Document EvEdzYBK87OR6DXGvOYGXkEz0

Tissue Response to (nfraperitonea! Asbestos with PrelirrjTrTary Report of Acute Toxicity of Heat-Treated Asbestos in Mice Jcbaj Jagatic, M. E. Rubnitz, 11. C. Godwin*, axd Robert IV. Weiskopf Laboratory Service, Veterans Administration Hospital, Hines, Illinois Received July IS, 1967 Intraperitoneal asbestos not only produced fibrosis, but a special and peculiar type of fibrosis which was proliferative, granulomatous and invasive, and histologically simi lar to mesothelioma, although morphologic evidence of actual malignancy was not obtained. Further, asbestos fibers exposed to high temperature produced a high degree of toxicity in experimental animals; this resulted in a 60% mortality rate. There has been recent clarification of an important association between as bestos exposure and neoplasia, including bronchogenic carcinoma (Selikoff et al., 1964) and mesothelioma of the pleura and the peritoneum (Wagner et al., 1960). Because this association was seen in a number of cases at the Veterans Adminis tration Hospital, Hines, Illinois, this investigation was undertaken. We wished to study znesothelial-cell response to asbestos fibers to determine whether this tissue might undergo malignant change or, if a neoplasm did not result, if there would be some other distinctive lesion that could be attributed to asbestos fibers. MATERIALS AND METHODS Two hundred white male mice were used (Garworth Farms, Hew York, New York). Thirty-eight died in the initial stages of the experiment from a variety of causes, possibly related to transportation, the new environment, etc., but not in any way related to the experiment. The remaining mice were divided into two groups. The first group of 136 mice, group A, were injected with a 50% suspen sion of asbestos powder in N:saline. The control group of 50 mice, group B, were injected with a 50% suspension oi talc in Nrsaline. (The talc was purchased from Octagone Process, Inc., Edgcwater, New York. 6505-147-0000 Talc, USP V 7023P-9108, Lot B 1842. No further analysis of the talc was made.) The com mercially prepared asbestos powder was obtained through the courtesy of Grant Wilson, Inc., Chicago, Illinois. The Research and Technical Services Branch, Occupational Health Research and Training Facility of the USPHS examined a sample of this material and found .. it to be roughly 50% chtysotile, the asbestiform variety of serpentine." * Chemical Analysis: "SiO,, 3S-42; MgO, 40-42; H0,12-15; FeO, Tr-6; Fe,Oi, Tr-6; A!>0>, Tr-3; CaO, 0-020; Cr:Oi. nil; sp. gr., 2.48-2.57." H:0 represents combined water. The material was mined in Quebec, Canada and was characterized as Quebec Standard Test 7D-275,7K-277, 7M-366,7R-3G6,7T-455, and 7RF-721. 1868 by Academic Bress Inc. 2X7 ASARCO ELP 0003130 218 J. JAGATIC, M. E. BOBJilTZ, M. C. GODWIN, ASD R. W. WEISKOPF The remainder was apparently . . composed largely of equant grains of the massive variety of serpentine known as antigorite. The average diameter of the chrysotile fibers is small, probably less than 0.5 microns.`Many very fine fibers are apparent at 400 X* This sample was found to consist of the chrysotile form of asbestos containing 2.5%> iron, 0.4% nickel, 0.1% chromium and 0.03% tita nium, each expressed as the element. Emission spectrographic examination con firmed presence of Mg and Si in the 10-20% concentration range. Free silica: 0.44%." Injections were given intraperitoneally in the left lower quadrant. Each mouse received 0.5 cc of suspension. Immediately following injection, all mice received a 5% solution of Terramycin in their drinking water. This was repeated daily for 5 days. Throughout the entire experiment, the animals were on Rockland Rat Diet ("D" Free) and Rockland Rat Diet (Complete). RESULTS Of the 162 mice studied, 118 were injected with asbestos and 44 with talc. The experiment lasted 343 days. Animals were sacrificed at six different time intervals. Postmortem examination was performed on each animal immediately after death, and several sections were taken from each for histological examination. The histological sections were stained with hematoxylin and eosin. The first group of 34 mice, originally scheduled to be sacrificed at 30 days, was examined 26 days after injection--these mice died unexpectedly following