Document ym3a6r670Mw11vLxXdZR4V1pn

HOW TO use THESE SEPARATORS Use one page fo r each separation * S elect appropriate ta b , add fu r th e r Id en tificatio n i f d e s ir e d , and co ve r It with scotch tape Cut o f f and d is c a r d a ll tabs except the one covered by tape* TABBED SEPARATOR SHEET For* HEM-69B <*-> */7 \M H O \XltNrvL HESEVHC'H 4. 49'V-011 l 1971) Experimental Asbestos Carcinogenesis1 Andrew L. Reeves, Henry E. Peno, Ralph G. Smith,- and Arthur J. Voiu alu1 Department of Occupational and Environmental Health, School of Medicine, W ayne State Unicertitij, Detroit, Michigan 48207 Received July 21, 1971 Laboratory rodent e r e exposed to one of three mineralogica! types of asbestos dust, am osite, crocidolite. or chrvsotile. Inhalation exposures vvtre accom plished in chambers w here ba'ilniillcd specim ens of these dusts were dissem inated for 4 hours daily, -1 days w eek]}, at a mean atm ospheric concentration of about IX m g 'm '. Vdditional animals were in a c ttd w.th these dusts intratracheal!}', intrapleurally. or mtrapeiitoneally. H istopathologic studies show ed a hbrotic reaction m ail species to all three types of ilu.ts, with am osite provoking the strongest such response espetiall} in guinea pigs. !w o pulmonarv cancers w ere produced in rats e \p >sed to the inhalation of crocidolite. Local m ic tio n into tin1 p ie o i.4 or pentoneal cavities ca.used 5 niesnthelionias in rats after chrysotile treatment and 6 mesotheliomas in rats and rabbits after crocidolite tieatimmt. G uinea pics and hamsters developed no tumors in this experiment, and with the do-e used, there were no tumors m am species in the amosite group. Ex or since Lynch and Smith in 1935 suggested the association between asbestos exposure and lung cancer in man. this possible factor in environmental carcino genesis lias continued to attract attention. During the next 25 years, over 100 cases - f Ai- , .i- il,Kll pi M idi * A*li A J U U J iU l V U U ` i G p O l l C A i itllU evidence accumulated to show that at least certain t>pes of asbestos, in addition to their fibrogenie property, apparently also possess carcinogenic potential (e.g., Doll, 1955). In I960 an additional aspect developed when Wagner and cowoikers reported 32 cases of pleura! mesothelioma, otherwise a rare tumor, among persons exposed to cTocidolite in South Africa. Subsequently, numerous other eases were dis covered in all parts of the world and connected in some cases to chrysotile ex posure as well ( e.g., Selikoff et a/.. 1965). A third type of neoplasia which showed association with asbestos exposure was peritoneal tumors, of which 11 were de scribed by Keal 1 1960 i and 9 by Enticknap and Smither (1964). It was specu lated that transfer ot the carcinogen to the peritoneum was by hematogenic spread, and the specific vulnerability of this organ resulted from a stimulus clue to movements between the peritoneal surfaces. Recently, possible association be tween asbestos exposure and cancer of the gastrointestinal tract ( Ilammond et ai. 1965' or of the hematopoietic system (Gerber. 1970i were also reported. Experimental work on the asbestos lesion was commenced by Gloyne as e;u!> as 1930 and continued on a larger scale by Vorwaid et a!. (1951). The early work showed the principal action of asbestos fibers to be the elicitation of a foreign- 1Supported by Grant No. 5-R O -l-E C 00210 from the U. S. Public Health Service. ` Present address. School of Public H ealth. University o f M ichigan, Ann Arbor, MI 48101. : Present address: 1741 Decker Avenue, Green Bay, W I 55302. 1972 by Academic Press, Inc. 496 K.v body reaction with gian bodies." leading to extern vervet monkeys, and gu in the stated order with to amosite. Holt and as various kinds of asbestos tion. and it was suggestc of asbestos. The situatioi 1967, 1970), who also pulmonary response to macrophages to giant cel Gross and DeTrcville ( and found the lesion nunp Tin's difference was atiril as well as to differential i ('1 the asbestos fiber was n glass under similar expo Tre' iHe, 1969). On the ot and occurred after differe their name (when the uati referring to their iron eon Attention was also dim length. There was much ev much of its harmful pub n lia'1-.pui tu'niiuy ( iltiseher . (1933) indicate that eon.sij inai result from grinding threshold ot effective met (1951). 10 n by Wagner ; et t1. (1966) considered fi for pathogenicity; he lot fibrogenie. the somewhat sin face area. In 1967. .MacXab and'I chnsotile dust, which wa: spondee! less well to tin' in mini, both of which prot (crocidolite, amosite, and a (Secchi and Rezzonico. 