Quantitative Studies of in Vitro Morphological Transformation of Syrian Hamster Cells by Inorganic Metal Salts

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[CANCER RESEARCH 39, 1008-1013, March 1979] 0008-5472/79/0039-0000502.00 Quantitative Studies of in Vitro MorphologicalTransformation of Syrian Hamster Cells by Inorganic Metal Salts

J. A. DiPaoIo1 and B. C. Casto

Laboratory of Biology, National Cancer Institute, Bethesda, Maryland 20014 (J. A. D.J, and BioLabs, Inc., Northbrook, Illinois 60062fB. C. C.)

ABSTRACT In the present report, 12 metal compounds were tested by direct application to cells that had been seeded for Morphological transformation of Syrian hamster embryo colony formation; 4 of these were tested also by a transpla cells was induced by direct exposure to some inorganic cental in vivo-in vitro approach (9). Interestingly, of the 12 salts of nickel, cadmium, chromium, beryllium, and arsenic metal salts examined, acceptable evidence for human car but not salts of iron, titanium, tungstate, zinc, aluminum, cinogenicity is available for only arsenic, beryllium, chro and nickel sulfide amorphous. Furthermore, no transfor mium, and nickel; these, plus cadmium, were identified as mation was observed in untreated controls. Lethality, mdi transforming agents. cated by a reduction in cloning efficiency, occurred with carcinogenic and noncarcinogenic metal salts. In addition, transformation of Syrian hamster cells was observed also MATERIALSAND METHODS after transplacental exposure to inorganic salts of nickel, beryllium, chromate, and cadmium which had been injected Cell Cultures. Primary HEC2 were derived from Syrian into pregnant hamsters. The transformed colonies had a embryos 13 to 14 days in gestation. Methods for cultivation variety of morphologies reflecting the cell types present in have been published in detail (7). Essentially, after trypsmni early passages of cells and were identical to those observed zation of the whole embryos, primary cultures were ob after organic carcinogens. Animal experimental data exist tamed by seeding 1 x 10@cells/100-mm plastic Petri dish. for the carcinogenicity of all positive inorganic metals Secondary cultures were prepared from the primary cul except arsenic. Epidemiological evidence indicates an as tures 3 to 4 days later. After medium was removed , the cells sociation between exposures to all positive metals except were trypsinized, centrifuged, suspended in complete me for cadmium, although there is a possibility of a relationship dium, counted, and seeded at 5 x 10@/10ml of complete betweenoccupationalexposuretocadmium and cancerof medium in 100-mm plastic Petri dishes. Mass cultures were the prostate. Although in terms of dose-response relation grown in Dulbecco's modification of Eagle's minimal essen ships cadmium and chromium were the most potent trans tial medium, supplemented with 10% fetal bovine serum, in forming agents, the comparative potency of the various incubators at 37Â°ina 10% CO2atmosphere. Total cell count metals cannot be determined since it would depend on the for the secondary cultures after 2 days ranged from 10@to ionization potential of the metal, the inorganic salt form, 1.4 x 10@ cells/dish. and possible biointeractions of the various metals. TestMaterials.The inorganicmetalsaltswerepurchased from Chem Services, Inc. , Westchester, Pa. , except for INTRODUCTION nickel subsulfide and NiS amorphous which were gifts from Dr. F. W. Sunderman and ferric oxide and titanium dioxide A number of inorganic substances are considered carci which were obtained from National Cancer Institute Chem nogenic. Various essential elements may influence the ical Repository. The BP which was used as a positive growth of autochthonous and transplantable tumors. 0th control was purchased from Eastman Kodak. All chemicals ers are considered to be environmental contaminants that were prepared in a darkened room. Ten mg of each chemi initiate cancer in humans and/or in experimental animals. cal were dissolved in 1 to 2 ml of acetone, added to 100 ml A number of reviews and meetings have focused on metal ofwarm complete medium, and mixed. The NiS amorphous carcinogenesis (11, 13, 19, 37, 42) and the problems arising was insoluble and was used in suspension. These stock from the fact that some metals can induce cancer. Some solutions were stored at 4Â°until used. The desired concen incriminated inorganic metals are often associated with a trations were prepared by diluting stock solutions with variety of environmental contaminants, and much of the complete medium. The final concentration of acetone was experimental data consist of the induction of tumors at the always less then 0.02%. site of injection. Recently, quantitative in vitro transforma Transformation. For the transformation assay in which tion was induced with leadacetate using fibroblasts derived chemicals were applied directly to cell cultures, 300 cells from Syrian hamster embryo cells (8). A dose-response from secondary cultures of HEC were plated in complete relationship for transformation was obtained, and the male medium with 20% serum in 50-mm plastic Petri dishes vance of the transformed cells to neoplasia was demon along with 6 x 10@HECcalls which had been irradiated as strated by the ability of cells derived from the transformed confluent monolayer cultures. The feeder cells ware pro colonies to produce progressively growing fibrosarcomas duced using a Picker portable industrial X-ray apparatus when injected into nude mice. (T55-433) at 100 kV and 5 ma, 3000/mm adjusted to deliver

