcdding the cellophane subcutaneously in the anterior abdom inal wall. By either of these methods, tumors were induced in approximately 35 per cent of the aniI mats (10). Further work soon showed (11, 12) that we acre dealing not merely with one more carcinogen, of which hundreds were already known, but that we had chanced upon an entirely new group of carcinogenic substances, the polymer films. In | 1M1 Turner (13) had observed, also accidentally, * that disks of Bakelite implanted subcutaneously in rats produced fibrosarcomas. Subsequent to our original report in 1948, similar results have been obtained by Druckrey and Schm&hl (4, 5), Zol linger (15), laskin, Robinson, and^Weinmann (7), ind Bering1. While, thus, there can be no doubt * Thi, investigation wu supported by a research grant, N'a. -19*0 (CM), from the National Cancer Institute, Notional Inatitutea of Health,' United Stntee Public Health Savin. 'E. A. Bering, Jr, pemonnl communication. Received for publication February S4,1059. as to the actual facts, the interpretation of these facts as regards the mechanism or mode of action of polymers in inducing tumors is still obscure. The present paper describes the investigations which were pursued in an effort to gain a better understanding of the processes involved in the car cinogenic action of polymers. A study was made of the effects of a number of polymers having dif ferent chemical structures. Also, because the plas tics used in our earlier work were commercial products often containing plasticisers, stabilisers, traces of catalysts, a residual monomer, etc., we have imbedded a number of samples of polymers specially prepared to assure their purity. The pos sible carcinogenic effect of several monomers has also been investigated, and studies have been made on the degradation of poiymcrs within the organism by means of polymers tagged with iso* topic carbon. METHODS AND RESULTS Imbedding Phocedubes In most experiments the animal chosen was the Wistar rat, but the Sherman strain was used in earlier tests, and for purposes of comparison ex periments have also been done on mice of the Longacre, Paris, and C57 strains. In most experi ments males were used, but, in some, females were employed with similar results. The animals were fed Purina Laboratory Chow and, occasionally, fresh carrots, and had free access to water. The general procedure was to use small squares or circles of film, averaging 1.5 cm. in width, which had been sterilized in Zephiran1 (1:1000 dlilution of the commercial 12.8 per cent solution) for sev eral hours, and washed in sterile saline. These were inserted subcutaneously one on each side of the abdominal wall, just ventral to the fascia. * In the early experiment!, (tarilimation woe in 80 per cent alcohol. In n few coaee film* or powden have been etcrilieed by beat. 333 ucc 041073 Cancer lleacurch "Willi ft'\v exceptions, each t-xiicriuictil consisted of AO intltcddiiigM; sometimes (lie mime material was imlteddetl on Ixitli aides of 2A animals, mid sometimes oih% form of the material wai imlicddcd on l bo side mill another on Lite lofl. The ani mals wore kept mu lor done observation for several weeks, until the iitrisiotis wore completely healed, and subsequently were examined weekly to deter mine the onset of tumors. I'MITIM ImIIKDDKD A Tost of the plastics imlieddcd were obtained through the kindness of various industrial firms. Tlio designation `'commercial" implies those man ufactured for ordinary industrial purposes, with a more or teas unknown content of nonjiolymeric material. Of the "pure" forms, some have been s|K'cinliy made for us liy the manufacturers and others prepared or purified in our own laboratory. The following arc the plastics which were im bedded: Cellophane A .*--a commercial sausage casing. Cello/thane If.--tile same material after extrac tion with alcohol lor 3 days. Cellophane C---Cellophane B after additional extraction with benzene. Cellophane D.--a special form employed for tis sue culture work. Cellophane 0.--a Cellophane of the highest pu rity obtainable which was kept in formalin and washed just before imbedding. Dacron, Kel-F, Pliofilm, Saran, Silastic, and Teflon.--all commercial products. Nylon.