196 the hemolytic action of chr ether sodium salt. It was ol the surface of chrysotile fib activity, suggesting that t h l membrane lipoproteins, or i l were the key properties i l l ; Schnitzer and Bunescu, I 9 7 (| { j 5 i 1 $ A..i*L<d Aw--- inesis1 n'd A r t h u r J. V o r w a l d 1 School of Medicine, mo7 i| l\p ex of asbestos dust, rom plislicd in chambers or -f hours daily, 4 days /u d . Additional animals v, or nitraperit.'ineally. cs to all three types of pecialK m guinea pigs nil,datum of crocidolite, o niesothclionias in rats rabbits after crocidolite in this esp eu m en t, and le amosite group. -iatiwii between asbestos environmental carcino25 years, over 100 cases oma were reported and of asbestos, in addition mogenic potential (e.g., and coworkers repotted among persons exposed . other eases were dis3 eases to chrysotile exlcoplasia which showed , of which 11 were der (1964). Jt was specum was by hematogenic ,ed from a stimulus due possible association bctiaet ( Hammond et a l. e also reported, ced by Gloyne as ear!' (1951). The early work, el citation of a forcign- ic Health Service. 1 Ann Arbor, MI 48101. j EXPERIM ENTAL ASBESTOS CARCINOGENESIS 497 body reaction with giant cells, granulation tissue, and appearance of "asbestos bodies," leading to extensive collagenous fibrosis. Wagner (1963) exposed rabbits, vervet monkeys, and guinea pigs and found these species increasingly vulnerable in the stated order with chrysotile; all three species responded equally severely to amosite. Holt and associates (1964-1966) exposed rats and guinea pigs to various kinds of asbestos. Phagocytosis of the dust led to "asbestos body" forma tion, and it was suggested that phagocytosis is a prerequisite to the fibrogenieity of asbestos. H ie situation was examined election microscopically by Davis (1963, i 1967, 1970), who also concluded that phagocytosis was a key event in the pulmonary response to asbestos, and postulated the coalescence of dust-laden macrophages to giant cells or their conversion to fibroblasts. Gross and DeTreville (1967) produced experimental asbestosis with chrysotile, and found the lesion nonprogressive in rats but progressive and lethal in hamsters. This difference was attributed to the relative efficiencies of pulmonary clearance as well as to differential reactivity of pulmonary tissue, ft appears that geometry of the asbestos fiber was not a paramount factor in pathogenicity, since filamentous glass under similar exposure conditions was essentially inert (Gross and De Treville, 1969). On the other hand, "asbestos bodies" appeared to be nonspecific ] and occurred after different kinds of fibrous dust inhalation so that the change of j their name (when the nature of the fiber was not known) to "ferrug nous bodies," * reierrinir to their iron rontent was suggested (Gross et ah, 1967, 1963). Attention was also diu cied at the correlation between fibrogenieity' and fiber length. There was much evidence to suggest that very finely ground asbestos loses much of its harmful potential, presumably because of its improved lymphogenic transportability (llilscher et al., 1970), but the studies of Oecella and Maddalon 1 (1933) indicate that considerable loss of crystallinity (up to S57 for chrysotile) ) may result from grinding, especially if accomplished by cutting action. The ] threshold of effective median filler length was regarded as 20 x by Vorwakl (1951), lO/i by Wagner (1963), and 2 i by Holt et al. (1964). However, Kogan et al. (J966) considered fiber length or indeed fibrous structure as nonessential for pathogenicity; he found chrysotile and associated serpentine rock both fibrogenie, the somewhat greater effect of chrysotile attributable to its larger surface area. In 1967, MacNab and Harington called attention to the hemolytic activity of chrysotile dust, which was comparable to that of quartz dust although it re sponded less well to the inhibitory action of polyvinylpvridine-,V-ox.ide or alumi num, both of which protected cells against lysis by silica. The amphiboles (crocidolite, amosite, and antophyllite) were much less hemolytic than chrysotile (Sceehi and Rez.zonico, 1968) and Schnitzcr and Pundsack (1970) observed that the hemolytic action of chrysotile could be inhibited by carboxymethyl-cellulo.se ether sodium salt. It was observed that substances which were well adsorbed on the surface of chrysotile fibers were the most potent antagonists of the hemolytic . activity, suggesting that the adsorptive capacity of the fiber possibly involving : membrane lipoproteins, or its chelation potential involving magnesium hydroxide, were the key properties in this phenomenon (MacNab and Harington, 1967; Schnitzcr and Bunoscu, 1970). 49S REEVES ET AL. The nr>! experimental production of lung tumors with asbestos apparently goes back to Nordmann and Sorge in 1941 and similar experiments were conducted later bv Lynch et al. (1957). These experiments were done on mice which in haled chrysotile dust, and the positive results consisted of accentuation of the natural tendency of these animals to develop pulmonary adenomas. More defini tive results were obtained with implantation of dusts into the pleural cavity of rats ( Wagner. 