1 To whom requests for reprints should be addressed. 2 The abbreviations used are: HEC, Syrian hamster embryo cells; BP, Received September 11, 1978; accepted December 14, 1978. benzo(a)pyrene ; CE, cloning efficiency.

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Downloaded from cancerres.aacrjournals.org on October 5, 2021. © 1979 American Association for Cancer Research. Transformation by Inorganic Metals a total of 4400 R in air at a distance of 22.2 cm. The HEC metal salts, farmic oxide, titanium dioxide, sodium tung were exposed to the metal salts 1 day after cells had been state, zinc chloride, aluminum chloride, and NiS amor seeded for colony formation. Chemicals as 2x concentra phous, were used at concentrations as high as 20 @g/mlof tions were added in 4 ml of complete medium so that each medium. At the highest concentrations used, the nontrans dish had a total of 8 ml of medium. Experiments were forming metal salts reduced the CE by 20 to 25% relative to terminated 7 to 8 days after the addition of the test metal; controls even though transformation was not observed; the medium was removed, and the cells were washed with however, with NiS amorphous (5 j@g/ml in suspension), Dulbecco's phosphate-buffered saline, fixed, and stained toxicity was even less (â€”5%). with Giemsa for examination. Colonies (a minimum of 2 mm The positive results with the transforming metal salts are diameter) were counted using a stereoscopic microscope summarized in Table 1. All metals caused transformation at at a relatively low magnification. The CE was determined by more than one concentration which was accompanied by dividing the average number of colonies per dish by the toxicity, as indicated by a reduction in CE of approximately number of cells seeded per dish multiplied by 100. The 25 to 50%. The carcinogenic metals were as potent as BP frequency of morphological transformation was expressed which was also used with 20% fetal bovine serum. The data as the average number of transformed colonies per dish indicate, on the basis of the absolute number of transfor relative to total number of colonies per dish and as the mation, that the transformation frequency was dose de average number of transformations per dish. Morphological pendant. Concentrations of metals higher than those given transformation is defined as an altered colony morphology in Table 1 resulted in further lethality and in some cases consisting of criss-crossing and piling up of cells not essentially eliminated all colonies. observed in the controls. Each experimental point was The largest absolute number of transformation observed, repeated 3 to 7 times with cells that originated from differ disregarding concentration, was obtained with nickel sul ant animals; but since the results were consistent, only one fate at 10 @tg/mImedium. The more potent metal carcino set of data is presented for each chemical. gens on the basis of dose-response were sodium chromate Additional experiments were done with some metals and cadmium acetate, which induced an average of 1.75 using a host-mediated in vivo-in vitro assay. This system transformations/dish at a concentration of 0.5 @g/mlof differs from the established quantitative in vitro assay by medium. When the concentration of cadmium acetate was including in vivo transplacental exposure of the fetuses to doubled to 1 pg/mI medium, there was a reduction in both the chemical or to its metabolites. Pregnant Syrian golden transformed colonies and the average number of colonies hamsters at 11 days of gestation were given i.p. injections per dish. Results obtained with sodium chromate indicate of the chemical in alcohol using 2.5 or 5 mg/100 g maternal that a transformation frequency in the same range was weight. The injected animals were fed water and standard observed with cadmium acetate but with less lethality, as laboratory