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American Journal of Industrial Medicine 19:629-636 (1991) Carcinoma of the Colon in Asbestos-Exposed Workers: Analysis of Asbestos Content in Colon Tissue Albert Ehrlich, mo, Ronald E. Gordon, PhD, and Steven H. Dikman, md Epidemiological studies have indicated an increased incidence of carcinoma of the colon in asbestos workers. The present study evaluated the colon tissue asbestos burden, by light and electron microscopic analytic techniques, in patients with a history of occu pational asbestos exposure and colon cancer. Asbestos fibers and/or asbestos bodies were present in colon tissue from 14 of 44 (31.8%) asbestos workers with colon carci noma (range 142,199 to 15,231, 543 fibers/g/wet weight, mean 2,517,823). Chrysotile was identified in 9 patients and amosite in 3 patients. Both amosite and chrysotile were found in the colonic wall in one individual. Other forms of asbestos (e g., crocidolite, tremolite, or anthophyllite) were not found. Asbestos fibers and asbestos bodies were not found in colon tissue from 20 control patients (colon carcinoma and no asbestos exposure). Asbestos fibers frequently enter and reside in the wall of the colon and are often intimately associated with tumor tissue at the site of colon carcinoma in workers with asbestos exposure and colon carcinoma. Key words: colon cancer, asbestos exposure, chrysotile, amosite, electron microscopy INTRODUCTION There is an increased number of deaths from colon cancer in asbestos workers [Selikoff et al., 1979; Miller, 1978; Puntoni et al., 1979; Newhouse and Berry, 1979; Hiltetal., 1985; Ehrlich etal., 1985; Frumkin and Berlin, 1988]. In a study of 17,800 asbestos insulation workers, where 38.1 deaths due to cancer of the colon and rectum could have been expected, there were 59 [Selikoff et al., 1979]. Asbestos was demonstrated by light and electron microscopy in the colon carcinoma and mesentery of an asbestos worker and asbestos bodies have been found in colon tissue from patients with occupational asbestos exposure [Ehrlich et al., 1985; Kobayashi et al., 1987], To determine the frequency, amount, and type of asbestos present in colon tumor and non-neoplastic bowel wall from patients with a history of occupational Department of Pathology, Mount Sinai School of Medicine of the City University of New York, NY. Address reprint requests to Steven H. Dikman, M.D., Department of Pathology, Box 1194, Mount Sinai Medical Center and Mount Sinai School of Medicine of the City of New York, One Gustave L. Levy Place, New York, NY 10029-6574. Albeit Ehrlich, MD. is deceased. Accepted for publication August 23, 1990. 1991 Wiley-Liss, Inc. 630 Ehrlich et al. TABLE I. Workers With Occupational Asbestos Exposure and Colon Carcinoma: Analysis of Colon Tissue: Positive Asbestos Burden Study* Case no. t 2 3 4 5 6 7 8 9 10 11 12 13 14 Colon tumor Bodies" Fibers* 90 NDb 40 ND 60 ND 150 1,129,104 amosite 10 ND BDL BDL BDL BDL BDL BDL BDL BDL BDL 121,000 chrysotile 227,000 chrysotile 5,000,000 chrysotile 211,444 chrysotile 3,026,000 chrysotile 15,231,543 chrysotile 142,199 chrysotile 2,219,913 chrysotile 201,959 chrysotile Normal colon Bodies* x Fibers* ND ND BDLC BDL ND ND ND BDL 10d BDL 185,886" amosite BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL ND 586,434 amosite ND BDL BDL ND ND Occupation Machinist Insulator Construction Cleaner R.R. Insulator Pipefitter Machinist Boiler maker Engineer Carpenter Pipe coverer Electric shop Boiler room Near pipe fitters Years exposure 30 16 24 22 32 29 29 30 20 37 40 37 42 26 *Case 5 previously reported [Ehrlich et al., 1985]. "Gram wet weight. bND = Not done. CBDL = below detection limits. "Mesentery. asbestos exposure and colon cancer, digested colon tissue was examined by light and electron microscopy for identification of asbestos bodies and asbestos fiber burden. Similar studies were performed on colon tissue from patients with colon cancer and no asbestos exposure. MATERIALS AND METHODS Occupational history data, medical records and colon tissue from 44 asbestos workers with colon carcinoma living in various rural and urban environments in the United States were referred to the medical center (Tables I and II). There were no other forms of asbestos-related neoplasia in this group of patients, e.g., lung cancer or mesothelioma. Twenty patients with colon carcinoma from the New York metro politan area, without, on careful screening, a history of occupational, hobby, or other exposure to asbestos, served as the control population. The histologic sections were examined in all cases to confirm the diagnosis of colon carcinoma and to exclude other types of malignancy. The histologic sections \1 i-V s. V, " V' \ Colon Carcinoma and Asbestos TABLE II. Workers With Occupational Asbestos Exposure and Colon Carcinoma: Analysis of Colon Tissue: Negative Asbestos Burden Study Case no. Occupation Years exposure Colon specimen IS Boiler maker 20 Tumor 16 Brake worker 24 Tumor 17 R.R.