1962; Wagner and Berry, 1969; Berry and Wagner, 1969; Donna, 1970). hamsters (Smith et al., 1965), or into the air sacs of fowls (Peacock and Peacock. 193-5 ). and in 1967 Gross and DeTrcville obtained pulmonary cancers in rats exposed to the inhalation of chrysotile. In this paper, we report two cases of pulnu nary carcinoma in rats after inhalation of crocidolite and several, pleural and peritoneal mesotheliomas in rats or rabbits after implantation of either crocidolite or chrysotile. MATERIALS AND M ETHODS D ust S pecim ens Commercial samples1of the three most important mineralogical varieties of as bestos, i.e.. amosite. crocidolite. and chrysotile, were ballmilled in glazed ceramic jars with stainless steel balls for about 10 days, and the resultant samples were forced through a 1 inch mesh screen for the disintegration of larger clumps. The dusts so obtained were examined by light and electron microscopy, x-iay diffrac tion. and atomic absorption spectrophotometry.'' Electron microscopic particle sizing gave the following range of values based un numerous counts for tile mean length and mean diameter of the several fibers, respectively; Amosite. 3-5 and 0.2-0.5/i; crocidolite, 3-6 and 0.4-0.5/i; and ehrvxotik. 6-15 and 0.2 a. The upper limit of fiber length, as determined with the light microscope, s'as 55-130 ,u, comprising 0.001-0.0157 of the dust. X-ray dif fraction studs g.tse the values shosvn m Table 1 as compared to those reported tor the standard reference samples of the International Union Against Cancer [UICCj , 1 imbrell et al., 1963). Table 2 summarizes the results of chemical analysis, showing that substantial amounts of nickel, and in some cases also of manganese and/or chromium but not cobalt, sserc added to the dusts during experimental preparation. Analysis svas also penformed for beryllium, shosving s alues under 1 ppm in all cases. Em u l a t i o n - E x p o s u r e s In three 12 x 1- ft rooms with 2 perforated walls, 206 rats. 106 rabbits, 139 guinea pigs, and 214 hamstcis. of both sexes and 2-3-months old, were about evenly distributed. One wall seised as the supply, and the opposite wall as the exhaust. The asbestos dusts were disseminated into these chambers ss ith the aid of hammer mill and fan systems essentially according to Holt et al. (1965), and * Am osite. \V -3 fibers; Crocidoiite, S-Blue fibers; and Chrysotile, 3 -T fibers. These sample* were obtained by courtesy of the Johns-Mans die Corporation. s R C. A. electron m icroscope, model EM U 3-E; C. E. X-ray diffraction appaiatus, Model XRD-5; and Perkin-Elmer atomic absorption spectrophotometer, M odel 303. exp F eatures of Asbestos type A m osite Pc X, t Crocidolite Chrysotile 4 5 l g 3 4 5 6 7 1 2 3 4 " In CuK a-nuliution, X = 1. 6 Principal diffraction angles ` Tnterplnn.'tr D u lc e -pacing.- Cum Asbestos type Elen A m osite Co Cr M Ni Crocidolite Co Cr -Mi Ni Chrysotile Co Cr Mn Ni " As shipped. h As collected on millipore filli 1 Tim brell a al. (li)GM). A*- , T.7i ifi f4.J* c* > ith asbestos apparently goes xperiments were conducted ^re done on mice which li sted of accentuation of the larv adenomas. More defini.s into the pleural cavity of and Wagner, 1969; Donna, sacs of fowls (Peacock and ibtained pulmonary cancels paper, we report two cases icidolite and several pleural 'ter implantation of either S uneralogical varieties of asvdlmillcd in glazed ceramic the resultant samples were ation of larger clumps. The n microscopy, x-ray diffrac- \ing range of values based uneter of the several fibers. ;, 3-6 and 0.4-0.5 p; and gth, as determined with the 15'4 of the dust. X-ray difompared to those reported nal Union Against Cancer s, showing that substantial e and/or chromium but not preparation. Analysis was ppm in all cases. 206 iats, 106 rabbits, 139 3-months old, were about d the opposite wall as the ese chambers with the aid to Holt et ul. (1965), and tile, 3-T fibeis. These sample' y diffraction apparatus, Model , Model 303. EXPERIM ENTAL ASBESTOS CARCINOGENESIS 499 TABLE 1 F e a t u r e S OF X-K \Y D l F I K WTIO.N D iauham o r A s b e s t o s D o STS 2 ^ d` Asbestos type N o ." UICC Std Exp. UICC Std A m osite Crocidolite Chrysotile 1 10 7 2 27 3 3 29 1 4 32 3 5 1 10.5 2 19.7 3 26 0 4 28.7 5 32 9 6 34.4 7 35 3 i 12.0 2 19.5' 3 24 3 4 36 5 5 60 2 " In CuKrt-radi'U'on, >. - 1.51 A. b Principal diffraction angles. r Interphmar lattice ..pacings in A. 10 7 -- 29 2 32 5 34 2 10 5 -- -- 28 6 32 9 -- 35 3 12.4 -- 24 7 36 8 60 5 8.26 3 27 3 07 2.77 -- 8.43 4 51 3.43 3.11 2.72 2.61 2.54 7.38 4.55 3 66 2.46 1.54 Exp. 8.26 -- 3.06 2.75 2.62 8.42 -- -- 3 12 2.72 -- 2 54 7 19 -- 3.62 2 44 1 53 TABLE 2 C hemical A nalysis of Ashestos S amples Concentration ppm Asbestos type E lem ent liaw" P r o c e s s e d 1' A m osite Co 13 Cr -- Mn 6S8-851 Ni 40 Crocidolite Co Cr Mn Ni 11 -- -- 70-73 Chrysotile Co 36-39 Cr 287-361 Mn 173-219 Ni 222-294 " As .shipped. 