chow, and the embryos were excised on Day 13 indicated by the higher CE. Nickel subsulfide (1 to 5 pg/mI of gestation, 48 to 60 hr after maternal injection. The medium) also showed a trend of increased number of preparation and culture of cells from these embryos and the transformations with increased concentration. The percent identification for transformation were as described above. age of transformations obtained with nickel subsulfide was the highest observed, regardless of the metal carcinogen RESULTS used. The average number of transformations ranged from 0.66 to 1 .75/dish or 1.5 to 11% on the basis of transformed The colonies formed by untreated HEC were either light colonies relative to total colonies scored. The absolute or dense as previously described and were made up of numbers of transformations per dish after beryllium sulfate various cell types reflecting the diversity of cells found in or sodium arsenatewere approximately 1 and 2/dish at 2.5 the embryo. Morphology of control colonies was constant and 5 pg/mI medium, respectively; but on a relative colony with regular cell arrangements and easily distinguished basis, in the experiments with 5 j.tg/ml, beryllium produced from those classified as having been transformed. BP a higher transformation frequency (6.4 versus 4.13) than treatment produced, in addition to colonies that appeared arsenic. similar to those in control, some colonies with a random or Four metal salts also produced transformation in the criss-cross orientation of cells. When 2.5 j.tg BP per ml of host-mediated in vivo-in vitro assay. When the salts of medium were used, the transformation frequency, on a nickel (sulfate), beryllium, chromate, and cadmium were colony basis, was 4 to 6% (data not shown). The relevance injected into pregnant Syrian hamsters, transformation was of the transformed phenotype as an indicator of neoplastic observed in cells cloned from the third subpassage. Beryl properties is that cells derived from these transformed hum, chromate, and cadmium were used at 2.5 mg/100 g colonies repeatedly have been shown to demonstrate the body weight, and nickel was used at 5 mg/100 g body ability to produce progressively growing tumors when in weight. Doubling the concentrations proved lethal to the oculated into appropriate hosts. Typical colonies scored as pregnant hamsters. transformed after direct treatment with metal carcinogen were identical to those obtained with other chemical carcin DISCUSSION ogens. These were classified as either light (Figs. 1, 3, and 4) or dense (Figs. 2, 5, and 6). Inorganic metals may cause cancer in animals and may Of the 12 metals studied, 6 produced transformation; 4 of have mutagenic properties in microbial systems. Some the 6 positives were also tested and were found to be metals may also be human carcinogens. Of the 6 metals positive in the transplacental assay. The nontransforming reported to be negative for transformation, only zinc salts

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Table 1 saltsPetriTransformation and cloning efficiency of Syrian hamster embryo cells treated with diverse inorganic metal (6x dishes (50 mm) were seeded with 300 cells from a secondary culture with an irradiated hamster feeder layer werescored1O@cells). Chemicals were added 24 hr later. Dishes were fixed and stained 9 days after seeding. Colonies blind by 2 observers.Av.Total

coloniesNo. Transformed no. of Transformed coloniesChemical of transformed colonies/ colonies/ CEa Total (%)NiSO4'6H20 pg/mI dishes colonies dish dish (%) 002.5 12 0 0 84.2 28.1 1.255.0 8 8 1.0 73.6 24.5 1.4410.0 8 12 1.5 61.5 20.1 5.45(CH3COO)2Cd 12 27 2.25 41.3 13.8 .H20 000.1 11 0 0 84.4 28.1 1.490.5 8 8 1.0 67 22.3 3.11.0 8 14 1.75 56.5 18.8 4.02Na2CrO4â€¢4H2O 8 9 1.12 28 9.3 000.1 12 0 0 84.7 28.2 0.70.5 12 7 0.58 83.4 27.8 2.101 12 13 1.75 83.4 27.8 3.48Ni3S@ .0 12 21 1 .8 51 .7 17.3

