Document 3eo5knv8723e7DVYe4XyQBkqE

T C^TXOSXEMAL USEAjtCH 35, 277-292 (1934) Malignant Mesothelioma Induced by Asbestos and Zeolite in the Mouse Peritoneal Cavity Yasunosuke Suzuki and Norihiko Kohyama >i ,ronmental Sciences Laboratory. Department of Community Medicine and Dtpartmtnt of fatbi'tof\. 'fount Sinai School of Medicine of the City University of New York, One Gustave L. levr Place. Sew York. Sew York 10029. and National Institute of Industrial Health. 21-1. Satgao 6 Chome. Tama-Ku, Kawasaki 213. Japan Received April II, I9S3 rbe carcinogenicity of asbestos (amosite and chrysolite) and zeolite I fibrous erionite, mordeute. and synthetic zeolite aA) were studied in the peritoneum of 536 BALB/C male mice after a single intrapcntoneai or intrubdomuui wall injection. As controls, 132 mice treated with and without saline solution were used. Both asbestos types and fibrous enomte frequently produced malignant peritoneal tumors after long latency: tumors developed in 93 of 394 animals (23.6ft) treated with asbestos or fibrous cnonite 7 months or more after v. administration. All of the induced peritoneal tumors were intimately associated with marked ' peritoneal fibrosis, in which asbestos or enomte fibers were regularly detected. Histopalholopcaily. 33 (73 fibrous, 9 biphaiic. and I epithelial) of 9) were consistent with malignant mesotheliomas. Other tumors consisted of 6 plasmacytomas. IJustiocytoma, I liposarcoma. I osteosarcoma, and I adenocarcinoma of the pancreas. Two of the cases of mesotheliomas were associated with plasmacytoma. In many Instances, the primary site of the mesothe liomas seemed to be multiple, the favorite sites being the omentum, mesentery, serosae of the gastrointestinal and genital organs, the diaphragm, the capsule of the liver and spleen, sod the abdominal wall peritoneum. In these cases, asbestos or erionite-tissue burden fol lowed by fibrosis was frequently observed. In.addition to the 93 peritoneal tumors. 3 extrapentoneal tumors 11 fibrosarcoma and 2 rhabdomyosarcomas') were induced by amosite which was probably accidentally injected into the exttapcntoneai connective tissue and the striated muscle tissue of the abdominal wall, respectively. These three tumors were also iiumatelv associated with focal fibrosis in which amosite fibers were detected. Among the three different types of zeolite, only fibrous enomte showed ltnking carcinogenicity and marked fibrogenicity. The enomte-induced mesotheliomas were similar to those induced by asbestos in exhibiting long latency, in gross appearance, in histology, and in close associ ation with fibrosis. Long-term persistence of asbestos or fibrous erionite around progenitor ceils of the induced tumors and the consequent fibrosis seemed to be an important precon dition of the malignant transformation of the progenitor cells. INTRODUCTION A single intraperitoneal administration of asbestos and other inorganic fibers is known as an effective experimental method for the production of malignant peritoneal mesothelioma in laboratory animals, including the mouse and rat. This oimai model was used to ^investigate pathogenetic problems of malignant periooeal mesothelioma, such as the development and course of the tumor after exposure to asbestos and zeolite, the incidence and latency of the tumor, favorite sites of the primary focus in the peritoneum, cell types of the tumor, and simi'arities and differences between the induced tumor and human malignant meso* 277 0O13-9351/W S3.00 Cfl*rC 1*4 fry Academe Pm*, (m. Ail njftts o{ripfodicw u y men**. 