1 As collected on millipore filters in chamber. r Timbrell it al. (BIOS'). 14 -- 824-990 97-108 12 -- -- 88-111 38-41 382-402 139-153 374-461 U IC C std 7-11 32-35 -- 34 2-9 16-22 864-870 13-100 45-46 316-490 443-510 795-990 i 500 REEVES ET AL. achieved fairly even distribution. To insure that all animals received equixahnt dosages, all cage racks were reoriented routinely and cages on the racks were re positioned with respect to their height from the floor. The used asbestos was ex hausted from each chamber into plastic collection bags. The chamber atmospheres were checked daily by membrane filter sampling technique throughout the experiment. The obtained values were remarkably uni form. giving the following means ( s d ) for the three rooms: amosite, 48.2 i 1.4. crocidolite, 4S.7 2.4. and ehrysotile, 47.4 1.7 mg/nri. Fiber count oy light microscope with a resolution of 0.8 x gave the following results: amorite 864. crocidolite 1105, and ehrysotile 54 million/nv. This included only particles with an axis/diameter ratio of about 3:1 or more, lu addition, there was also sub stantial nonfibrous component in the dusts. The exposures to these atmospheres were conducted for 4 hour/day, 4 days/week, with each Friday reserved as clean-up day. I n je c t io n s Intratracheal, intrapleural, and intraperitoneal injections of all three types of asbestos dusts were given to certain additional animals in order to study the tis sue reaction. For these purposes, the dusts were prepared according to Badollet and Gantt (1965) by treating the specimens in a Waring Blendor, sieving, and regrinding in an agate mortar for about 15 minutes. 4 he asbestos wars then sus pended in isotonic saline at a concentration of 20 mg/ml, autoclaved, and ad ministered in simple doses as shown in Table 3. N ec r o psies a n d A t ir it io x a l M o r ta lity Periodic killings were scheduled at 1, o. 6. 12. IS and 24 months after injection or commencement of inhalation, consuming about one-half of the total animal com plement in each group. The other half of the animals was allowed to live to an age of approximately 3 s ears. Total eumulatixe exposure hours in the chambers were 1496 (am osite1'. 15SS (crocidolite), and 1644 (ehrysotile). Attritional mortality was varied with the 4 animal species under study,1' the rats TABLF. ;5 I n j e c t i o n 1Iv - a u k - ok A s b e s t o s I ) i> t .v Intratracheal Intrapleural Intraperitoneal ml N o. anim;ils ml N o. animals ml N o. amnia: Species dose a i. c do'e a b c dose a b Rats 03 Rabbits 05 G uinea pit's 0 3 16 12 16 13 15 15 15 It 15 0.5 0s 05 15 13 11 13 13 17 12 14 11 10 -- -- 11 --- 13 --- H:iiru>tt'rs 0 2 13 10 -- 0 5 14 12 9 0 .5 --- --- " a-- Amosite, b --Crocidolite, e-- Chrvsotile. ' Charier River C D rats; Shankin Farms D utch rabbits; L ighlner-IIartley guinea pi"s; ain! Lakeview Syrian Golden hamsters. ` expel ( being apparentlx the hart . some endemic pneumonia ' numbers. Guinea pig loss ) early and the typical stir vontive medication was *. together with the total m . The attritional mortalit ' examined. It appears that injurious (one-year survis crocidolite (0.840). Sim Strongest effects were se haling amosite vs croeiclt cant at the 99.97 confiden a | i In view of extensive p under study, the liumbet post'd animals was small Ten such rats, 10 rabbit received no exposure or rat, at about 1 year of monary tissue; and one the terminal bronchiole in bit- coutiol colony. Pi 1971) showed that entle bronchiolitis in rats and Pulmonary fibrosis was not observed among sex here in the past. T he All three types of ash tiocytes as the earliest p This was followed by' animals. Squamous met the crocidolite group, xv hyperplasia (Fig. 1). B metaplasia, and out of longer, 5 displayed pr< killed at the end of cr lung.j Fig. 2). There xvt i i ` N o survivors at 1 year i ii'i uwvwrfif >Si f i # ;---wi. iwt sak'. t 1 Jinimals received equiv.ff ,> cages on the racks wen- i< The used asbestos was i \ membrane filter sampim lalucs were remarkabh un rooms: amosite, 4S 2 1.; /m;'. Fiber count In liA/ bvving results: amosite Shi licluded only particles Idition, there was also sui. bsures to these atmoxphen each Friday reserved ,w ctions of all three types <! Ils in order to stud}- the !e bared according to Badolh * laring Blendor, sieving, .uh! ITlie asbestos was then mis lg/inl, autoclaved, a n d .id PVTALITY hd 24 months after injirtmu half of the total animal com was allowed to h\e to .e sure hours in the chamb< u hrysotile). rccies under studv.' the i d' lnlr.a|M 'ri(oi!c:il m l N o. jnmu c dose - ---------- -- 2 ]0 % --- 1-- U o r> a U -- -- br Hi * . - _* EXPERIM ENTAL ASBESTOS CARCINOGENESIS 501 being apparently the hardiest and showing the best surv ival record. Rabbits had some endemic pneumonia but they also survived to their senescence in sufficient numbers. Guinea pig losses