001.0 12 0 0 84.2 28.1 1.512.5 12 8 0.66 43.7 14.5 5.175.0 12 12 1.0 19.3 6.4 11.5BeSO4 12 21 1.75 15.2 5.1

002.5 10 0 0 78 26.0 1.855.0 12 11 0.92 49.2 16.4 6.4Na2HAsO4 10 23 2.3 36.1 12

002.5 6 0 0 87 29.1 1.745.0 12 14 1.16 67 22.2 12 25 2.08 51 17.5 4.13

a Determined by dividing the average number of colonies per dish by the number of cells seeded per plate multiplied by 100. are reported to induce tumors. Tumors were obtained by Inhalation of nickel sulfide induced malignant lung tumors injection of zinc salts into the testes of fowl (2, 27) or in rats (32), and nickel subsulfide caused a carcinogenic hamsters (16) but not by s.c. or i.m. routes (43). Zinc does synergism when given with nickel oxide or carcinogenic not affect the fidelity of DNAsynthesisin vitro (35),but zinc polycyclic hydrocarbons (20, 26). Transformation of HEC by chloride or sulfate are weak enhancing agents for SA7 nickel subsulfide was found by Costa3 but not with amor transformation of HEC (5). phous nickel. No tumors occurred in matsafter amorphous A positive correlation exists between the capacity of nickel(44). metals to cause transformation in vitro and tumor formation Occupational exposure to cadmium may be relevant to in animals with all metals tested except arsenic. carcinoma of the prostate, kidney, and of respiratory cancer Although most human carcinogens produce some type of (21, 43) and provides an example of the apparent increase cancer in animals, arsenic is unique because attempts to of risk in men who smoke and work in high-risk occupations produce arsenical cancer have failed. Skin cancer in hu compared to men who do neither (21). Rats have developed mans results from arsenic in drugs, drinking water, and sarcomas after s.c. injection of the chloride, sulfate, sulfide, occupational environment (3, 30, 35, 46). Although the and dioxide of cadmium and with cadmium powder (43). incidence of deaths from lung cancer among metallurgical When soluble cadmium salts (sulfate and chloride) were workers incriminates arsenic (40), the role of sulfur dioxide injecteds.c.intomatsormice,interstitialtesticulartumors must be considered (24); the lung cancer mortality data associated with testicular atrophy occurred (19). However, reflect an increasing average dose of arsenic in terms of lung adenoma incidence did not increase in A/St mice time and concentration (28). treated i.p. (41) or in Swiss mice (38) when cadmium acetate Epidemiological evidence for the carcinogenicity of was administered in the drinking water. Dose-dependent nickel compounds is convincing. Cancer of the lung and necrotic events were caused by cadmium in confluent nasal cavity occurs in nickel refinery workers (19, 42). The human cultures (Wl-38) (17). Maintaining the culture in the increased incidence of respiratory tract cancer suggests log growth phase prevented such morphological changes. that metallic nickel, nickel subsulfide, nickel carbonyl, and The ability of cadmium salts to produce tumors systemically nickel oxide may be responsible (45). A variety of inorganic in animals and the presumptive data in humans are further nickels produced fibrosarcomas and/or rhabdomyosarco mas when injectedi.m.intomice or mats(19,42, 44). 3 Costa, personal communication.