failure of the air conditioning system in the laboratory. Twenty-two had been injected with asbestos and 12 with talc. Postmortem examination revealed similar findings in both groups. The peritoneal cavity in each animal w'as studded with-l-2-mm diameter nodules on the serosa of the gastrointestinal tract, while in some a large abscess lay between loops of the small intestine. Microscopically, the nodules were seen as foreign body granulomas; more prominent fibroblastic activity appeared in the asbestos injected group. Asbestos particles were either engulfed by foreignbody giant cells or were lying free. Formation of characteristic coated asbestos bodies was not noted (see Figs. 1 and 7). The second group was sacrificed 57 days after injection. Animals in this group and all subsequent groups were not specially selected but picked at random. There were 20 mice in group A and four in group B. On gross examination, a difference between these two groups was seen. In group-A mice, the previously described nodular lesions had changed into fiat placque-like structures covering the serosa of the gastrointestinal tract. These placques, composed primarily of fibrous tissue were large and firm and not only coated the surfaces, but they also penetrated tht .outer intestinal layers as well as the capsule of the liver. Group-B lesions wer localized as abscesses or small foreign-body granulomas. Microscopically, lesion of the two groups were even more dissimilar. In the group-A mice there was nc only progression of the previously noted fibrosis but, in addition, there wer fibrous patches in areas some distance from the sites of injection. Grossly, the: had suggested the appearance of metastatic lesions. In the control group, t! foreign-body granulomas did not show the same behavior, either grossly microscopically (Fig. 2). ASARCO ELP 0003131 TOXICITY IX ASBESTOS-TREATED MICE A third group was sacrificed 112 days after the injections. All 20 animals in group A exhibited marked evidence of fibrosis. Microscopically, they showed the previously described aggressive and proliferative fibrous tissue. In the control group of five animals, only one animal demonstrated similar marked fibrosis. An interesting observation was that all mice in both groups wore found to be in excel lent physical condition (Fig. 3). The fourth group was sacrified 147 days after injection. AH animals in group A showed both gross and microscopic evidence of marked proliferation of fibrous tissue. It was noted at this point that the most prominent mesothelial cell and/or fibrous tissue reaction was concentrated in the vicinity of the asbestos particles. Although there was diffuse fibrosis coating the abdominal organs, the most dense tissue surrounded the uncoated small asbestos fibers. From this central point, growth of fibrous tissue could be traced toward the periphery of these granulo mas. In the control group there was hardly any gross evidence of fibrosis. Micro scopically, there was mild foreign-body granuloma formation devoid of the ag gressive appearance seen in the asbestos-treated mice. Remarkable growth of animals continued to the point where most of the animals had doubled in weight and size (Fig. 4). The fifth group of animals was sacrificed 170 days after the experiment started. All 20 animals in group A showed marked coating of the abdominal cavity with fibrous placques. Fifteen exhibited granulomas penetrating into muscle, liver, and serosa of the intestine. Microsections revealed scattered mitotic figures in some of the spindle-shaped fibrous tissue cells. Such mitoses indicated proliferative activity, not necessarily malignant. The placques were composed of mature fibrocytcs and showed a histologic pattern characterized by an effusive prolifera tive overgrowth of tissue in intimate association with the intestinal serosa. The foreign-body granulomas in such areas appeared to be pushed aside by these placques, and were composed of lymphocytes, plasma cells, and foreign-body-type giant cells which engulfed small particles. It was interesting to note that small asbestos fibers were seen in heavily proliferative serosal placques, while larger unidentified particles were found within the foreign-body granulomas (Fig. 5). The sixth and final group of animals was sacrificed 