--the purest form obtainable, said to con tain no additives. Polyethylene A.--a commercial film. Polyethylene B.--specially prepared for us by the manufacturers, said to contain no additives and only a trace of catalyst. Polyethylene Bil (high molecular).--had all the low molecular weight fractions removed. Polymethyl methacrylate A.--cost from a com mercial product. Polymethyl methacrylate D.--prepared os follows lows: freshly distilled monomer, methyl methac rylate, was sealed in an evacuated glass tube at 4 X 10~* mm. pressure and kept at 80 C. for weeks. The resulting polymer was purified by so lution in methyl ethyl ketone and three reprecip itations with methanol. The film was made by casting from solutions of methyl ethyl ketone. No catalysts or additives were employed in this prep aration. The designations "A." "B" **C," "D," sad TO" are arbitrary and for convenience of reference only. "I'oTxpex.."-- nn Knglisli |>ri'|iurut lot, of jmly. With methyl methacrylate, said to Ik; |>c<:iully pure. definite , Polyvinyl cldoritle A.--a commercial product, I found c known to contain some additives. ! which p I'oli/vinyl chloride I).--socially prcjMired for ui between by ultraviolet t*lymcrization of vinyl chloride. firmly in Contains no plasticizers, catalysts, or other addi In cuy tives. stages co Polystyrene A.--a commercial form. ing arour. Palyxlyrene D.--prepared from its monomer, were usu. styrene, by a process similar to that used for poly, more in *, methyl methacrylate D. It likewise contains no appearan catalyst or additives. been imb Ivalon sponge and Vinyon N.--both manufac wall, the > tured for surgical use. aseptic ci Silk film was mode from natural silk filler hy vivc, in o dissolving it in an aqueous solution of lithium bro of a turno mide, removing the latter hy dialysis, and casting growth of on glass.4 same site, As will be seen from the accompanying tables, growths v these films differed greatly both in thickness and stances in flexibility, varying from the delicate pliable mem- j were allov branc of polystyrene, only 0.01 mm. thick, to the | poses of gi rigid disk of "Perspex," with a thickness nearly ; in axillary 40 times os great. j were rare!; Further variations in physical form were intto- j The tun duced by using some of the polymers in the shape l subcutanei of textile fabrics (fibers), perforated films (6S 1 to have pe holes per square inch), granules, sponges, and pox- , wall, or, v dera. Many of these experiments, and other modi. ' muscle int fications devised to test one theory or another. : stances, a 1 have been too recently begun for any conclusions 1 wards thro to be drawn. In most Bwnm or Imbedding The first demonstrable effect of imbedding i plastic film was found to be the encapsulation of ` the film in a sac or pocket of connective tissue of varying thickness (Fig. 1). With some films, eg. j Cellophane, the pocket wall was thick and dense j and sometimes even contained calcareous areas: i the film m times a por ing from t grown only It is not* no fibrous c were seen (Fig. ), soi while with other films, such as Pliofilm or po|y- ; styrene, the pocket wall was thin and soft This encapsulation was evident within -3 weeksafter j imbedding, and was found in animals of all ages j except in coses where a tumor was induced. t The film was never adherent to the pocket bn! could be easily removed, leaving the pocket nil intact. Thin, pliable films were sometimes fouiw sometimes c the inner (n tumor had g connective side. Nothin ncctive tissu of the film.. rolls, or the folded or rolled, but always in a pocket, sometime with fibrous membranes between the folds. At | autopsy brittle films would occasionally be fouoi : broken, with the broken pieces either all in on j pocket, or, less often, encapsulated separately- 1 nior tissue g |ay be dest instance whi Appeared, ti /him) side o 4 For till* film vo are indebted to Dr. Peter Aimed* ^ : its outer sid< the Cheater Beatty Beeeatch Institute, London. grated that. ucc 041074 l for us Idoridc. 'r addi- inomcr, or poly, nins no nnufac- iber by uni btt>. casting : tables, icss and 1c mem. to the i nearly re intro, ic slinpc ii.m (035 ml |iow. cr inodinnotlicr, elusions tiding a lation of tissue of ms, e.g., id dense is areas; or polyjft. This eks after all ages cket but ,-ket wall es found imetimes 'olds. At be found 11 in one tely. launder of Oi'i'KNukimKit cl al.