278 SUZUKI AND KOHYAMA theltoma. Answers to these pathogenetic prohiems were possible bycpm of detailed anatomical and histopathoiogicai observation of the mouje neums which were investigated between 0 and 23 months after exposure to bestos or zeolite. ' MATERIALS AND METHOOS A. Asbestos and zeolite. As minerals, the following were used: Amo:__. (U.I.C.C., milled 120 sec) and two types of chrysotile [(i) U.I.C.C., chrysotifet- (milled 20 sec) and (ii) Calidria of Union Carbide Co.) were selected as asbesfei. samples. Two types offibrous erionite, (natural fibrous erionite from the Resour$t International Company, Denver, Colo, and natural fibrous, less-contaminated ionite taken from Needle Peak, Pershing County, Nev.), mordenite (a mixture fibrous and granular types from the Resource International Co.), and synthetic' zeolite 4A (from Union Carbide Co.) were selected. In this paper, we call the former erionite Erionite 1 and the latter erionite Erionite 11. Size distribution and chemical analysis of all but one sample (Calidria ebry? sotile) were carried out by one of the authors (N.K.) by transmission electro* microscopy and analytical electron microscopy. Size distribution of these minet? samples can be summarized as follows: (l) Amosite. Length; 93% were shorter* than 7 urn and 6% were longer than 7.5 pm. Diameter 99.9% were smaller tfc 1 pm in diameter. (2) U.LC.C. chrysotile B. Length: 94% were shorter than pm. Diameter 98% were less than 0.1 pm. (3) Erionite L Length: 90% wenA shorter than 8 pm and 6% were longer than 9.5 pm. Diameter: 82% were test than 1 pm and 8.7% were greater than 1.4 pm in diameter. (4) Erionite II. Length: 95% were shorter than 8 pm and 4% were longer than 9.5 pm. Diameter. 82%- were less thaa 0.5 pm and 100% were less than 1 pm. (5) Mordenite. Length: ,. 94% were less than 3 pm and 4% were longer than 38 pm. Diameter. 89% were less than 1 pm and 6.25% were longer than 1.4 pm. (6) Synthetic zeolite 4A. 2.4 pm in average length and 2.24 pm average diameter. Details of size distribution in each of the samples are shown in Charts l to 6.' ' We did not characterize Calidria chrysotile. However, size distribution of this- chrysotile has been reported by Langer et al. (1) as 92% of the fibers shorter that* 3 pm and 4.6% longer than 5 pm in length, with almost ail of the fibers freely ^ separated from each other (250 to 400 A in diameter). Chemical analysis of singlej fibers of the six samples was done by analytical electron microscopy. Results of the chemical analysis are shown in Table 1. B. Doses of the mineral samples. Various doses of these mineral samples wete used: 0.J mg (Erionite II). 2 mg (amosite, U.I.C.C. chrysotile. Calidria chrysotile.. and Erionite II), 10 mg (amosite. Erionite I. Erionite II. mordenite, and synthetic zeolite 4A), and 20 mg (amosite and U.I.C.C. chrysotile). These samples were suspended in l ml saline solution to be given to animals. Type and dose of samples which were used in each subgroup of animals are described in Table 2. C. Animals. Seven hundred and sixty-eight male BALB/C mice were used m this study, and consisted of 291 asbestos treated. 295 zeolite treated, and 182 1 The shaded ban m Charts 1. 3, a. and 5 are composite! representing fibers of a diameter or lenttk treater than the number which appears above the bar in each case. - ASBESTOS- AND ZEOLITE-INDUCED MESOTKEUOHA )'** ft UtO* vr iu*OA*o Mvuno* & r- & ii #' fe. ( I kQ i'-t-r. 280 L O0Of* AV JO* 411 17* Ml 00 iM `1TAM0A40 OCVtATIOM SUZUKI AND KOHYAMA *4 1V" > * J '- Chaxt 3. Erionitt l. <V 312. controls. Age of these animals was 5 to 6 weeks old when exposed to asbestos or zeolite. As shown in Tables 2 and 3, these mice were divided into 16 groups including two controlled ones. Except animals of both Groups 7 and 8. treated mice received a single intraperitoneal injection of asbestos or zeolite. Mice of Group 7 received a double intraperitoneal injection (a single injection of 2 mg, U.I.C.C. chrysotile and a single injection of 2 mg amosite). Mice of Group * j received a single intraabdominal wall injection of 10 mg amosite: instead of theT mesothelium. the submesotheiial connective tissue of the abdominal wall was selected as the site of exposure to amosite. Inspection of these animals was done twice a day. To avoid postmortem changes, very sick animals were sacrificed. ' D. Pathological analyses of the animats. All animals sacrificed or found dead ' between 0 and 23 months after the injection were systematically necropsied. , Gross anatomical and histological observation were done in all of the animals including the controls. In addition to hematoxylin eosin. Masson's tnchrome. Prussian blue, and Van Gieson were