343 days after injection. As listed in Table I, all 15 of the asbestos-treated mice developed extensive fi brosis and foreign-body granulomas, while 10 of the 15 also demonstrated the serosal placques and invasive granulomas, as described above. The 12 control animals also all developed foreign-body granulomas, but none showed any of the gross or microscopic changes seen in proliferative invasive granulomas or placques. (Figs. 6 and 8). In another experiment, we observed an unusual reaction to a specially prepared asbestos. Commercially obtained asbestos powder was exposed to a temperature of 1000 C for 3 hours; cooling took place overnight. On the following day 200 mice, divided into three groups, were injected. One group of 100 mice was injected with the unheated asbestos powder previously described (0.5 cc of 50% asbestos sus pension in N:saline); a second group of 50 mice was used as a control group and injected with talc (also previously described); and the' third group was in- u-- ASARCO ELP 0003132 220 1. JAGATIC, M. E. RUBNITZ, M. C. GODWIN, AND R. W. WEISKOPF Group* TABLE I. CROUP OF ANIMALS INJECTED WITH ASBESTOS - IIS MICE Animals Injected wife Asbestos Days alter Injection Flbrojii Gross and Microscopic Abcess Largo F.B. Spleen Granu Tumor and LK lomas Invas Ive Gran ilomzs No. % t. 22 2. 20 26 ST 3. 20 112. 4. 21 14T 5. 20 170 6. IS 343 22 20 19 19 20 IS i i 1 S 22 10 45 7 20 17 SS 17 ' 20 IS 75 15 21 8 40 17 20 15 75 IS IS 10 55 2/3 TABLE 2. CONTROL GROUP INJECTED WITH TALCUM - 44 MICE u 12 2S 3 12 12 2. 4 57 1 4 4. 3. s 112 21 4 S1 4. 6 147 1 3$ 5, S 170 is s. 12 243 12 jected with a 50% saline suspension of asbestos which had been exposed to the high temperature. This group of 50 mice was injected with the same tech nique of intra-abdominal injection as the first and second groups (cf. Table II). Within 24 hours, the 50 mice injected with the previously heated asbestos all showed severe signs of toxicity, and nine had died. Thirty-six hours after injection 26 mice were dead, and after 43 hours, 30 mice were dead. The remaining 20 mice in this group showed a toxic reaction for approximately 4 days and subsequently recovered. There were no deaths in either of the other two groups. The surprising and unquestionable relationship between the injection of the asbestos exposed to high temperature and death was immediately noticeable. Samples of the heat-treated asbestos powder were submitted to the Physical and Chemical Analysis Section, Research and Technical Services Branch of the TJSPHS where they were examined using X-ray diffraction procedures. Findings were reported as follows: The X-ray diffraction pattern of the heat-treated material, as received, was compared with the X-ray diffraction pattern obtained from a sample of Lab. No. 40339 which we reheated at 1000C for 3 hours as well as with a diffraction pattern of a sample of your previously submitted as bestos powder, Lab. No. 34622, which was also heated at 100QC for 3 hours. These diffraction patterns were then compared, with one obtained from a sample of Johns-Manville 4-D, Chrysotile powder heated at 1000C for 3 hours. All four samples gave the same_X-ray diffraction pattern. . The X-ray diffraction pattern of your last sample. Lab. No. 40339, in ASARCO elp 0003133 TOXICXTY IN ASBESTOS-TREATED MICE 221 v,," v-r/:a. j Flo. 1. Gross photograph of mouse 26 days after injection with asbestos. Large white area (arrow) is an abscess. Microphotograph (a) shows normal intestine in the right lower comer. From the intestinal serosa toward the left upper comer there is fibrous tissue and the abscess. 6X. In for left upper comer, dark black spots are necrotic debris and the asbestos particle. High enlargement (b) from the edge of the abscess shows foreign body gaint cells with en gulfed particles. 47X. ASARCO ELP 0003134 J I ita. 2. Gross photograph of mouse 57 days after injection with asbestos. Arrow points to fibrous placque formation which coats intestine. Microphotograph (a) shows histiocytic pro liferation in upper right corner with small black dots represnting asbestos fibers. 6x* En largement (b) shows foreign body giant colls in right comer and marked fibrocytic prolifera tion. 47X- 222 ASARCO ELP 0003135 TOXICITY IX ASBESTOS-TREATED MICE 223 v- Jj 1 r-- VaV. Q )i - 'it 7 q 62?'