--Polymers as Carcinogenic Agents 335 With iierforalt'd films and also with textiles, no definite |x*rket was formed, but the material was found enmeshed with connective tissue fibers e-liieh jienetrated through the fierforiitious, or between the textile threads, holding the plastic firmly in place. In eases where tumors were produced t he early stages could lie |ml|Nitcd as a thickening or swell ing around the film. The tumors grew rapidly and were usually large enough for removal (3 cin. or more in diameter) in 2 nr 3 weeks after their first appearance. Since in most cxiicrimcnts films had been imbedded on Iwth sides of the nlidntninnl wall, the first tumor to np|icnr was removed under aseptic, conditions and the animal allowed to sur vive, in order to give nn op|>ortunity for growth ot a tumor on the opposite side. Sometimes a regrowth of the first tumor occurred at or near the tame site, and in three of these cases metastatic growths were found in the lung. In one or two infiances in our early experiments, where tumors were allowed to grow for many months for pur poses of gross demonstration, mctastascs occurred maxillary lymph nodes; but, in general, metastases were rarely seen. The tumors were usually located entirely in the sulicutimcous layers, but were occasionally found to have iiciiclnitcd the outer layers of the muscle wall, or, very rarely, to have grown through the muscle into the ]>eritonenl cavity. In a few in stances, a tumor was found to have ulcerated out wards through the skin. In most coses the tumor was found to surround the film more or leas completely, though some times a portion of the film would be found project ing from the tumor, or the tumor would have grown only on one side of the film. It is noteworthy that when a tumor developed no fibrous capsule could be found, but tumor cells were seen immediately adjacent to the plastic (Fig. 2), sometimes surrounding it on both sides, sometimes on one side only, which might be either the inner (muscle) or the outer (skin) side. If the tumor had grown on only one side of the film, then a connective tissue wall was present on the other side. Nothing definite is known as to how the con nective tissue capsule disappears from the vicinity of the film. Its cells may be converted into tumor cells, or the capsule may be pushed away by tu mor tissue growing between it and the film, or it may be destroyed by pressure infiltraton. In one instance where the capsule had not entirely dis appeared, tumor tissue was found on the inner (film) side of the capsular remnant, as well as on its outer side and enmeshed within it. This sug gested that, in this particular instance, the cap sule whm in the process of destruction ami replace ment by infiltration and pressure. Tin*, iituultcrs of malignant tumors obtained by these imliedding experiments are given in Tables 1 and 2. Since these arc all long-term experiments, with a latent i>criod varying from 1 to 2 yenrs after im bedding lieforc the appearance of tumors, mnny of the experiments arc still unfinished, and merely the nmnlicr of tumors produced to date can tic re corded. Many other experiments are in progress, but since in these no tumors have so far been pro duced no results can lie given. Table 1 shows the completed experiments, with the number of tumors produced and the percent age production calculated from the number of ani mals surviving the minimum latent period for tu mor appearance. Only tumors arising around, or in direct contact with, the imbedded polymer were included as having been induced by the plastic. Any other tumors appearing in the experimental animal were interpreted as "spontaneous," and these appeared in about 2 per cent of the animals. Including the kidney-wrapping experiments (10), our observations show 275 primary malig nant tumors induced by plastics. All of these tu mors were mesenchymal in origin: the large ma jority (over 85 per cent) were fibrosarcomas, but other types, particularly osteogenic sarcomas and rhabdomyosarcomas, were also obtained (Figs. 3a to 6). The complete list ot types obtained fol lows: Fibrosarcoma................................. 