used for staining. Histochemistry (colloidal iron with and without hyaluronidase and periodic acid Schiff with and without diastase) and electron microscopy were also used to investigate the induced peri toneal tumors. RESULTS As shown in Table 2. peritoneal tumors were not induced by asbestos or zeolite before 7 months after exposure to these minerals. Before 7 months, a large * i ASBESTOS- AND ZEOUTE-INDUCED MESOTHELIOMA UD *** a9u 10* M `SUHOAMOOfVUnOM 281 J4 tQ 5I Cha*t 4. Erionit* l|. N m 410. r ItK 5T 282 SUZUKI AND KOHYAMA UN o. oa* >u i a ) ir av. 1in*4 rotf*i CHAAT6. Zeolite 4A. N 1J. number of treated animals (192 of 586) were found dead or very sick. Pentoaea fibrosis, followed by intestinal obstruction, were the most common disease re sponsible for death of these treated animals. As shown in Table 2. sigruncan incidence of death or serious sickness was seen in animals exposed to high dose of amosite (43 of 58 in Group l and 11 of 28 in Group 2), U.I.C.C. chrysotiie (2 of 55 in Group 4) and Erionite 11 (67 of 75 in Group 10). Since mineral fibers vser histologically identified in the fibrotic peritoneum, peritoneal fibrosis seemed ti TABLE 1 Chemktal Analysis sr Analytical Election Microscopy or Sows Asiistos and Zeolite* SO, A1.0, F.0, F0 MoO M(0 co N*.0 K.0 H.O Total utcc chryscik i.V :) A2.5lrO.il i.h=o:i 0 l 4 irO.ll -- _J9 91 =0 J) _ (151 55 1 UtCC imotit* \N - $) EnooUft iN 51 a.5(=0 6) t t (=o:i 0 >7 J ( =0.1) 2.1 (=0.21 7 91=07) __ (51 *69 61.9 ( =2.1) 1461=0.5) 0 0.7 (=0.2) -- _0 7 1=0.') 5 1 (=0.9) 1.1 ( =0 6) ox 52.1 Enomtt II l.V 5) 61.0 (=0.71 14.61=0.)) 0 0.4 (=0.1) 1 4 ( =0.4) i ro.:i -- 2.4 ( =0 4) no 82.0 Mocdcmi* i.V u 70.l( = l h 12.1 < =0 6i 0 0 & I =0.41 -- 0 ) ( =0 }) 2.0 ( =0.6) -- 0 2 ( =0.1) (Ul 56.0 Zeoiite 4A t\ * n u : i = 0 a) l| ;JII 0 -- -- -- -- ;* 1 . :l -- n 4 ViJtm rtprttcnt prcentiii. * asbestos- and zeolite-induced mesothelioma (* VI P4 Ifl V M Sa?2S35!^5ooco 283 ~"3-------- 55 -3------- > ? 5 :3r333~w.2-l||33rj^tj aa *8* 2sSSS32R3a.J:-S:a.a a-.8* ,r a ,; 84 - 3 35 II w 3 % 3 U-sU-j-sijj C4 u- S* 231*33 *3*2 if 3 -- o IIiiiisiSSISIaaa * S -- s t t 2 assassasassassaa.gg ja5|a SSs-5 iJlT J.JL1 i II * 0*J.SS5 J3 Mlllliil 31111I J`2 3 w fo|4-">w*r|d p***`w* '<* s u m w o *j5 q K X W *itn fq ty j j p> utx*f |i|tt|t'tf:| 1 p** '***; rmrtidffl f| 'orjoj <m>Kp ) | > Vk * W fV O - C Vi ir.zrr-ntr* F shfiiiKiMtt! sK"???Is;!**4k E ?*i*i*sei* Ifc *>N 2 I --occo2w-or w e C v. -- wKer*e*v>4A 3gs;5 i:ww > 00*>MN^V00wt9# Is I* i? -- eoeotBe.V4 s & -- ceoteBBe>BB OB6eBOOOeOBB M*e R (8 >r InL w^ vwvahos qnv ixnzns *8Z -\ J ASBESTOS- AND ZEOLITE-INDUCED MESOTHELIOMA 285 be caused by fibrogenic effects of the asbestos and zeolite. Fibrogenic effect of ' synthetic zeolite 4A (Group 14) seemed to be mildest since peritoneal fibrosis zs minimal in animals of thie group. Mordenite-treated mice (Group 14) also <uffered from peritoneal fibrosis followed by intestinal obstruction. However, jeverity of fibrosis seemed to be milder than that of asbestos and fibrous erionite. Between 7 and 23 months after exposure, malignant peritoneal tumors devel oped in animals of all but 6 groups, the U.I.C.C. chrysotile 2 mg (Group 5). the iniraabdominai wail amosite 10 mg (Group 8), the mordenite 10 mg (Group 13). the synthetic zeolite 4A (Group 14). the saline control (Group 15). and the un orated control (Group 16). Malignant peritoneal tumors developed in 93 of 385 treated mice 124.29c) which were sacrificed or found dead between 7 and 23 months. Incidence of peritoneal tumor production varied among the treated jroups. As indicated in Table 2. the order of incidence was as follows: (l) 60% m the double treated (2 mg U.I.C.C. chrysotile and 2 mg amosite; Group 7), (2) 54.3% in the erionite II 2 mg (Group U), (3) 50% in the erionite I 10 mg (Group 9). (4) 40.5% in the amosite 2 mg (Group 3), (5) 37.5% in the erionite II 10 mg (Group 10). (6) 33.3% in the erionite II 0.5 mg (Group 12), (7) 26.7% in the amosite S mg'(Group I). (8) 25% in Calidria chrysotile (Group 6), (9) 23.5% in the amosite 19 mg (Group 2). and (10) 18.2% in the U.I.C.C. chrysotile 20 mg (Group 