-Mechanic 25 Tumor 18 Electrician-Navy 35 Tumor + nontumor 19 Insul/pipe fitter 13 Tumor 20 R.R. insulator 29 Tumor + nontumor 21 Insulator 30 Tumor 22 Shipyard 43 Tumor 23 Air conditioning 24 Tumor 24 Electrician 21 Tumor 25 Cook-Navy 20 Tumor 26 Electrician 34 Tumor 27 Shipyard 39 Tumor 28 Boiler maker 28 Tumor 29 Insul. locomotive 30 Tumor + nontumor 30 Sheet metal 30 Tumor + nontumor 31 Steam eng. refitter 40 Tumor 32 Fireman-turbine 27 Tumor 33 Plumber 39 Tumor 34 Salvage recovery 29 Tumor + nontumor 35 R.R.-Machinist 44 Tumor + nontumor 36 Railroad 38 Tumor + nontumor 37 Ship fitter 35 Tumor + nontumor 38 Shipyard-boiler 35 Tumor + nontumor 39 Sheet metal 34 Tumor + nontumor 40 Shipyard-welder 30 Tumor 41 Machinist 44 Tumor + nontumor 42 Asbestos removal 29 Tumor salvage 43 Scrapped steam 40 Tumor + nontumor engines 44 R.R.-insulator 11 Tumor 631 were utilized to select paraffin blocks containing bowel wall infiltrated by colon carcinoma and colon tissue uninvolved by tumor. The paraffin-embedded tissue was cut from the paraffin blocks, the paraffin was melted, and the tissue was washed in xylene and rehydrated. The tissue wet weight ranged from 0.1 to 0.8 g with a mean of 0.4 g. In case 5 and the 20 control cases, 5 g of formalin-fixed surgical tissue from the tumor and non-neoplastic bowel wall were utilized. The deparaffinized and for malin-fixed tissues were diced into small pieces and digested in 5% potassium hy droxide (KOH) for 1-3 hours at 70C. The digested material was washed in distilled water and centrifuged for 15 minutes 3 times at 14,000 rpm. The digested material was resuspended in 10 ml of distilled water. Ten microliters of the 10 ml suspension was placed on formvar-coated nickel grids. The specimens were given a code num ber, and a minimum of 40 grid spaces on 3 different grids were counted for fibers by 2 investigators utilizing a JEM 100 CX II STEM electron microscope equipped with TN 2000 Tracor Northern Energy Dispersive Spectroscopy Unit. The detection limits were determined utilizing a modification of published procedures for air samples 632 Ehrlich et ai. Fig. I. Asbestos bodies found in digests of colon carcinoma in cases 1-5 (original magnification x 320). [Yamate et al., 1984], Cytocentrifuge specimens were prepared from .2 ml samples placed in right-angle cytofunnel plastic containers with a 6 mm diameter round opening and centrifuged (Shandon-Cytospin No2) at 1,200 rpm for 10 minutes onto a slide clamped against the opening [Ehrlich and Suzuki, 1987], The sediment on the glass slide was examined for asbestos bodies by light microscopy. Cases 1-3 were examined only by light microscopy. RESULTS Asbestos bodies (Fig. 1) were found in digested colon tissue in 5 cases (11.3%) and asbestos fibers in 11 (25.0%). The number of asbestos bodies ranged from 10 to 150 bodies per gram wet weight, with a mean of 72 asbestos bodies per gram wet weight. The number of asbestos fibers ranged from 142,199 to 15,231,543 fibers per gram wet weight with a mean of 2,517,823. The detection limits were an average of 7,565 fibers per gram wet weight for the control group and 94,563 fibers per gram wet weight for the asbestos-exposed group. Chrysotile (Fig. 2) was identified in 9 patients (cases 6-14) and amosite (Fig. 3) in 3 patients (cases 4, 5, and 11). Both amosite and chrysotile were found in case 11. Other forms of asbestos were not \t Colon Carcinoma and Asbestos 633 B Fig. 2. A: Electron photomicrograph of 2 chrysotile fibers (arrows) in colon carcinoma of case 6 (original magnification X 13,000). B: Fibers were identified as chrysotile by energy dispersive spectros copy. Fibers with the same morphology and spectrum were seen in cases 7-14. found, e.g., crocidolite, tremolite, or anthophyllite. Asbestos fibers were seen by electron microscopy in digested colon wall tissue from a site uninvolved by carci noma in 1 patient (No. 11). Asbestos bodies and fibers were not found by light or electron microscopy in the digested normal bowel tissue from the 20 control patients. DISCUSSION The Standard Mortality Ratio (SMR) of asbestos-exposed workers with colo rectal carcinoma has been reported as 2.5 to 3.0 times higher than that of the general population [Selikoff et al., 1979; Miller, 1978; Puntoni et al., 1979, Newhouse and Berry, 1979; Hilt et al., 1985; Frumkin and Berlin, 1988], Some epidemiological studies report the contrary, i.e., no