235 Osteogenic sarcoma...................... 12 Rhabdomyosarcoma.................... 8 Mesenchymoma........................... 6 Liposarcoma................................ 5 Reticulum-cell sarcoma................ 5 Myxoma...................................... 2 Plasmoeytoma............................. 1 Histiocytoma (malignant)............ 1 275 The evidence of malignancy was based on his tological findings, including the frequency of mi toses, on transplantability, on occasional metasta ses, and on frequent local recurrence after removal of a primary tumor. The somewhat infrequent occurrence of metastases from the primary fibro sarcomas may be partly explained by the fact that the tumors were usually removed 2-3 weeks after their first appearance. The tables show that tumors are induced wheth er the film is thick or thin, flexible or rigid, and that a plain film appears to induce more tumors than other forms such as perforated films, textiles, or powders. So far we have obtained no. turnon 336 Cancer Research ! t with plastics in powder form, hut only one of these experiments is coinph'tecl, and little significance should be attached to the observation os yet. Controls As control experiments we imbedded a variety of nonplnstic materials, including glass covcrslips, slips of wood and mica, pellets of paraffin, cotton "loiters" (tile fillers from which our Cellophane A ' was mndc), surgical cotton, gloss cloth, and a num. tier of metal foils. Two natural polymers, kentio (foetal nails) and a thin collagen film, were also im. bedded. Of the completed controls, as previously i*. j ported (12), linters and surgical cotton produced no tumors, but with the gloss coverslip there m , s single fibrosai viving rat of fif experiment will tile (fiberglas) > tumors were obt siv still unfinish tfhort a time to TABLE 1 Results or Imbedding Plastics Subcutaneously in Rodents* ConpleUd Experiment! SUnaiML unumwa CEIJX)1`1IANE: A plain film A plain film A plain film 1) plain film C plain film U plain film D perforated film uairiwa or nut IIUMa twin.) Flexibility 0.04 0.04 0.04 0.04 0.04 0.01 0.01 Flexible Flexible Flexible Flexible Flexible Very flexible Very flexible Mo.un fluiiiiim muna uian mmae Lanorr ------ (ante) 42 406-779 sat 246-408 m 309 44 829-086 30 380-708 10* 423-821 22 804-604 StuautT No. Parent 16 86.7 8 22.8 1 , 4.6 20 46.4 18 40.1 3 16.8 4 18.2 DACRON: Plain film Perforated film Textile 0.02 0.02 0.06 Very flexible Very flexible Very aoft 41 330-008 42 327-081 38 8 10.6 2 4.8 0 0.0 IVALON STONGE Soft, flexible 34 687-867 8 8.8 KELP; Plain film 0.02 Flexible SO 269-681 7 23.8 NYLON: Plain film Perforated film Textile 0.00 0.00 0.08 Flexible Flexible Soft 28 441-861 31 611-738 S3 7 27.0 2 8.6 0 0.0 PLIOFILM: Plain 0.01 Soft, pliable 48 369-708 8 16.0 POLYETHYLENE: A plain film 0.06 Flexible 80 892-722 10 12.6 B plain film 0.02 Very eoft, pliable 66 386-742 11 20.0 B plain film 0.02 Voy aoft, pliable sot 343-646 3 10.3 B perforated film 0.02 Very aoft, pliable 41 407-784 0 14.0 B textile 0.16 Rather (tiff lex* 40 407 1 2.6 me HM plain film 0.07 Flexible 34 362-683 8 8.8 Powder 42 0 0.0 POLYMETHYL METHACRYLATE: A plain film 0.14 Rigid, brittle 20 681-868 4 20.0 POLYSTYRENE: A plain film 0.01 Soft, pliable 27 860-668 7 26.0 POLYVINYL CHLORIDE: A plain film A perforated film SAIIAN: Plain film 0.04 9.04 0.02 Soft, pliable Soft, pliable Soft, pliable 44 180-727 17 38.8 27 0 0.0 42 300-847 6 11.0 SILASTIC: Plain film 0.26 Rubbery TEFLON: Flain film Perforated film 0.02 0.02 Flexible Flexible * Empl wlwn mUd U tL cottnij, d f Albina (limftni) mkm hmL t BUefc COT) nb mu --i m WbUr nti 36 84 32 800-000 430-748 626-728 14 40.0 8 23.6 0 18.7 j The possibilii of the polymer; entrapped in the ! mer breakdown. ! D,n 10/17/ a 2/18/53 6/5/53 1/23/63 1/14/SS 3/28/53 3/8/5S 9/10/63 10/1/63 4/20/63 12/2/63 periments were carcinogenic eff 1. Ten rats w with methyl m months. 