4). ' it was noteworthy that unlike asbestos or fibrous erionite (Erionite C and II), both mordenite and synthetic zeolite 4A did not induce peritoneal tumor sug gesting that those subtypes of zeolite lacked carcinogenic effect for the peritoneal (issue. Gross anatomically, hemorrhagic ascites were almost constantly observed in the induced mesothelioma cases. As shown in Fig. 1, neoplastic nodules of var ious sizes seen in the peritoneum were usually multiple at the site of fibrous or xopiastic adhesion. Since these macroscopic .findings were also observed in the peritoneal tumors other than mesothelioma, diagnosis of the induced tumor diould be confirmed by histopathoiogical investigation. There were certain "favorite" sites of tumor production in the peritoneum; the abdominal wail peritoneum corresponding to the site of the injection, the dia;hngm. the capsule of liver and spleen, the connective tissue of pancreatic cap sule. the omentum, the mesentery, serosa of the stomach and intestine, the fatty tapsuie of the kidney, the tunica vaginalis of the testis, and serosa of the seminal tcsicle. _ As shown in Table 3. histopathologicaily these 93 peritoneal tumors were clas hed as S3 malignant mesothelioma, 6 plasmacytoma. I histiocytoma, I liposar.'oma* 1 extraskeietal osteosarcoma, and t adenocarcinoma of the pancreas. The xi type of these 83 malignant mesotheliomas was divided into 73 fibrous, 9 Hphasic, and l epithelial forms. Coexistence of fibrous malignant mesotheli"ea with plasmacytoma was*recognized in two cases in the Erionite l 10 mg Group 9). Figures 2, 3, and 4 represent fibrous, biphasic and epithelial forms seen in the aduced mesotheliomas, respectively. Unlike human epithelial mesothelioma, ep ochal cells seen in this study did not show tubuiopapiilary or cord-like fashions. I I 286 SUZUKI AND KOHYAMA ASBESTOS- AND ZEOUTE-INDUCED MESOTHELIOMA 287 addition to (he 93 peritoneal tumors, 3 extraperitoneal tumors, which were --jo assumed to be induced by asbestos (by amosite), were observed. These three tumors consisted of one fibrosarcoma in the subcutaneous connnective tissue of a mouse (Group (: amostte 20 me) and two rhabdomyosarcomas in two mice of tbc intraabdominal wall 10 mg (Group 8). Since these three tumors contained coated and uncoated amosite fibers and the peritoneum of those three animals cas completely free of fibrosis, it was strongly suggested that the injected amosite fibers were accidentally administrated into either the subcutaneous connective dssue or the striated muscie of the abdominal wall resulting in malignant trans formation of local cells such as a fibroblast and a striated muscie ceil, (n Fig. 5, ja asbestos body (arrow A) and partially coated asbestos fibers (arrows) are &>wn in a part of the subcutaneous fibrosarcoma tissue, and in Fig. 6, an as bestos body (arrow A) is seen in rhabdomyosarcoma tissue in which bizarre eopiastic ceils with large vesicular nuclei are present. ii ifistochemically, a large majority of the induced mesotheliomas did not show lrigh productivity of hyaluronic acid. It is known that the hyaluronic acid pro ductivity is usually poor in human fibrous mesothelioma; accordingly, mouse fibrous mesothelioma seen as a major cell type in this study may also be not rally active in producing the substance. __ectron microscopically, fibrous cells of the induced mesotheliomas were sira- to those of human mesothelioma; as shown in Fig. 7, the cell to cell attach- 'wot of two adjacent cells and well-developed rough-surfaced endoplasmic retie* were frequently seen in this cell type. Rarely, numerous microvilli were jfcowit in epithelial cells as seen in Fig. 8. although well-differentiated epithelial fit* were not seen in the induced mesothelioma. To differentiate plasmacytomas ion mesothelioma, electron microscopy seemed to be a useful method; ultra structure of the former was similar to that of plasma ceils with frequent presence of C particles. .. FU. t. A macroscopic photograph showing an abdominal cavity in which perttonesl tumors were aSKcd. taken from an animat of the amostte 20 mg group (Group t). Neoplastic nodules