Document ybNX7RZE5379qBzXq2NG1OvrD

ility. Bull. Environ. Cont,,m . of No. 2 fuel oil on common i Marine Ecosystems and or on mallard egg hatchabilit>. j 2 fuel oil on hatchabilin >; il, 7-10. environmental research 22, 315-321 (1980) RECEIVED MAR 0 4 1985 S. A. RASMUSSEN The Activity of Environmental Samples in a Cell Culture Test for Asbestos Toxicity1 -ioL-X ' -1 M Betti Reiss,* James R. MiLLETTE,t and Gary M. Williams* *Naylor Dana Institute for Disease Prevention, American Health Foundation, l Dana Road. Valhalla, New York 10595, and *Field Studies Division, Health Effects Research Laboratory, Cincinnati, Ohio 45268 Received January 15, 1980 The inhibition of colony-forming efficiency of cultured human embryonic intestinederived epithelial (1-407) cells was utilized in order to assay the toxic potential of six coded samples of particulate matter provided by the United States Environmental Protection Agency (EPA). The results of the assay indicated that the most toxic of the EPA samples tested "blindly' ' was amosite which was equal in toxicity to the amosite used, by chance, as a positive control. The toxicities of the particulates from drinking water were approximately 100-fold less than the amosite; of these, the order of toxicity of the samples was San Francisco > Seattle > Duluth. The samples of attapulgite clay and taconite tailings dis played approximately half the toxicity of the drinking water particulates. These results indicate that this assay provides a sensitive and accurate method for screening asbestos and asbestiform contamination for potential toxicity. i K INTRODUCTION ; Occupational exposure to airborne asbestos fibers has been implicated in the production of pulmonary asbestosis, lung cancer, and pleural and peritoneal mesotheliomas (Selikoff, et al., 1964, 1972; McDonald el al., 1971; Enterline et al., 1972). Data are also suggestive of a relationship between exposure to asbestos and the development of various forms of gastrointestinal cancer as a result of swallowing inhaled fibers or ingesting food and beverages containing asbestos fibers (Hammond et al., 1965; Selikoff et al., 1974). Since asbestos fibers penevtrate the mucosal lining of the gastrointestinal tract, they may also lead to the : production of tumors in remote organs (Westlake et al., 1965; Storeygard and ( Brown, 1977; Cook and Olson, 1979). f These possibilities have caused concern about the presence of asbestos fibers in (. municipal water supplies in this country (Masson et al., 1974; Levy et al., 1976) o and in Canada (Cunningham and Pontefract, 1971). The discovery in 1973 of ^ asbestos fibers in Lake Superior, the source of municipal water for Duluth, Min > ncsota, prompted investigation of Duluth's drinking water (Cook et al., 1976; Kramer, 1976). Studies revealed the presence of 600 million amphibole asbes- * tiform fibers per liter in samples of Duluth tap water (Cook et al., 1976). Contami^nation of the water supply with asbestos apparently began in 1955 as a result of the flumping of taconite mine tailings containing amphibole impurities into the lake o ro cn o 'This work was supported by U.S. Environmental Protection Agency Grant R803998-01; parts of ^ this study are available in Research Report EPA-600/1-79-023 (B. Reiss, J. H. Weisburger, and G. M. Williams, "Asbestos and Gastrointestinal Cancer: Cell Culture Studies"). Vf~tiv-', 315 0013-9351/80/040315-07S02.00/ 0 Copyright 1980 by Academic Press. Inc. All rights of reproduction in any form reserved. (United States of America vs Reserve Mining Company, 5-22 Civil 19). Although,^ to date, surveys have demonstrated no consistent increase in cancer incidences* (Masson et al., 1974; Levy et al., 1976) or mortality (Wigle, 1977) that can bej| specifically attributed to exposure to asbestos in drinking water, with the excep-W tion of one study which did indicate an increased incidence (McCabe and Millette,J| 1979), ongoing cancer surveillance in regions with high levels of asbestos fiber# contamination in the drinking water has been initiated. Furthermore, it should bej| borne in mind that among persons occupationally exposed to asbestos dust, the .rf increases in death rates from cancer of the stomach, colon, and rectum do not 4 reach measurable proportions until