2. Ten mice neck with a 50 sene 3 times aw S. Ten mice eent benzene & one of the comr In none of tJ although some t Considcratio that the active free radical cat this idea pellet; and 3 per cent 1 ] both sides of ! i I ucc 041076 T Jloph.un* \ >il tinuin. b. keratin tv also int- iously rei produced there was OrrKNiiKiMKit cl at,--Vnlymcrx ax Carcinogenic Agcnlx 337 , duple lihrosiircoilin, up|N*ariug iu the Inst stir,jving rat of fifty, 050 days after imlN'ddiug. An rsperiuteiil with tin foil and one with a glass tex tile (filtorglns) have recently terminated, and no tumors were obtained; hut the other cx|>criments lie still unfinished, and have Won in progress too ihort a lime to have any significance. Monomkhm The possibility that the carcinogenic nctivity of the polymers may W due to some monomer entrapped in the film, or formed as a result of polyoer breakdown, cannot be disregarded. Three ex induced so l>y far these materials, hut the experi ment is not terminated. Pellets which were re moved from the animals three or more months after imbedding contained no lxmzoyl [s:roxidc. This indicates that the material hail l>ccn com pletely ahsorlicd, decomposed, or loth, within this period. Conclusions which may l>c drawn from these experiments arc suggested in the "Discus sion." Kxnmnwn wim Taooko Poltmkhh To ascertain whether plastic films, although so inert, undergo any changes in the animal body, Date MTimgn 10/17/S* t/18/53 5/5/53 1/23/53 1/14/53 3/M/53 3/6/53 8/10/53 10/1/63 4/80/53 It/S/53 .TABLE * Results of Imbedding Plastics Subcutaneously in Rodents Experiment* in Program No* KAU8* Matoul ainHB CELLOPHANE a Perforated film CELLOPHANE 0 Plain film PERSPEX POLYMETHYL METHACRYLATE D Plain film POLYSTYRENE D Ilain film POLYVINYL CHLORIDE D llnin film SILK FILM VINYON N Plain film POLYKTHYLENE-C1' POLYMEIBYL METHACRYLATE-C*4 POLYSTRYEN&C44 nhAaw C--) or HIM FUiibiUtr 0.04 0.03 0.30 ,, Flexible w Slightly stiff Rigid MtfT tUKlM www vr o 1/1/M ' 0 0 8 0.06 0.04 Rigid Flexible 11 8 0.03 Irregular 0.0* 0.08 0.10 0.07 Flexible Brittle Flexible Flexible Flexible Flexible 4 6 * 7 < * linmttw unme narae (bat.) 553 413 474 447 380 533 313 440 37* 350 *08 periments were conducted to study the possible metabolic studies were made of rats imbedded carcinogenic effect of monomers: with polymers tagged with CM. 1. Ten rata were painted on the back of the neck Radioactive polystyrene was prepared by heat with methyl methacrylate 3 times a week for 4 polymerisation of styrcnc-/J-Cu(C*niCII--C,4II*) months. in a manner analogous to that used for polysty- 2. Ten mice were painted on the back of the rcnc D. Films of this polymer (55.0 mg.), contain- seek with a 50 per cent solution of styrene in ben- ing 2.04 X 10T cpm/mg, were imbedded subeu- sene 3 times a week for 4 months. taneously on both sides in the anterior abdominal S. Ten mice were painted similarly with a 1 per wall of 25 male Wistar rats. Tagged polyethylene cent benzene solution of hcxomethylcne diamine, (--CIIj--CMH|--), having 8.6 X 10* cpm/mg, one of the component* of nylon. and polymethyl methacrylate (--CHiC(CII,)-- In none of these cases was any tumor induced, COOC*4H*) having 3.6 X 10* cpm/mg, were ob- ilthough some skin irritations resulted. tained from their manufacturers, and small pieces were imlicddcd in the usual manner in rats. Dehxotl Pxmoxidw Tho different tissues and the feces were sub- Consideration was also given to the possibility jeeted to the Van Slyke wet combustion procedure that the active carcinogen might be some residual (14), converted to BaCOj, and counted as such, free radical catalyst in the polymer film. To test Respiratory COj was trapped in 10 per cent NaOII this idea pellets containing 0.5 per cent, 2 per cent and converted to BaCO,. Urine samples were con- nd 8 percent benzoyl peroxide were imbedded on ccntratcd and filtered, and 1 ml. was plated dircet- both sides of 30 animals. No tumors have been ly. Background values for the latter, 28-32 cpm/ *1 ; 1 j ) j < ] ucc 041077 338 Cancer Research u\l, were obtained with noniml noitrndiniiclivc and polystyrene urine treated in the mine nmimer. With the polystyreiic-iiiilioddcd rats, no radio- activity was detectable ill the excreta or tissues for SI weeks after the imlicdditig. At the end of tlda time, however, a anmll nmnimt of radioactiv ity was found in the urine (41.3 rpm/24 hr excre tion). This low level of urinary radioactivity has continued up to the present, i.c., for 