were heavier, and hamsters, especially, tended to die early and the typical survival for the latter species was 13-15 months. No pre ventive medication was given. The inventory of live animals at different times, together with the total malignancy incidence (cf. Results) is collated in Table 4. The attritional mortality of each species in the three chambers was statistically examined. It appears that in terms of overall lethality, amosite inhalation was most injurious (one-year survivorship ratio 0.765),r followed by chrvsotile (0.775) and crocidolite (0.S40). Similar trends were seen in the injected animals also. Strongest effects were seen in guinea pigs, where the comparison of animals in haling amosite vs crocidolite (X- ~ 10.02) showed a difference that was signifi cant at the 99.97 confidence level. RESULTS C o n t r o l A n im a l s In view of extensive previous departmental experience with the animal strains under study, the number of controls set aside from the same shipment as the ex posed animals was small, in order to allow maximum numbers to receive exposure. Ten such rats, 10 rabbits, 12 guinea pigs, and 8 hamsters served as controls and received no exposure or treatment. Tlic.se animals survived up to 1-3 years. One rat, <it ,ibuut 3 >ear of age, showed epithelial hyperplasia and necrosis ot pul monary tissue; and one rabbit, at 10 months, showed histiocytic proliferation at the terminal bronchiole with inflammation. There was no other pathology noted in the control colony. Previous experience with these animal strains (e.g. Reeves, 1971) showed that endemic conditions unrelated to any exposure include puiulent bionchiolitis in rats and alveolar epithelial hvperplasia in rats as well as rabbits. Pulmonary fibrosis was infrequent, and metaplasia or neoplasia in the lungs was not observed among several thousand unexposed animals of each species studied here in the past. T h e R espo n se to I n h a l a t io n E x posures All three types of asbestos caused accumulation of hemosiderin-conta'ning his tiocytes as the earliest pulmonarv response, associated with inflammatory reaction. This was followed by fibrosis in the region of the terminal bronchiole in a lew animals. Squamous metaplasia in tin's area was also often prominent especially in the crocidolite group, where in some rats it progressed to a pseudo-epitheliomatous hyperplasia (Fig. 1). Both crocidolite and chrvsotile also induced columnar-tvpe metaplasia, and out of 40 rats which survived chrvsotile inhalation for 2 years or longer, 5 displayed prominent benign pulmonary adenomatosis. Out of 31 rats killed at the end of crocidolite-exposure, 2 had squamous-cell carcinoma of the lung (Fig. 2). There was no neoplasia in the amosite group. 'h t n e r - I l a r t l o y Kiiim .i p u t1 1N o survivors at 1 y e a r/N o . all animals at start. v m s e t s-t*-.> -rt v TAHi.K 4 Mo k t u .itv \ \ i> M W.KIN \M'Y s.'im .M 'i: or A m m w .s Kmcosm, r o Amu.stos Amosite Crocidolite Chrysotile Inventory nventory Inventory I 0 12 24 Incidence 0 0 12 24 -- O -- -- -- Incidence of 0 ( 12 24 -- ------- . ----- -- Incidence of 3 Species lioule months mtilijriiancy nionths miiliffnancy mimt lis imilifti umey lints Ihlmlul ion 77 (7 Oli 42 on 01 01 lil 2 pill, cn." both nt 00 40 40 40 __ 20 mo. .1 Tnl nil rneheiil 10 10 0 1 - H! f) li I 1.7 S 0 0 .--- Ililnipleiiriil ir. (1 4 0 Hi 5 f 0 1 pint, meso/' at l(i mu. 12 0 4 1 2 jileo, nesu,'' ui, 0 lnl mperiloiieul ii :i 0 0 14 mo. Hi 1 4 0 1! perl, m e so / at 12, Hi, Hl 7 0 0 Il |ier. m eso / al 7, & 17 Inn. 11 mo. Rabbits Inhalat ion Ini tat rneheiil lilt r:i pleural :ts Vj 20 12 it) s Hi 4 2 Hi 0 J -- -- H2 21 IS 1.1 15 S s l IUI GO 20 21! 14 0 4 Hi 0 li 0 2 pioti, meso.'' at 22 & 14 r, 4 2 24 mo. __ -- -- Guinea pigs Inhalation 4.1 20 17 5 - - 50 li.l lili 4 -- 44 21! 21 0 __ lilt rat unheal 10 S 4 ;; 1.1 7 .*> 2 __ 1.1 0 ;} 0 Intrnpleuinl 11 ) > 0 -- 17 {) 1 0 -- 11 2 i 0 -- Hamsters Inhalation 711 57 50 0 -- t>7 51 4.7 0 IiUrutruehenl Hi 0 <> 0 -- 10 2 *) 0 74 40 0 0000 __ -- Intrapleural 14 s 0 0 12 > 1 0 -- 0220 -- lut rape uloneid 0 (1 0 0 0 000 So 0 0 -- * Pulm onary carcinoma (squamous cell p a tte rn ) 'PuM iial m esotheliom a (fibrosarcom a p atte rn ). r Peritoneal luc'it!hehoinn (fibrous, pupillary, osteogenic, or epithelial patterns). KT AL. Am"j EXPERIM ENTAL ASBESTOS CARCINOGENESIS 303 Cc-Ci o *f i-z -- ^ o :r. x T-- --1 iI r t 'M C CO 10 i CCO I* Cl i Cl f*ig 1. Specim en \ o . lOIiZ-oO. J-im,f of fem ale Kit. Age 1-iO w eek ', evr*G*-ed to inhalation c uf u iA io u m r lor ioSS eunm iative Roms. Jlie hronthiolar epithelium sh<rvc iiiminnm pia^M. X2J0. tc N--i 'f Cl LOO Cl oO X Cl 1- ^ X O <0L3> bAC 5, -5 -A , i. ' W ' -. % : f.-iX ;j . . f' ' jb * 4* Vs*"* V*. * sf .> V' v -C vV. X. t i c . 2.^ Specim en No. 1012-67. Lung of fem a'e rat. aye 1-10 week, exposed to inhalation crocidoiite for loSS evm mlathe houis. Portion of 5 mm lesion, cavitating center. Squamous ^11 earrinoma. w ell-differentiated. X 2 0 0 . 