many years after the onset of exposure. Thus. J the lapse of time since Duluth water was first heavily polluted with mineral fibers ^ is not yet sufficient to have produced a significant increase in death rates from ^ these cancers, particularly if the risk for residents of Duluth is not as great as foriK workers occupationally exposed but still of important proportions. If increased risk exists, it will become apparent within another 5 to 15 years (National Re- search Council, 1977). *|| In order to study the cellular effects of asbestos, we have developed a& cytotoxicity assay that measures the inhibition of colony-forming efficiency of T cultured human embryonic intestine-derived (1-407), adult rat liver-derived - epithelial (ARL-6), and mouse colon-derived epithelial-like (MCE-1) cells (Reiss et al., 1980). Using this assay we were able to quantitate the cytotoxic effects of = the chrysotile and amphibole (amosite and crocidolite) forms of asbestos. The 1-407 ; line was found to be the most sensitive of these cell lines to the toxic effects of all forms of Union Internationale Contre Le Cancer (UICC) asbestos (Timbrell and Rendall, 1971-1972) that were tested. This cytotoxicity assay was utilized in the present investigation to determine "in a blind fashion" the toxicity of six coded samples of particulate matter provided by the EPA. Among these samples were, particulates extracted by filtration from the drinking water of three large American cities, San Francisco, Seattle, and Duluth (the Duluth sample was obtained prior to the installation of a filtration plant), as well as taconite tailings, attapulgite clay, and the amosite form of asbestos. The results of the assay indicated that the most toxic of the EPA samples tested "blindly" was the amosite which was equal in toxicity to the amosite used, by chance, as a positive control. The toxicities of the particulates from drinking water were approximately 100-fold less than the amosite control; of these, the order of toxicity of the samples was San Francisco > Seattle > Duluth. The toxicities of the attapulgite clay and the taconite tailings were approximately half that of the drinking water particulates. Thus, the test displayed a wide range in sensitivity to particulates of different composition, and, of these, the asbestos sample was the most toxic. ! MATERIALS AND METHODS Samples of Particulates Six samples of particulates were provided by the EPA. At the time of testing, the identity of the samples was unknown; however, following the completion of the assays, the EPA supplied us with the following descriptions: A. Sample /. i prior to the amphibole c line fraction B. Sample 2. P No filtration chrysotile a: C. Sample 3. 1 water. The analysis is s D. Sample 4. S which had b phibole fibei E. Sample 5. S eral which c chrysotile as from the mi checked by some water generally les bundles in di F. Sample 8. A ingested asbc tute of Envi; Administrate I he UICC referenc ;i MVr decrease in th <D25 g/liter also pro c\ lotoxicity assay. Before weighing, tl exposed to 13D erg/m contamination in a m diemical properties o inaccuracies in weig Analysis by Ei Sample Duluth San Francisco Seattle Attapulgite Single crystal shapes am t nvular/1977 IC-8751. A02508 UCC 017896 22 Civil 19). Although, se in cancer incidence igle, 1977) that can k water, with the exccp (McCabe and Milieuc. evels of asbestos fiber rthermore, it should k d to asbestos dust, the 3n, and rectum do not iset of exposure. Thus, ted with mineral fibeis se in death rates from th is not as great as tot ^portions. If increased 15 years (National Re .ve have developed : '-forming efficient-\ .