40 weeks. The rata iudieddcd with jxdycthylene C4 liegnn to excrete radioactive material after 2G weeks, and those indicdded with polymcthyl methacry late after 54 weeks. When the film was removed, in any of these cases, the urinary radioactivity disa|>- (--CUf--tU--) all have the polyethylene "Iwickhonc" hut are sub. stituted at various points. In short, they all sit vinyl or acrylic polymers. Nylon J? (--C: (CII,)r---CC--NH (CH*)NH--...), peared. The urinnry radioactivity cannot be due to any residual monomer in the films, since no ra dioactive material appeared in the urine immedi ately upon imbedding but only after an extended interval. on the other hand, is a polyamide, while Dacron1 ?? (... CC*H*--C--OCjHi--CHj--0 .,.) No radioactivity was detectable in the expired is a polyester; Silastic (... (CH|)--SiO ...), i* air, in any of the tissues, or even in the tumors substituted silicate, Cellophane * which have already resulted in some cases (sec Table 2). It would seem that the degradation product released from the polymer is very mi nute and is rapidly removed and excreted. This is the same metabolic pattern already demon strated in previous experiments with styrene (3). The nature of the breakdown products of the poly mers is still unknown; experiments to investigate these are in progress. DISCUSSION a polysaccharide, and Ivalon a cross-linked poly vinyl alcohol The carcinogenic polymers enumerated in Tables 1 and 2 differ widely in their chemical struc --) ture. The simplest one is polyethylene, which is essentially a pure paraffin, . , . --CH*--CIIi-- ... ; there is evidence of branching of the chains (6), but otherwise it differs from paraffin only in its higher molecular weight, or chain length. Poly vinyl chloride (. . . CHC1--CHj-- . . .), Saran (--CCl*--CHr-CHa--CH.--), Teflon (--CF*-- CF), VinyonN (--CH--CHj--CH--CHi--...), The only common denominator in all these sub stances is that they are polymers, i.e., molecules of high molecular weight, containing units which repeat themselves. The possibility that the carcinogenic agent in these experiments is not the actual plastic but some low molecular weight impurity (plasticizer, additive, or even residual monomer) was seriously OCOCH, CN considered. Pure polymers (polystyrene D, poly mcthyl methacrylate D, polyvinyl chloride D, and Kel-F (--CFiCFCl--,.,), polymethyl methacry late Cellophane 0) which contain no additives and in which the amount of residual monomer, if any, it extremely minute, were therefore imbedded. These experiments arc still incomplete, and no final per centages can be given (Tabic 2). However, the fact that numerous tumors have already been induced with these pure plastics docs demonstrate that the Pliofilm primary carcinogen is the macromolccule itself, rather than any additive or impurity which may be present in the commercial film. Furthermore, the completed results with various forms of Cello phane an* lationship duccd an* absence o: renc, met diamine n are the nr, polymer*, the carvin' the poJym The pri carcinogcr they arc ii colly rath* and cell c< ise. That t ical irritat tin foil, a produce in genic. Moi carcinogen film, since roorsto th< would be ical irritat In view our exper shown that oliaed in tl of breakdo olites have the possibi interaction tion produ the organi polymer tl ways. In t tion produ mal polyn esses procc it is reaso; logical bre radical nai depolymer tain extent biological * pear to in\ is possible I ence of th< arc in the ] more, the f> dcgradatioi processes. ' would thus of the degr Secondly ucc 041078 >nt lire nibhey nil are It--...), Iiilc Dacron -0...) iO ...), is a -O...) -linked poly- ill these sub.e., molecules g units which jenic agent in al plastic but y (plasticizer, i was seriously xene D, polyhloride D, and ditives and in mer, if any, is bedded. These d no final perwever, the fact f been induced istrate that the nolecule itself, ity which may . Furthermore, forms f Cello- Qi'PKNiikimkr rt al.--Polymers as Carcinogenic Agents 839 1,liam* and |mlyel.bylene show l.lint there is no re- reactive centers in the polymer itself, as a result l^tinnsliip Ix'lwcon the inmilxT or Mireotunx pro- of I,lie degradation. These "active centers" would (Infi'd and the. decree of purily'nf the tilin. 