504 REEVES ET AL. T h e R espo n se to I ntratrac h e a l I n je c t io n s Generally, the early response was similar to that seen after inhalation. In addi tion, necrosis of the bronchiolar epithelium in the region of the fibers was present in most animals, especially in the chrysotile group. Giant-cell accumulation fol lowed in some instances. Several guinea pigs also showed terminal bronchiolar epithelial proliferation. One rat of the chrysotile group showed focal fibrous ad hesion to the diaphragm. There was no neoplasia in any of the groups. T h e R espo n se to I n tr a pl eu r a l I n je c t io n s Histiocytic and foreign-body response was typical of all groups especially among rabbits. Fibrosis followed in most eases, occasionally with benign proliferative fea tures. One rat ( 4S6 days after croeidolite injection) and two rabbits (666 and 72S davs after croeidolite injection, respectively) had spindle-cell mesotheliomas of the pleura. Two rats (278 and 419 days after chrysotile injection, respectively) had fibromatous mesotheliomas of the pleura (Fig. 3) with invasion of the dia phragm (Fig. 4). presenting as dense proliferative lesions attached to the dia phragm. There was no neoplasia in the amosite group. T h e R e s p o n s e t o I n t r a p e h it o n e a l I n je c t io n s The response was generally similar to that seen in the pleura after the intra pleural injections. All animals showed initial inflammatory response associated with histiocyte and giant-cell accumulation, followed hy fibrosis. The fibrosis was thelioma presenting a dense, fibrous-appearing adhesion to the diaphragm. X 53. bVg N",\ \\Ni- *"i ' > 'A';. "A >Af'-Vv.' -.y-, '-< y V,. ;;V; v , X J ; > 'A t AjC \ .A A .- .v -v /; ' i . s A , :v -" A:;-:,- A ii- '" -1t v - *;*\ tic;. 4. Same spot paiuVuIailv advanced i; croeidolite injection, sir survived croeidolite inji follows: One with fibre with osteogenic sarcoim him- and spindle-cell sa: group, there1 were also -6orcI day. respectively, of muscle ); and one, on animal, on the 261st da neoplasia. There were n Among the 13 cases o among more than 500 i cf. Table 4 ), 2 were pv to the inhalation of croc deuce' of hmg cancer in and elsewhere, the cause dear, but it must be c ^ group) vs 0 in 40-42 ( f different according to the #te8B&Ww. IN JE C T IO N S |en after inhalation. In addilion of the fibers was present piant-cell aecinnulation folpowed terminal bronchiolar |ip showed focal fibrous ad- of the groups. JIT .T IO N S all groups especially among ifh benign proliferative fead two rabbits (666 and 72S ndle-eell mesotheliomas of tile injection, respectively) ) with invasion of the diasions attached to the dia- VJ ECTIOXS the pleura after the intramtorv r("snnns'f' ^^oclatcd U n J u n S. 1 l i e )I1)I'(JMS w a s fcS" i ;J * t*<i, f y -A ,* if~ ` age 76 weeks. Micnficed 119 face contains a fibious hicm>idpliragm. ,-'50. EXPERIM ENTAL ASBESTOS CARCINOGENESIS 505 t i c . 4. Sane specim en, diaphi,is>ni. lm a sio n of timior iroin the pleura > 50, particularly advanced in animals of the amosite group. One rat, 263 davs after erocidolite injection, showed mesolhelial proliferation and 3 out of 4 rats which survived erocidolite injection one year or longer lmd peritoneal mesotheliomas as follows: One with fibrous and papillary pattern on the 366th day (Fig. 5); one with osteogenic sarcoma pattern on the 372nd day (Fig. 6); and one with reticu lum- and spindlc-ccll sarcoma pattern on the 521st day (Fig. 7). Jn the chrvsotile group, there were also 3 cases of mesothelioma. Two of these, on the 211tli and 263rd day, respectively, had spindle-cell sarcoma pattern ( the latter with invasion of muscle); and one, on the 333rd day, was of solid epithelial type. One additional animal, on the 261st day, had atypical fibrosis suggestive of but not confirmed as neoplasia. There were no tumors in the amosite group. D ISC U SSIO N Among the 13 cases of malignant neoplasia which were observed in this study among more than 500 animals of various species with adequate survival record (cf. Table 4), 2 were pulmonary carcinomas. Both of these were in rats exposed to the inhalation of erocidolite. In view7 of the known lack of spontaneous inci dence of lung cancer in the Charles River CD rat as observed in this laboratory and elsewhere, the cause-and-effect relation between exposuie and cancer seems clear, out it must be emphasized that the incidence of 2 in 31 (in the erocidolite group) vs 0 in 40-42 (in the amosite and chrvsotile groups) is not significantly diffcient according to the chi-square test (X- - 2.12). Theiefort, it cannot he said 503 REI VES E T AL. v'tC p - *..*". - V .-,*!-J?*>^!. ' *" '* '' `. &' " ' '\ ' '*-V' k v T :'iV-jje^*,*.'*'. > -J- _J ' .............. <r% -' * ; 5 . 'g. % ' * 4 -* ' V : *' \ & < : v*x-- X -v ^ * '--.. '\ ^ i'" .