were formed a various sites of the peritoneum. Large tumors were seen on the surface of both the liver (arrow l > wd tbe omentum (arrow 2). Arrow 3 indicates marked expansion of the intestine, caused by severe 2rooc and neoplasuc adhesions of the peritoneum. Fta. 2. Showing a relatively early stage of peritoneal mesothelioma (fibrous form). Spindle-shaped --ceils are seen m the omentum which adhered to the fibrotic pancreatic capsule. Taken from t aous&pf the Caltdna chrysolite 2 mg group (Group 6). Hematoxylin tosin. x 305. Fn. 3. Showing the biphastc form of malignant mesothelioma. Both poorly differentiated epithelial ailtsbowa in the upper part of this picture) and fibrous cells (in the tower part) coexisted in a tumor -see. taken from a mouse of the amosite 2-mg group (Group 3). Hematoxylin eosin. x 490. Fes. 4. Epithelial mesothelioma ceils are shown. The cells are poor in polarization. These cells are hrawf a aest of sheet-ltke fashion. Tkkea from a mouse of tbe Erionue t 10-mg group (Group 9). Soatnxylin eosto. * 305. fta. 5. A part of an cxiraperitooeiAlumortBbfosarcoma) induced in a mouse of the amosite 20-mg mg (Group t). .An asbestos body (arrow A) and aggregated asbestos fibers (arrows) are seen in tbe amor tissue. Colloidal iron stain. x490. no. 4. Showing a pert of a rhabdomyosarcoma usiuc seen in the abdominal wall of a mouse of the ^s-tbdonunai wail amosite 10-mg group (Group 9). A short asbestos body (arrow A) is teen. Giant etanie cells with vesicular nuclei are shown. Hematoxylin eosin. x 490. 288 SUZUKI AND KOHYAMA FW. 7. Ultnstructurt of fibrous mesothelioma cells seen in a mouse of the imotite :o mg (Croup I). Well-developed endoplastic reticulum, direct contact of a cell with another cell, elocpted nucleoli, and lipid granules are seen, x 4160. FtO. I. Epithelial mesothelial cells, equipped with numerous microvilli art seen. TaVen from ia animal of the Enomte 1 10 mg group (Group 9). *9300. . ASBESTOS* AND ZEOUTE-INDVCED MESOTHELIOMA - COMMENTS . Carcinogenic and fibrogenic effects ofasbestos and zeolite and the -V'*'*-" v tad course of malignant mesothelioma induced by these miner* ~'v' ' jUidied in the mouse peritoneum. A single peritoneal adrmmstra.v-* ''' fibers at various doses was used, followed by long-term observatu'* *v ' toneum. This animal mode] was relatively simple to use and it pr.v.v'A j reproducible results in the induction of tumors and tibrosis. | A high incidence of peritoneal tumors {mainly malignant mesrevv'-' ' I observed ut animals injected with either asbestos or fibrous enorrv v * "d ^ 3fter exPsuret 39 of 192 (20.3%) m the asbestos pro-- >* ' 112 (4e.,f) m the fibrous erionite group. It is noteworthy that the Iwv v''' the induced peritoneal tumors did not seem to Nt related to eifiv* :v ' fibers or the dose; regardless of the type or doses no tumor was wre* ' * oaths after exposure. This is different from that of the mouse V'-.V'.'-''' ' aoor, which can be induced at a high rate by chemical carcinogens. v *'v artier either when a stronger chemical carcinogen was used andor *''v ^ jate of a chemical carcinogen was larger (2. 3). The asbesios-iodived famas were similar to those induced by fibrous enretite in exhibiting *** ' htgross appearance, in histology, and in close association with fbr**** N> Mceamesotheliomas were also similar to human malignant mesccSefrre** ^ exhibiting long latency and in gross appearance. A disrinct dtffcre<ve At animal and human mesothelioma, however, is the cell type re' :Nr **1$**' ssesotheiioma. The ratio of epithelial, biphasic, and fibrous terms **> '' <s` * induced mouse mesothelioma, while it was 67:2&7 in human m.