'J'ltc then lie capable (if binding proteins or other liasic ,|)5<.|ioe of onroinogi'iiieily in the monomers sty- tissue constituents and consc'iucntly might impair (jno, methyl methacrylate, nitl hexamclhylenc the mclalxilisin of the adjacent cell. diamine rules out the-)>ossil>ility that monomers On the basis of the long latent period it appears *rc the active agents, nt least in thcSc particular that the production of tumors would require the polymers. It therefore appears fairly certain that presence of free radicals in a specific area for an the carcinogenic nelivity of plastics is inherent in extended period. This may he the reason for the the polymer itself. absence of carcinogenic activity in our imlicdding The primary difficulty in comprehending the cx|icrimcnts with Ixinzoyl jx;roxidc. The latter is rarrinogenie netion of "plnsl ies** is the fact that relatively unstable and dccoin|>oxcN in a comfuirn- they art' insoluble in lupieous systems ami chemi tively short jained. Furthermore, tumor jmxlue- cally rather inert. Aliy interaction Isitween them tiou may depend ii|xn a free radical of a very hjkj- and cell components is, therefore, hard to viminl- cific nature and stability which is pcriiaps not iu>. That their nel ivity is li result of mere mechan chaructcriAic of lamsoyl jxiroxidc. ical irritation by friction is hardly prolwdde., since In any case, tlic fact that jxilymcrs break down tin foil, cotton lintcra, and {Munflin should also to some extent allows for the possibility of their produce irritation; these, however, are not carcino chemical interaction with orgunic constituents genic. Moreover, there is no correlation between and a resultant carcinogenic activity, whereas, carcinogenicity and the stiffness or rigidity of the. without this evidence, one was reduced to assum film, since soft, thin, pliable films often induce tu ing damage due to metabolic interference by mere mors to the same extent as the stiff rigid ones which physical obstacles, a not very satisfactory hypoth would be expected to cause far greater mechan esis. Of course, it may possibly be a combination ical irritation. of physical restriction plus chemical metabolic In view of this, it may be very important that interference. our experiments with tagged molecules have The extreme length of the latent period before shown that the polymers are degraded and metab the appearance of the tumors could be correlated olized in the body, at least in the rat. The amount with the length of time necessary for polymer of breakdown is extremely minute, and no metab breakdown and the slow rate of release of the olites have as yet been identified. This re-opens breakdown products, since carcinogenic activity the possibility of a chemical or physico-chemical would then be a cumulative function of this deg interaction between the polymer or its degrada radation. If this be true, one would expect the la tion products and some basic cell constituent of tent perjod to be shortened when more "reactive" the organism. The carcinogenic activity of the plastics, such as polymer hydroperoxides or even polymer may thus arise in at least two possible partially degraded polymers, are imbedded- Stud ways. In the first place, it may be the degrada ies along these lines will be included in the work tion products which are carcinogenic. Since nor of this laboratory in the near future. mal polymer breakdown in various aging proc esses proceeds via a free radical mechanism (9), SUMMARY it is reasonable to assume that some of the bio 1. Malignant tumors were induced in rodents logical breakdown products may also be of a tree by subcutaneously imbedding the following poly radical nature- Free radicals are known to effect mer films: Cellophane, Dacron, polyethylene, depolymerization of nucleic acids (ft) and to a cer polyvinyl chloride. Silastic, Pliofilm, Nylon, poly tain extent to produce tumors (1). A number of methyl methacrylate, polystyrene, Saran, Ivalon, biological oxidations and enzymatic reactions ap Kel-F, Teflon, and silk. pear to involve odd electron intermediates (8). It 2. The polymer films were always found encap i possible that polymers are degraded in Die pres sulated