- *~~,v,v*{T : : ''' " ' tJ.' ' /T' * . ' r?. **; Vf f ^ ' ~^ \ 1 - ' / <'-'*fV'**1...?'/*'irv\ y>-{" S Va's > i-iSi ; / ' - ? ' * . ' ' 1V(.-*, -srgi > v \-. - 1 ,1 F ig . 6. Specim en X o. 9S1-11. Peritoneum of n u 'c rat, age 64 w eeks, sacrificed 372 d.i" after an intraperitoaeal injection of erocidolite, show ing firm, pale, nodular lesions on da peritoneal surfaces. M esothelioma, osteogenic sarcoma type. X 5 0 . K X I'l *** . i n iri*---. - m . 1fit< % ^ '<%Va^'>V i ' t i , X X- ' j rV#{ , -w ** *%' -f ' *^ rAV T >* T 'v . i i v vs.* * * '*i " * \ ;'* \ , / i| p" f !> -\ '.i>, f l ' :1'i'r'>"?> ' ; >` r SJ_A *. K" r; ST- -* r f- - , *\ , r yv f|t-Wa ,7' V #<-; - ' y !'; t a ~ ^ '. ~V ,. * ;.x. ^ ' . ,,<*-**' &3 " . ' i l * . J* K r*, * I,, / " Kt. ; an iUiUUm] mjrcuij show ed clt'ii v fibro us adlu^KiHs, nn t!it' basis of these lesulJ tnan either amosite or ehr>| Xo mesotheliomas were piodueed in the pleura aj Tables 3 and 4). This res! they included rabbits as v.l animals treated with eipcil duced with amosite. The I 1,1 '`'^) shows a difference I T95). Jhe.se studies there ciiiooenie following local i| troc.dolitt* or chrvsotile, wL These observations are in i| uas niore fibrogenic in till pectalh; in guinea pigs tlianl Xo neoplasms were obt{ the number of animals so t] The opeiative principle 'Peculations in the recent ]J 'IM 1 .-*A! filai EXPEKLMEXTAL ASBESTOS CARClXOGENESiS 507 X X W ;e '.i- X \ ; ' 3 'W \ w ex-''- -/ w . " ." v ^ V 'V . -r-'; - W -\ . :. .* 7 , ^ vV fc**** G4 weeks, sacrificed 3,3 pale, nodular lesions on "* c '"- *' v.. , .- 1 * *" -- \ *~`*4 . ' -V- i ( . S - ' .A ,, ` ; a*"' V - ' X T *V>V ',*O . Jf V W.; > .7*;Vr *C, ' ' 'X ` l 'r ' ^ w " |1 f -: i - V ` v * 5- ; rN.W*$\, 'j\ \K \ 3' v - Xw w ./ r'Av1- X vx %*. # ^ ^ ^r f ti r.y:i i ^ t. / -' s l' . A ' X X , , , ' `j O 8 - X ^ 7** *. \ ;?' > > , * > i. V' <>J* f t W i . : .- 5 F f - i s, ; T ,v p *` f ' ` ' ^ fr : Vjr r.v f^5 <k , *\ X * -* X ' & iv ' 1ft- *v *' ,, . X , J . -4T ' - .O* * i' h ' (,a * "V * .3* ` - %* " ' *' t ~ . *{ ' f f '* ,, i V ^' S. ,' r V y\ X v-Xx 1 s i* \ ,i ..V ;:3 `/ya rl V -~ Fk;. 7. Sjjccmu'n No. 981-13, l 1! iilonc'iim of ukuo iat, aur 85 wei-kv, s.fcnfk-pd 521 da>s aflcr an m tiapcTitonoa] injection of crot idolite. M esotheliom a, spindle cell sarcoma type, which showed dom e filnous adiicsions 1/ctween loops. X 200. on the* basis of these results that crocidolite is more carcinogenic upt>n inhalation than either amosite or chrysotile. Ko mesotheliomas were obtained after inhalation, but 5 such neoplasms were produced in the plema and 6 in the peritoneum after local implantation (ef. Tables 3 and 4). This response occurred as early as 7 months alter treatments, they included rabbits as well as rats, and were about evenly distributed between annuals treated with crocidolite and chrysotile. Ko neoplastic lesions were pro duced with amosite. Ihe statistical comparison of these figures (5-6 in 32 vs 0 in 33) shows a difference that is significant at the 93!f confidence level (X- -- 5.95). These studies therefore support the conclusion that amosite is less car cinogenic following local implantt orr into the pleura or peritoneum than either crocidolite or ehrvsotile, which was also observed by Wagner and Berry (1969). These observ ations are in interesting contradistinction to the finding that amosite "'as more fibrogenic in this study and caused much higher early mortality es pecially in guinea pigs than either crocidolite or chrysotile. No neoplasms were obtained after intratracheal injection. It is poss ble that the number of animals so treated ( 10-16 of each species) was too small. The operative principle of asbestos carcinogenesis was subject to numerous speculations in the recent past. The theory of purely mechanical iiriiai.on ( "Op 'd 5 SOS REEVES ET AL. penheimer effect ) was considered but ruled out by Ilaringcon and Roe ( 196b) and there is no evidence thus far that a crystallographic or chemical property in herent in the silicon-oxygen lattice of asbestos fibers is the essential carcinogenicentity. On the contrary, it may be noted that the two amphiboles, amosite and crocidolite. are not equally effective in this respect. Even though they both form harsh, relatively acid-resistant, and truly chain-structured fibers, amosite appears to be a weaker carcinogen than crocidolite. On the other hand, ehrysotilc which is mineralogicallv quite different from the amphiboles and is soft, acid-soluble, and not a chain-structured fiber but rather a microtubule, is apparently compa rable in its carcinogenic potential to crocidolite. Current thinking favors the hy pothesis that labile cations attached to the silicon-oxygen lattice, metallic con taminants. or organic compounds which may become adsorbed on the fibers either during their geological genesis or during commercial handling, may he the key factor in the carcinogenic potential of asbestos. In 1962. ilarington reported that certain virgin samples of asbestos contained cyclohexane-extractable oils composed of aromatic hydrocarbons, one of which was 3.4-benzpyrene. Furthermore, it was later pointed out that in addition to these natural trace constituents, asbestos may contain contaminants acquired dur ing production, storage, or transport, such as the jute oils derived from storage bags. Tests for the tumor-promoting activity of these oils on mouse skin yielded considerable positive results with at least one variety of jute oil ( ilarington and Roe. 1963: Roe ct /.. 