>! dult rat liver-deri\ed e (MCE-1) cells (Reiss he cytotoxic effects ot s of asbestos. The Mir ) the toxic effects ot til asbestos (Timbrell and ;say was utilized in the e toxicity of six coiled ng these samples were )f three large American pie was obtained pnoi ilings, attapulgite cla>. he EPA samples tested > the amosite used. b> :es from drinking watei ; of these, the ordei ot lluth. The toxicities of mately half that of the ; range in sensitivity a* bestos sample was the At the time of test me. ving the completion ot ptions: ASBESTOS TOXICITY OF ENVIRONMENTAL SAMPLES 317 A. Sample 1. Particulates filtered from Duluth, Minnesota drinking water . prior to the installation of the filtration plant at Duluth. Known to contain amphibole crystals, red clay, and biological fragments. From the crystal line fraction, the particle analysis is shown in Table 1. B. Sample 2. Particulates filtered from Seattle, Washington drinking water. No filtration is used in water treatment. The sample is known to contain chrysotile asbestos. The particle analysis is shown in Table 1. C. Sample 3. Particulates filtered from San Francisco, California drinking water. The sample is known to contain chrysotile asbestos. The particle r- analysis is shown in Table 1. r D. Sample 4. Sample of the less than 2-/am-size fraction of taconite tailings which had been prepared by a sedimentation separation procedure. Am* phibole fibers were identified among the particulates in sample 4. E. Sample 5. Sample of attapulgite (palygorskite) clay, a nonasbestos min eral which consisted of fibers of the same or smaller diameter range as chrysotile asbestos. The attapulgite was obtained in relatively pure form from the mine in Attapulgus, Georgia. The purity of the sample was checked by X-ray diffraction. Attapulgite fibers have been identified in some water supplies in Georgia and Florida. The attapulgite fibers are generally less than 2 pm in length and tend to clump and form small bundles in drinking water. F. Sample 8. Amosite fibers currently l>eing used to study the effects of ` ingested asbestos on rats and hamsters sponsored by The National Insti tute of Environmental Health Sciences (NIEHS), The Food and Drug Administration (FDA), and the EPA (Moore, 1978). The UICC reference sample of amosite at a CDso (cytotoxic dose that produced a 50% decrease in the number of colonies formed in the cytotoxicity assay) of 0.025 g/liter also provided by the EPA was used as a positive control for the ' cytotoxicity assay. | Before weighing, the coded samples and the known sample of amosite were ^exposed to 130 erg/mm2/sec ultraviolet light for 2 hr in order to suppress viable contamination in a manner that would be least likely to alter the physical and chemical properties of the particulates (Reiss et al., 1980). In order to eliminate inaccuracies in weighing caused by the electrostatic character of asbestos, TABLE 1 Analysis by Electron Microscopy of the Crystalline Fraction of the Samples of Particulates Sample Fibers" (%) Equants (%) Aggregates .m Acciculars m Duluth San Francisco Seattle Attapulgite 4.2 7.7 2.0 100 61.1 37.4 80.9 Trace 9.3 18.1 1.5 0.9 0.0 0.0 Single crystal shapes and aggregate classification as described in the Bureau of Mines Information #fcular/1977 IC-875I. A02509 Percentage in h ib itio n o f colony form ation 1 318 REISS, MILLETTE, AND WILLIAMS aliquots of the coded samples or amosite were weighed in a known weight of wat or culture medium immediately prior to use. Cell Cultures 1-407 cells were used for the cytotoxicity assay. These cells were maintained^ 37C in Williams' Medium E (Williams and Gunn, 1974; Flow Laboratories Rockville, Md.) supplemented with 10% fetal bovine serum (Flow Laboratories^ and containing 50.0 units/ml mycostatin (Gibco, Grand Island, N.Y.) and 1Q. /ig/ml gentamycin (Schering, Kenilworth, N.J.). ^ Cytotoxicity Assay The cytotoxicity of the coded samples was quantified as described previously' (Reiss et al., 1980) by measuring the inhibition of 1-407 epithelial cell colony formation following exposure. For this assay, 10 1-407 cells/cm2 were inoculated into 25-cm2 culture flasks. Twenty-four hours after the cells were seeded, the ' medium was replaced with medium containing an aliquot of the sample. Following 3 days of exposure, the cells were washed twice and reincubated in fresh medium. Between 1 and 2 weeks after the initiation of treatment, the cells were fixed in 10% formalin and stained with Giemsa for the determination of colony formation. ^ RESULTS | The dose-response for each unknown was determined over a range of concen* trations from 0.001 to 5.0 g/liter. Testing was initiated by adding between 0.01 and 0.05 g/liter of each sample to the cultures. Depending upon the level of toxicity that the samples exhibited in these early experiments, a range of concentrations was chosen at quarter-log intervals in order to obtain