in a pocket of connective tissue, except ence of these biological free radicals just as they when a tumor was induced. ere in the presence of organic peroxides. Further 8. It does not appear that the carcinogenic ac more, the free radical fragments.arising from these tivity is a result of the presence of impurities, since degradations may inhibit enzymatic free radical tumors were induced by pure polymers as well as processes. The carcinogenic activity of polymers by commercial products. would thus stem from the free radical reactivity 4. The monomers, styrene, methyl methacry of the degradation product- late, and hexamethylenc diamine, were not carcin Secondly, we might visualise the creation of ogenic in rodents when painted on the skin. r- ucc 041079 340 Cancer Research . Studios willi logged |K>lyincm showed tlint they deeontj>oaeri nt n minute rule wlicn left in the organism, (5. A possible mecliatiism hy which polymers mny exert their carcinogenic uetivity, Imscd on the observed degradation, is suggested. ACKNOWLEDGMENTS We take pleasure in ai'knowkxlgiiig our indebtedness to many wlu> hnve given us help iuhI ciipnumgcmcnt ill the course >( lliis work, including Dm, Alexander llnildow and 1`etcr Alexander of (ho Cheater lleatly llcwnidi Institute, laHnlon, l)r. Arthur L. Walpole of Imperial Chemical Indus tries, Mnneliester, England, Dr. William Iftiepcr of the National Cancer Institute, and Dr. Seymour lidiemun of Coluutliin University, as well as to many commercial firm* ml their representatives, who have so generously donated to us many of the plastic materials we needed. These include the Dow Chemical Co. aud its affiliates, the DuPont Co, Good* year Tiic k llubbcr Co, the Monsanto Co, and Union Carbide and Carbon Co. REFERENCES 1, Biota, A. M, and Bauson, E. S. G. Biochemistry of Cancer. Ann. Rev. Biochcm., 30:380-66, MSI.* t Bonn, J. A, V, and Conwat, B. E. Effect of Oxygen on the Degradation of Nucleic Acids by X-Rays. J, Chon. Soc., pp. 3418-16, I860. 3. DAnioninner, I., and Wiluutk, M. Metabolism of Styrene. J. Biol. Chen, 211:549-63, 1934. Dtocun, H.. and SenMini, D. Csncerogene Wirinmg von Kunststoff Folien. Ztachr. Naturiendi, 76:333-61, 1333, 6. --------- Cniicerngeiic Wirkung von Anorganischen i organiselien polyrocrcn Sulmlnnscn bci ItaUen. Act*. 10:110-24,1064. ^, 0, Fox, J. J., and Maictin, A. E. Investigation of Jnfn. ' Iteil Spectra. Determination of C-II Frequencies (--3ooq 1 cm-1) in Faraffins and Olefins, with Some Oletcrvationi : on `TolyLhenos.'' l'roc. Itoy. Soc., A, 176:208-40, isu. j 7, Baskin, D. M.; Rooinhon, I. B.; and Wkinmann, J. f ' Kx|)crimciiliil Production of Sarcomas hy Methyl Melba 1 crylate Implants, l'roc. Soc. Kxper. Biol, tt Med, 37:31). 32, 1034. B. I.KAcn, S. J. Mechanism of Knsymic Oxidoiedortus. Advances in Knaymoiogy, 16:1-47, 1934. 9. Mark, II. F, and Mwuiobian, It. B. Effect of Oxygen n ' Physical and Chemical Properties of Polymer*. Ann. Re l'hys. Chem, 1:326-30, I960. 10. OmaniaiMKa, B, S.i OppiNniiMKR, F,, T.; and Sroct, A. P. Sarcomas Induced in Rats by Implanting CeBo! phsoe. Proc. Soc. Kxper. Biol. & Med, 67:33-34, lots, 11. -------- . Sarcoma* Induced in Rodents by Iroheddn, Various Elastic Films, /bid, 79:300-09,1932. 12. Omwiwn, B. S.; Oppiniikikkr, E. T.;Stout,A. and Damduibtokt, I. Malignant Turnon Resulting fnss Imliodding Plastics in Rodents, Science, 118:305-6, ng. 13. Tuknkb, F, C. Sarcomas at Sites of Subcutaneously !. planted Bakelite Disks. J. Nat. Cancer Inst, 2:81-63. 1941. 14. Van Slyik, D. D.; Plamn, J.; and Wmtoa, J. B. Reagents for the Van Slyke-Folch Wet Carbon Com. bastion. J. Biol. Chan, 191:899-304,1931. 15. Zouanoib, H. 17. Experimentelle Eneugung moligne Nierenkspaeltumcren bei der Ratte durch Drufaw (Plaatie-Kspscln), Sebweis. Ztadbr. Ally.. Path, luu, 16:963-71,1969. 7*p* i ip - V Fro. 1.--Connective tissue pocket surrounding polystyrene D film 3 months after imbedding in male Witter rat. The film was removed before sectioning. XS30. Vm. 2.--Pocket or deft in a fibrosarcoma induoed by polyroethyl methacrylate D in 492 days, Rat No. 2922. The turno cells completely line the deft which coitained the film (removed before sectioning), XS30. * m * * f< Vi*' A y-4 ** .* * l ucc 041Q80