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Downloaded from cancerres.aacrjournals.org on October 5, 2021. © 1979 American Association for Cancer Research. Transformation by Inorganic Metals reinformed by the transformation of HEC after either direct A (DNA repair) gene, neither was mutaganic (31). Similarly, application or i.p. injection of cadmium acetate into preg negative reports were obtained with E. coli phage T4 with nant hamster. chromate and lead (6). The latter produced neoplastic A high lung cancer risk is associated with occupational transformation of HEC(8). A number of metals were tested exposure to chromium compounds in chromate-producing for potential mutaganicity using recombinant-deficient plants (1) and in the chromate pigment industry but not in strains of B. subtilus rec (31). Arsenic, cadmium, and chromite ore mines (23). The evidence points toward an chromate were more inhibitory for reck cells. Among the exposure-cancer relationship for haxavalent chromium metal compounds that showed no mutagenicity were the compounds (18). Since haxavalent chromium can be ra chlorides of beryllium and nickel. Fidelity of DNA synthesis duced to the trivalent state, such compounds may also be decreased with the salts of most metal carcinogens, includ carcinogenic in humans. Both metallic chromium and its ing beryllium and nickel (39). With the Salmonella reversion hexavalent compounds produce sarcomas by a variety of assay, which depends on histidine revertants, iron (4) and routesin rats,mice,and rabbits(43).Carcinomasand chromate but not arsenic(25)were mutagenic,but iron adenocarcinomas in lungs of matsoccur after intrabronchial neither induced transformation nor affected the fidelity of implantation of pellets of calcium chromate (22) and in DNA synthesis (39). Sodium arsenita added to E. coli after mica after chronic inhalation of calcium chromate (29). UV radiation caused decreased survival attributable to the Calcium chromate putatively transformed BHK 21 calls, but inability to correct excision repair. The decreased mutation the possibility of spontaneous transformations could not be frequency by arsenite after UV in bacteria containing rec excluded (12). It is not surprising that transformation fol and lex@genes indicates inhibition of mRNA (34). In mam lowed sodium chromate treatment of HEC or in cells derived malian cells, however, alkaline sucrose gradient analysis from fetuses following transplacantal exposure. indicates that not all metals induce breakage of DNA (8). Until recently, epidemiological data on the incidence of Whereas nonlethal concentrations of arsenic and cadmium respiratory cancer among beryllium workers was consid caused breaks,berylliumornickelproduced no breaks ered equivocal. Questions existed: could the methodology even at concentrations that caused 100% cell death (5, 8). of analysis be responsible for the excess cancer incidence Therefore, it is probable that metals are very broad in their found in patients with berylliosis; and might the lung cancer activity and that the resulting carcinogenicity is due to a mortality among beryllium workers be explained by correct number of different mechanisms. ing for cigarette smoking (18)? Recent data demonstrated that a higher percentage of noncigarette smoking workers ACKNOWLEDGMENTS exposed to beryllium developed cancer than did members of the United States white population (49). Beryllium is the We thank R. L. Nelson for his excellent assistance during the course of this study. lightest element known to cause cancer. Inhalation of various beryllium salts caused cancer in rats (33, 36, 48). Injections of beryllium salts i.v. into rabbits and mice REFERENCES induced osteogenic sarcomas (14). 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Fig. 1. Portion of a dense, control colony (acetone, 0.02%) at 9 days. Contiguous fibroblasts exhibiting orderly growth pattern. Fig. 2. Light, transformed colony (sodium chromate, 5 @g/mlmedium), 9 days old. Long spindle-type cells have random growth pattern including overlapping cytoplasmic elements. Giemsa, x 63. Fig. 3. Portion of a light, transformed colony (beryllium sulfate, 1 j.tg/ml medium), 9 days old. Relatively large stellate cells are not oriented. Giemsa, x 63. Fig. 4. Portion of a light, transformed colony (nickel sulfate, 2.5 @.&g/mlmedium),9 days old. Cells are of short spindle type with random orientation. Giemsa, x 63. Fig. 5. Portion of a dense, transformed colony (sodium arsenate, 5 .tg/ml medium), 9 days old. Spindle cells are densely piled up towards the center and randomly oriented towards the edges. Giemsa, x 63. Fig. 6. Portion of a dense, transformed colony (cadmium acetate, 1.5 @.tg/mlmedium), 9 days old. Colony consists of small stellate cells that are not oriented. Giemsa, x 63.

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Transformation by Inorganic Metals

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MARCH 1979 1013

Downloaded from cancerres.aacrjournals.org on October 5, 2021. © 1979 American Association for Cancer Research. Quantitative Studies of in Vitro Morphological Transformation of Syrian Hamster Cells by Inorganic Metal Salts

J. A. DiPaolo and B. C. Casto

Cancer Res 1979;39:1008-1013.

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