several values of toxicity approximating a CDS0 for each sample. The results of the cytotoxicity assay (Table 2) demonstrated that by assaying the inhibition of 1-407 cell colony formation, the cytotoxic levels of samples coded only by reference number could be easily and reliably evaluated. The order of toxicity of these samples, as determined by the assay, was: sample no. 8 > 3 > 2 > 1 > 5 -- 4. The most toxic sample with a CD50 of approximately 0.01 g/liter W3S sample 8, amosite. The similar toxicity of this amosite sample to the amosite control indicates the reliability of this assay. The sample from San Francisco's drinking water (sample 3) was found to have a CD5a between 0.75 and 1.0 g/liter. The CD50 of the Seattle drinking water sample (sample 2) was between 1.0 and 2.5 g/liter and the CD50 of the Duluth drinking water sample (sample 1), taken before installation of a filtration plant, was be tween 2.5 and 5.0 g/liter. The CD50's of both the samples of attapulgite clay (sample 5) and taconite tailings (sample 4) were at concentrations greater than 5.0 g/liter. Because of the difficulty of obtaining a homogeneous suspension at high concentrations, the assay was not performed at concentrations above this value. DISCUSSION The cytotoxicity assay described previously (Reiss et al., 1980) and applied n this study is a rapid and efficient test which is shown to be useful for the screening AU251C o *- -1. V.-,a* . . . .. r.,,y ; _.,* * _ r X. known weight of w aier :ells were maintained at 74; Flow Laboratory, im (Flow Laboratories Island, N.Y.) and as described previous )7 epithelial cell colon;. ells/cm2 were inoculated : cells were seeded. the of the sample. Follow mu:ubated in fresh me do nr. ie cells were fixed in ! of colony formation d over a range of coiu-cn adding between OdM and apon the level of to\utt\ a range of concentration* several values of to\tci> nstrated that by assa>mg ic levels of samples coded f evaluated. The order ot vas: sample no. 8 ' roximately 0.01 g/liter w.o ite sample to the amo^tc iple 3) was found toh.oc -* ttle drinking water "ample Djo of the Duluth drink** a filtration plant, wa" he amples of attapulgitc ;entrations greater than * * ;eneous suspension at hX itrations above this \ >'uc el a!., 1980) and apr1'1^ ,r > be useful for the screens ASBESTOS TOXICITY OF ENVIRONMENTAL SAMPLES .' . V>-: . ;.'i REISS, MILLETTE, AND WILLIAMS of samples of particulates from drinking water or other sources for the presence oSf asbestos. Although all of the samples tested were of similar densities, large differ-! ences in the toxicities of these samples were found using this assay. Furthermore,^ these toxicities correlated closely with the asbestos content described in they analysis of the samples provided by the EPA after the completion of the assaysyf The results of this investigation suggest that asbestos exerts its toxicity through% specific physicochemical mechanisms and possesses a cytotoxic potential not - found in nonasbestos samples composed primarily of attapulgite clay or taconite y tailings. ^ This bioassay system developed for asbestos can be used as a rapid and accu-y rate method for comparing the relative cytotoxicities of commercial fiber samples, with fibers and particulates from relevant environmental samples. Among' mineralogists there is considerable debate as to whether the amphibole fibers found in the water of Lake Superior should be called asbestos or asbestiform. Although the basic chemistries and crystal structure of the lake fibers are the same as a commerical form of asbestos, a number of mineralogists propose that there" may be some critical differences which would change the hazard potential. Similar questions have been raised about the chrysotile associated with serpentinite. quarry samples. The results of our assay suggest that the 4.2% fibers that are in Duluth drinking water (sample 1) have a toxic potential and behave like true, asbestos; in contrast, the attapulgite clay containing 100% fibers displayed only minimal toxicity in this assay and is not asbestos-like. Thus, this in vitro assay, enables the screening of asbestos- and asbestiform-contaminated environmental samples for hazard potential, an undertaking that would be exceedingly difficult and require