*Vv tsotheiioma (4). It is not known why, unlike human mesothelioma. ;hec^v^* TM ts to rare and the fibrous form so common in the induced rarece Soma, although the severe focal fibrosis which constantly occurred awv ajesothelium might be related to the high incidence of fibrous mahgrwn. *N'' : beijotna. Both asbestos and fibrous erionite were able to induce tumors reber tv*` " ^nut mesothelioma. In the present study, plasmacytoma, liposar.vrv* orcoma. histiocytoma, pancreatic cancer, subcutaneous fibrosarccwM. s ''- s jocyosarcoma ail were induced peritoneally or extrapenioneaih bx tlx-*.' ''0' rae tumors have not been reported to occur as spontaneous iwpis'*'"' m'ce- C-particies were frequently seen in the neceisstv 'vS ' ae induced plasmacytomas. It is possible that both C-panicles and ts- " ^ eets participated in the induction of the plasmacytomas as eocarcuvgo*' that human osteogenic sarcomas rarely developed extrasUHetidb tfeori matru of the meupltstic connective tissue. A single case of fvrSO'X'* 'wrosireoraa induced by amosite (Group 3) may be classified as this vxv ' ^ar. since osteoid formatioti was not rue in ftbtvtic lesions induced by erionite. Another single case of pancreatic cancer (Group 11' '**' * jested to be erionite*related since focal scarring associated *h enre*^'' was seen in the pancreatic parenchyma. The subcapsular pancre.*' ' < 290 SUZUKI AND KOHYAMA renchym* was frequently involved in a fibroplastic process induced by. nOg and fibrous erionite which were intraperitoneally administered. . * Our study strongly suggested that asbestos and fibrous erionite had the p3 tiality for inducing neoplasms in various tissues other than the mesotheihnifl lung, if these fibers could reach the various tissues and remain there for |d periods. 3 Epidemiological studies have shown that, in addition to lung cancer and maS nant mesothelioma, various cancers such as cancer of the larynx, renal cell cj cinoma. and gastrointestinal cancer are present with increased frequency asbestos insulation workers (5,6). To confirm whether individual human cues* these various cancers are caused by asbestos or not, the detection of both bestos fibers and tissue response (such as focal fibrosis) should be attempted! the primary neoplastic focus of the individual cases. There is good reason k supporting this approach; in our study using the mouse as an experimental modd both of these conditions are met in ail of asbestos* or erionite-induced turnon: The mineral fibers which were intraperitoneally injected were located in d| animal peritoneum in certain "favorite" sites. Subsequently, peritoneal fibred followed by the induction of malignant mesothelioma occurred in these "favorite sites. The diaphragmatic peritoneum, one of the favorite sites, is known to bvi microvilli-rich mesothelium and well-developed stomata through which fbreq substances can be drained out from the peritoneal cavity into the lymphaii system of the diaphragm (7). Wang (8) has described a similar anatomical cha acteristic in the parietal pleura, known to be the original site of pleural mesoth lioma. What is not known, however, is whether all of these "favorite" sites a equipped with such a unique physico-anatomical characteristic. This may be key question as to why tumors develop in certain "favorite" sites of the pent neum. Baris *t al. (9, 10) have suggested that fibrous erionite (a type of zeolite) is tl etiological factor in the development of malignant pleural mesotheliomas whk occur endemically among inhabitants of se- \1 villages in south-central Turks Later, Rohl *t al. (11) reported that in adaiuon to the erionite, asbestos Gbe (chrysolite and tremolite) were found in both the environment and in lung tissu of these mesothelioma patients. A question is raised therefore, whether the: mesotheliomas were induced by fibrous erionite or by asbestos or by both. 0 present and preliminary (12) animal studies clearly indicate that, like asbestc fibrous erionite is also clearly carcinogenic, inducing malignant mesothelioma a high rate in animals. Accordingly, even if asbestos fibers coexisted with i erionite in the lungs of the TUrkish patients with mesothelioma, enorute can 5: not be excluded as a sole or cocarcinogen responsible for the induction of hum mesothelioma. Fibrosis was strongly suspected to be an important precondition for the indt tion of the neoplasms by asbestos and fibrous erionite; all neoplasms induced these minerals 193 peritoneal tumors, 2 rhabdomyosarcomas of the abdomit wall, and l fibrosarcoma of the subcutaneous connective tissue) were miimait associated with