<a . Vo\ ' cJ ;-<<1!#f<*>, 4"" , *<*/":* 'a Vo ' -I,.?4a- V* *V V jy' ,A * ^ 1..-\b q o;.x?sj. 4^ ;: > "* *3b i>~ > J*`M Fia. 3. Gross photograph of mouse 112 days after injectioa with asbestos. Arrow points to marked aggressive fibrosis. Microphotograph (a) shows aggressive granuloma of tissue of peritoneum between intestines. 6X. Enlargement (b).shows center of the aggressive granu loma with three pieces of unidentified material. 47X. ASARCO ELP 0003136 J - - ----* s' i - \4 ASARCO ELP 0003137 .TOXXCITT IX ASBESTOS-TREATED MICE 225 PINK Era. 5. Gross photograph of mouse 170 days after injection with asbestos. Arrow points to fibrous placques which completely encase all abdominal organs. Bight side of microphoto graph' (a) shows nonobstructed intestine. On the left side there is marked, mature fibrocytic proliferation. 6X. Enlargement (b) shows a microscopic picture most' closely similar to malignancy. Sprouting of mesothclial cells from serosa in the left comer is obvious. 47X. ASARCO ELP 0003138 226 J. JAG.VTIC, M. E. RUBXITZj M. C. GOD\VTXf AND R. V. WEISKOPF v y i *t . '.V/.-v.1 - Vic, : I | ! 1 ! : !! F^'i i i j !'!: '{I': / ! * I l 1 i s 1* t J * * I * * *. . t * 1 1 * 1" 1 : 1 i .. a \6 .'centimeters PINK l---^-^er' --_ , Fia. 6. Gross photograph of mouse,343 days after injection with asbestos. Arrow points o solid fibrous coating of abdominal organs. There is still no sign of intestinal obstruction. Snlorgement (a) 6hows the aggressive granulomas in center of liver without continuity from he surface fibrosis. 6X. Enlargement (b) shows the same fibrous granuloma. 47X. ASARCO ELP 0003139 TOXICITY IN ASBESTOS-TREATED MICE 227 vrrrry- VTT^Ty-r:-; f! f]'! :: *: M ! 'T: rTVT JfTTT7fiTTHniTTtTT' ,r.. .; : `"T CENTIMETERS WHITE Fw. 7. Control Group No. 1. Gross photograph of mouse 26 days after injection with talc. Moderate reaction of foreign body granulomas adjacent to liver visible as a white spot below the section of the liver. Microphotograph shows fibrous tissue and, in upper part, necrotic calcified center. Such calcification was not observed in mice injected with asbestos. 6X its as-received state, showed the major constituent to be a member of the Olivine Group aud is identified as Forsteritc (Mg~SiO). The minor constituent .was identified as a member of the Pyroxene Group (Orthopyroxene), namely Enstatitc, [Mg(SiO)|. The diffraction patterns of the reheated material, Lab. No. 40339, heated sample Lab. No. 34622, and the heated Johns-Manville 4-D also showed Forsteritc to be the major constituent and Enstatitc as the minor ASARCO ELP 0003140 228 J. JAGATIC, SI. E. RUDNITZ, >1. C. GODWIN', AND R. W. WEISKOPF . CfcN rtMfc: I K`3 :i WHITE 6Vi..-W0v < " VoO /J " " A ^ ;t A . v* * s- .- SS^ 5*;0 '9rO . * '''tvS -r> < > n?0 0 tv* ./ S 'Vv.lt/ p 3V ; : a'-: <-Tk *c- } a0o-.v0^ ' a. .- . VJ .? o c O * C <? % w- *>. , o_ VjG .-.j} rsv f'j ^ vi-.** A Fro. S. Control Group Aro. U. Gross photograph ot mouse 343 days utter injection with talc. Very mild fibrosis in the area ot mesentery is obvious. There is do coating on serosa of intestinal tract. There is cluster of talc surrounded by giant cells anil fibrous tissue. 6X. constituent.'The amount of Enstatitc was very much less in Lab. No. 40339, as received, than in the other three samples. The X-ray diffraction patterns gave no indication of crystalline free silica in auy of its forms. For your information, (cf. Deere, Howie, and Zussman, 19C6) chrysotile and serpentine are converted to olivine by heating. This conversion occurs around 750C. The reaction which occurs is probably (Deere et al., 1966): 2 ASARCO ELP 0003141 . TOXICITY IX- -ASBESTOS-TREATED MICE 229 ^Ig3Sis03tOII)4--3 Mg-SiO* -f SiO, + 4H:0 with SiO, being present as amorphous silica which gives no X-ray diffraction patterns. i DISCUSSION Since this study couccmcd the possible relationship between asbestosis and the development of mesothelioma, it might be well to review the criteria necessary for .the diagnosis of mesothelioma. Cliurg et al. (1965) stressed as an initial requirement tliat care bo taken to determine that no primary tumor be present that could produce superficial growth along the serous surfaces, with or without shallow invasion of underlying tissue. Another, of course, is that the tumor con form to qnc of