prolonged exposure in vivo. ACKNOWLEDGMENTS The authors wish to thank Mrs. Sondra Solomon for her skilled technical assistance and Mrs. Bette Meyer for preparation of the manuscript. The polygorskite (attapulgite) sample from Attapulgus. Georgia, was provided by Mr. Kenneth Wallingford, Field Investigations Unit, NIOSH, Cincinnati. Ohio. The sample of taconite tailings was provided by Dr. Philip Cook, EPA, Duluth, Minnesota. REFERENCES Cook, P. M., Rubin, I. B., Maggiore, C. J., and Nicholson, W. J. (1976). X-ray diffraction and electron beam analysis of asbestiform minerals in Lake Superior waters. In "Proceedings of the Interna tional Conference on Environmental Sensing and Assessment, Las Vegas, Nev.," Vol. 34, pp. 1-9. Cook, P. M., and Olson, G. F. (1979). Ingested mineral fibers: elimination in human urine. Science 204, 195-198. Cunningham, .H. M., and Pontefract, R. (1971). Asbestos fibers in beverages and drinking water. Nature (London) 232, 332-333. Enterline, P., DeCoufle, P., and Henderson, V. (1972). Mortality in relation to occupational exposure in the asbestos industry. J. Occup. Med. 14, 897-903. Hammond, E. C., Selikoff, I. J., and Churg, J. (1965). Neoplasia among insulation workers in the United States with special references to intra-abdominal neoplasia. Ann. N.Y. Acad. Sci. 13 519-525. Kramer, J. R. (1976). Fibrous cummingtonite in Lake Superior. Canad. Mineral 14, 91-98. Levy, B. S., Sigurdson, E., Mandel, J., Laudon, E., and Pearson, J. (1976). Investigating possible effects of asbestos in city water: surveillance of gastrointestinal cancer incidence in Duluth- Minnesota. Amer. J. Epidemiol. 103, 362 - 368. A025 1 2 UCC 017900 ASBES \1 --on, T. J., McKay, F. relation to cancer moi M. i .the. L. J., and Millett water. In "Proceedin I rancisco." In press. M. f Kmald, J. C., McDona rality in the chrysotile M. re. J. (1978). NIEHS ( ment Methods," NBS VwhmkiI Research Counci Acad. Sci., Washingtc Ho". B., Solomon, S., V different forms of astx SclikolT, I. H., Churg, J., ; Wed. Assoc. 188, 22- vl.koff. I. J., Hammond, inviron. Health 25, K vi;koff, I. J., Hammond, tnited States. Insulati Si.weygard, A. R., and Bn libers. Mayo Clin. Pro Im.hrell V., and Rendall, dimples of asbestos. /' Westlake, G. E., Spjut, H. particles: An electron i Wiele. D. T. (1977). Cancel >.//. Health 34, 185-11 w ams, G. M., and Gunn. AY.*. 89, 139-142. or the preset: sities. large v! say. Furthcrn described irtion of the ;t' is toxicity th; oxic potenti;i te clay or 1;k i a rapid and :rcial fiber \;r samples. A i amphibolc os or asbeM fibers are the propose tha: d potential. S 1 with serpe' 7c fibers tha! d behave lik )ers displayo this in vitro . ated environ n xceedingly d ssistance and Mt ample from Ait. nit. NIOSH. C< Duluth. Minn. y difTraction and jceedings of ihc egas. Nev..*' N> in human urine iges and drinkm to occupational c insulation workc m. S.Y. Actut mral 14. 91 ^ 6). 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H., and Williams, G. M. (1980). Comparative toxicities of g* different forms of asbestos in a cell culture assay. Environ. Res. 22, 109-129. Sehioff. I. H., Churg, J., and Hammond, E. C. (1964). Asbestos exposure and neoplasia. J. Amer. Med. Assoc. 188, 22- 26. 'Sekioff. I. J., Hammond, E. C., and Churg, J. (1972). Carcinogenicity of amosite asbestos. Arch. Environ. Health 25, 183 -- 186. : Sdikoff. I. J., Hammond, E. C., and Seidman, H. (1974). Cancer risk of insulation workers in the . United States. Insulation Hyg. Progr. Rep. 6, 1-8. v Jhoreygard, A. R., and Brown, A. L. (1977). Penetration of the small intestinal mucosa by asbestos i;; fibers. Mayo Clin. Proc. 52, 809-812. Timbrel! V.. and Rendall, R. E. G. (1971-1972). Preparation of the U.I.C.C. standard reference qj. .samples of asbestos. Powder Technol. 5, 279-287. Westlake, G. E., Spjut, H. J., and Smith, M. N. (1965). Penetration of colonic mucosa by asbestos > particles: An electron microscopic study in rats fed asbestos dust. Lab. Invest. 14, 2029-2033. ligk,' D. T. (1977). Cancer mortality in relation to asbestos in municipal water supplies. Arch. Envi% ron. Health 34, 185--190. ; 'WShams, G. M., and Gunn, J. M. (1974). Long-term culture of adult rat liver epithelial cells. Exp. Cell Res. 89, 139-142. ~~ Zd-*T ` UCC 017901 AU25 I 3