increase in the incidence of gastrointestinal cancers in asbestos-exposed workers [Levine, 1985; Morgan et al., 1985; Doll and Peto, 1985; Edelman, 1988], However, when cohorts of published epidemiologic studies of asbestos exposure and gastrointestinal malignancy are stratified by dose, there is an elevated SMR for gastrointestinal cancer [Frumkin and Berlin, 1988]. Doll and Peto indicated that, although they concluded that there was no increase in the 634 Ehrlich et ai. Fig. 3. A: Electron photomicrograph of an amosite fiber (arrow) in colon carcinoma of case 4 (original magnification x 26,000). B; Fiber was identified as amosite by energy dispeisive spectroscopy. Fibers with same morphology and spectrum were seen in cases 5 and 11. SMR of gastrointestinal cancer in asbestos workers, "this conclusion would be weak ened if further evidence indicated that substantial numbers of carcinogenic fibers reach certain organs" [Doll and Peto, 1985], The present study of workers with occupational exposure to asbestos indicates that asbestos fibers reach the colonic wall tissue in substantial amounts and are not found in the general population with colon carcinoma. The fibers remain in the colon wall for extended periods as evidenced by the formation of asbestos bodies. vw ' \r. Colon Carcinoma and Asbestos 635 Asbestos bodies and asbestos fibers were not found in the control group even though the tissue samples were approximately 10 times as large as the samples from the asbestos-exposed subjects, with detection limits of 7,565 fibers per gram wet weight vs. 94,563 fibers per gram wet weight, respectively. In addition, the control group consisted of urban dwellers where the air concentration of asbestos tends to be higher than in the combined urban and rural areas from which the group of asbestos workers was drawn [Langer et al., 1971]. A prior light microscopic study of the digest material from 27 specimens of colon adenocarcinoma from the general public did not find asbestos bodies [Rosen et al., 1974], Kobayashi found an average of 0.6 asbestos bodies per gram of non-neoplastic colon in 7 of 19 autopsies of asbestos- exposed workers using light microscopy, and no asbestos bodies in similar digested colon tissue from 8 unexposed workers [Kobayashi et al., 1987], The observed higher frequency of asbestos bodies found in normal colon of asbestos workers by Kabayashi may be due to the much larger samples utilized (5 g). . Asbestos fibers can readily enter the gastrointestinal tract by swallowing bron chial mucus. A considerable proportion of the dust deposited in the respiratory tract after inhalation is transported on the mucociliary blanket lining the airways which moves entrapped material upward to the buccal cavity where it is swallowed. As much as 25--50% of inhaled fibers enter the pharynx [Selikoff et al., 1979]. After swallowing, asbestos penetrates the bowel wall as has been shown in rats [Westlake et al., 1965; Amacher et al., 1974]. Asbestos bodies and fibers were demonstrated in the serosa of an asbestos worker with colon carcinoma which could have reached this site through the bowel wall [Ehrlich et al., 1985]. The incidence of peritoneal me sothelioma exceeds that of pleural mesothelioma suggesting that considerable quan tities of asbestos fibers reach the abdomen, possibly through the gastrointestinal tract [Selikoff et al., 1979]. Animal experiments, however, have failed to produce gas trointestinal cancer utilizing asbestos although penetration of the mucosa and in creased synthesis of DNA were shown in the colon of rats following gavage admin istration of chiysotile fibers [Amacher et al., 1974]. It has recently been shown that the introduction of exogenous DNA on the surface of asbestos fibers into eukayrotic cells could cause mutations and contribute to the induction of oncogenesis [Appel et al., 1988], ACKNOWLEDGMENTS The technical assistance of Mr. Norman Katz and Ronald Uson is gratefully acknowledged and secretarial assistance by Marva Barbee is appreciated. REFERENCES Amacher DE, Alarif A, Epstein SS (1974): Effects of ingested chrysotile on DNA synthesis in the gastrointestinal tract and liver of the rat. Environ Health Perspect 9:319-324. Appel J, Fasy TM, Kohtz DS, Kohtz JD, Johnson EM (1988): Asbestos fibers mediate transformation of monkey ceils by exogenous plasmid DNA. Proc Natl Acad Sci USA 85:7670-7674. Doll R, Peto J (1985): "Asbestos: Effects on Health of Exposure to Asbestos." London Health and Safety Commission, pp 4--8. Edelman DA (1988): Exposure to asbestos and the risk of gastrointestinal cancer: a reassessment. Br J Ind Med 45:75-82. I 4 i A 4 }it *?! 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