the several histologic patterns described for mesothelioma. Metastases of mesotheliomas to distant sites arc generally not the rule, so they certainly need not be present for the establishment of the diagnosis (Godwin, 1957). In 195S SclnnUhl (195S) reported that he had induced the development of a sarcoma after subcutaneous injection and intrapcritoncal application of asbestos. Wagner, in 1902, induced pleural mesotheliomas by inoculation of various dusts directly into the pleural cavities of rats. Smith cl al. (1965) have also presented excellent experimental evidence of the carcinogenic effects of asbestos. Explanations have been offered for these carcinogenic properties by a number of observers. Some feel that the asbestos fiber is not the responsible agent, but that accompanying 3-4-benzypyrcne, which has been isolated from erocidolite, is responsible. Others have blamed the iron content in some types of asbestos. Still others feel that .possible radioactivity in sisbostos exerts direct effect on genes. The Oppenhcimcr theory, which has been accepted and rejected by several authors, suggests that prolougcd residence of inert material in the tissues produces the reaction, since the material is iucapablc of being rapidly removed. Enticknap and Smithcr (1964) collected eleven cases of abdominal tumors associated with asbestosis. They did not classify these tumors as mesotheliomas, but allow the possibility that they might actually be some type of mesothelioma. Figures 1, 2, 4, and G in their paper closely resemble the histological findings we have observed in mice. Review of their data strengthens our conviction that the fibrosis produced by asbestos fibers is completely different from fibrosis produced by other foreign material. The aggressively proliferating invasion granulomas and serosal plaequcs both grossly and microscopically bear a definite resemblance to one or two of the types of mesothelioma, although it must be emphasized that unequivocal neoplastic growth has not yet occurred iu our experimental animals, nor have the animals exhibited such signs of malignancy as cachexia. Nevertheless, isolated largo mesothelial cells have becu observed sprouting from serosal surfaces in some of the animals. These cells arc ovoid, have large hypcrchromatic nuclei, and occasionally exhibit mitotic figures. They arc usually embedded in dense fibrous tissue. Such proliferating mesothclial cells intimately associated with actively growing mesen chymal tissue might well eventually result in development of mesothelioma in more prolonged experiments. ASARCO ELP 0003142 230 J. JAGAT1C, M. E. RUBN1TZ, M. C. GODWIN*, AND B. W. WEISKOPF SUMMARY Experimental studies with intraperitoneal asbestos demonstrated an extensive fibrous tissue reaction of a particular type which was proliferative, granulo matous, and invasive. This tissue response was quite different from that pro duced by talc. In another experiment, heated asbestos produced acute toxic reactions. Those mice that survived the toxicity showed good recovery and were alive seven months later. REFERENCES Cm.no, J., Roses*, S. H., as*o Moolten, S. (1965). Histological characteristics of meso thelioma associated with asbestos. Ann. N.Y. Acad. Sci. 132; 611-622. Deere, Howie, and Zossman. (1966). "Rock Forming Minerals." Vol. 3. p. ISO. Wiley, New York. Enticknap, J. B., and Smithes, W. J. (1961). Peritoneal tumors in asbestosts. Brit. J. Indmtr. Med. 21, 20. Godwin, M. C. (1957). Diffuse mesotheliomas with comment on their relation to localized fibrous mesotheliomas. Cancer 10, 23. Scumaiil, D. (195S). Canccrogenc wirkung von asbest bci implantation on ratten. Z. Krebsforsch. 62, 561-567. Sklikoff, I. J., CiiUBC, J., and Hammond, E Cvyu:k. (1961). Asbestos exposure and neoplasia. J. Amer. Med. Assoc., April 6,142-146. Smith, W. E., Miller, I.loxas, Esasskr, R. E., and Hubert. D. D. (1965). Tests for carcinogenicity of asbestos, Ann. N.Y. Acad. Sci. 132,45C-4SS. Waoner, J. C. (1962). Experimental production of mcsotheliai tumours of the pleura by implantation of dusts in laboratory animals Nature 19G, ISO-181. Wagner, J. C., Slecc3, C. A., and Marchand, P. (1900). Diffuse pleural mesothelioma and asliesto* exposure in the north wc.-tem Cape Province. Bril. J. Induslr. Med. 17, 260. 1 ASARCO ELP 000314-3