fibrosis. This intimate relationship of the fibrogeniciiv with t carcinogenicity was best shown in the zeolite samples: both Erionite l and showed sinking carcinogenic and fibrogenic effects while mordenite and s> nthe ASBESTOS- AND ZEOUTE-INDl/CED MESOTHELIOMA 291 zeolite 4A did not induce-any turnon, and this was accompanied by mild and ounimai fibrogenicity. Another interesting finding was that animals receiving large doses of asbestos or fibroug erionite died of peritoneal fibrosis followed by in testinal obstruction before the tumor was induced. To perform long-term obser vations of the development and course of experimental peritoneal mesothelioma, ijoses of the carcinogenic mineral fiben must be carefully chosen. Stanton and Wrench (13) have hypothesized that the carcinogenicity of as bestos. fibrous glass, and bracite was due to the durability of those fibers within a certain range of size distribution (8 wm and longer in length and 1.5 jim and smaller in width) and that the chemical nature of the fibers is not relevant for the induction of mesothelioma (13, 14). Similar results have been reported by fott et (15. 16). Stanton's hypothesis, however, has been disputed by other investiplors (17). In the present study, for example, a large majority of asbestos and gbsous erionite fibers were short and thin, as indicated in charts 1 and 6. However g a still not known whether the short, thin fibers were responsible for the high ate of mesothelioma production or whether the long thin fibers present as a naority in the mineral sample played a role. To elucidate this, the range of size Attribution should be studied in the fibers detected in the primary neoplastic fgcus since this range may be different from the original sample. " Mitogenetic mechanisms suggested by studies on experimental mesothelioma aay not be directly applicable to human mesothelioma since there are some differences in pathogenetic conditions of the human tumor as compared with the tumor. For instance, the life span of laboratory animals (mouse, rat, and famster) is approximately 2 years, much shorter than that of humans. The long i fine latent period (20-40 years) seen in the development of human malignant esotheiioma (5.6) cannot be reproduced in these animals. Therefore, to induce mai mesothelioma, larger doses of the mineral fibers must be used and on any occasions, investigators must apply the artificial method of fiber exposure oxh as inoculation or injection (12-16, 18-21). In addition, anatomical differ- races, such as the widely differing range of the length and diameter of the respi ratory airway in human and animals, may also result in different data concerning .ht critical size range of the fibers responsible for the induction of the tumor. la spite of these differences, however, animal experiments have provided valu able information on the carcinogenicity of asbestos and other inorganic mineral rbccs as well as on histogenetic problems of malignant mesothelioma. . ACKNOWLEDGMENTS TW tntbori express thetr appreciation to Dr. 1.1. Selikoff for his review and Dr. 3. Frank for her ottrat assistance. The authors also thank Mr. R. Ashley. Mi. A. Calderaro. Mr. 3. Yuea. and Mr. I t- Pefler for their technical assistance, and to Mrs. I. Roberts for secretarial assistance. This work *ee appocted by Research CnatCA 29433 and CA 24311 bom the National Cancer Institute. REFERENCES I tenter, A. M-. Wolff. M. 3., Rofai. A. N., and Selikoff, I. J. (1971). Variatioa of properties of cfarysotilc asbestos subjected to milling. J. Toxicoi. Environ. Htalth 4, 173-18*. - Jfcweit, H. L.. Dunn. T. 8., SueR, K. C., and Dennjar, M. K. (1979). Timor of the respiratory tract. U "Patholo*y of Tutors in Laboratory Animals. Voi. II. Tutors of the Mouse." (U.S. . Tarasov tt at.. Eds.), pp. 131-247. IARC Scientific Pub. No. 23. 292 SUZUKI AND EOBYAMA 3 3. Suzuki, Y. (19*3). Neoplastic effects of vinyi chloride at mouse lun*--low dom am<3 t*rot exposure. Environ. Rts. 32. 91-103. ^9 * Suzuki, Y. (19*1). Petbolocy of human mili|i-- mesothelioma. Stmin. Oncol, t, 268-2t]3 J. 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