1966 ). The work of Miller et al. (1965), Smith et al. ( 1970) as well as Pyiev c! al. (1969) confirmed the substantial and to some degree specific adsorption of 3.4-benzpyrene on asbestos fibers. However. Ilarington et al. in fcv.c.d that r--mo'-al <4 these oils apnarentlv decieased but did not abolish the carcinogenic activity of asbestos. An alternate carcinogenic principle that may be present in or associated with asbestos arc metallic elements. These may be covalently bound to the fiber, they may be attached to it by hydrogen bonding, ionic bonding, or adsorption. In many of these cases the metal may be detachable and/or exchangeable during in eico interaction. The major metallic components present in asbestos are alu minum. magnesium, and iron; trace levels of chromium, nickel, and cobalt are also regularly found. Among the major metallic components, iron has attracted interest. It was re called that while ion itself is not regarded as carcinogenic, experimental tumors were produced by iron-clextran complexes where the bonding of iron to the polymer may bas e been similar to that in asbestos ( Richmond, 1959). Trace levels of chromium and nickel may play a role as these metals are sus pect as environmental carcinogens. It is apparent from the results of chemical analysis (Table 2) that the dissemination methodology' employed in this experi ment caused substantial enrichment of the dusts in these metals, and similar fac tors were also noted in the experiments of Gross et al. (1937). Indeed, Cralley et al. (1967) pointed out that such exposures may be sustained by workers employed in the manufacture of asbestos textile products. Dixon et al. (1970) reported re cently that Fe ~ and Cr,;+, both of which were extractable from a sample of chrys'tile, inhibited the action of benzpyrene hydroxylase, a detoxifying enzyme that E .\l presumably' protects tin induction of this enzyn (1967), suggesting into genesis by certain metal The authors are indebted consulting; to Kenneth R. construction and operation tenance and necropsies; and B a d o l l e t , M. S., and C a n . use. Ann. X. V. Acad. Sci. Berry, G., and W agner, J. times of occurrence of n Cancer 23, 582-86. C r a l l e y , L. J., K e e n a n , R. facture of asbestos textile D a m s, J. M. C. ( 1 9 6 3 ). lung. Biit. J. Exp. Pathol. Davis, J. M. G. ( 1 9 6 7 ). tamed m organ culture. Br D a m s , j M. G. (1 9 7 0 ). dust injected into the pleu D o l l , R. (1 9 5 5 ) . Mortah 81-86. D o n n a . A. ( 1970). T,,,,,, ratto. Med. Lavoro 61, 1-3 Pa t i o . n a p , J. B., a n d Smii Med. 21, 20-31. G e r b e r , M. A. ( 1 9 7 0 ) . .' Amer. J. Clin. Pathol. 53, 2! Cio y x f , s. R. (1 9 3 0 ). M, a n d p'eurae. Tubercle 12, ; (.Ross, P., D eT r e v il l e , R. T Experimental asbextosis-- T of d m sotile d u st. Arch Em C ross, P., and D eT reville Health 15, 638-19. C ross, P., C r a l l e y , L. J., . nonspecificity. Amer. Ind. II C ross, P., D e T r e v i l l e , R. bodies, their development i concentration. Arch. Pathol. Cross, P., and D e T r ev ille, to high concentrations of c; Health (T o kyo ), pp. 18.5-7. H ammond, E. C., Selikoff, workers in the U. S. with : Sci. 132(1), 51 9 -2 5 . Ha r i n o t o n , J. S. ( 1 9 6 2 ) . 'tances m ashesto,. Xalurc 1 aU6tiu~.v rington and Roe (1965) or chemical property in he essential carcinogenic amphiboles, amosite and n though they both" form d fibers, amosite appears >r hand, chrysotile which and is soft, acid-soluble, le, is apparently compat thinking favors the hygen lattice, metallic eon- adsorbed on the fibers dal handling, may be the lies of asbestos contained rocarbons, one of which out that in addition to ntammants acquired der ails derived from storage Is on mouse skin yielded f jute oil ( Harington and 965), Smith et al. ( 1970' id to some degree specific rver, Harington et al. in -ased but did not abolish sent in or associated with y bound to the fiber, they Hiding, or adsorption. In 1/or exchangeable during L'sent in asbestos are ala li, nickel, and cobalt aie acted interest. It was reenic, experimental tumors bonding of iron to the mond, 1959). e as these metals are susn the results of chemical ' employed in this experi,e metals, and similar fac1937). 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