(CANCER RESEARCH 39, 193-198, January 1979]0008-5472/79/0039.0000$02.00

Enhancement of Viral Transformation for Evaluation of theCarcinogenic or Mutagenic Potential of Inorganic Metal Salts1

Bruce C. Casto,2 Judy Mey•rs,and Joseph A. DiPaolo

BioLabs, Inc., Northbrook, Illinois 60062 fB. C. C., J. M.J, and Biology Branch, Carcinogensls Program, National Cancer InstItute, Bethesda, Maryland20014(J. A. D.J

A different approach for the in vitro assay of potentialcarcinogens and mutagens in which the capacity of chemicals to enhance viral transformation is determined in SyrianHEC3 has been described by Casto (6, 7) and Casto et a!.(10—12).This system appeared useful since suspect carcinogenic hormones (33) and metal salts (13), that are detected poorly on not at all in microbial systems, are activewhen assayed for enhancement of viral transformation.

This communication presents the results of viral enhancement assays with 38 metal salts, showing that thosewhich are carcinogenic or mutagenic increase the frequency of adenovirus transformation.

MATERIALSAND METHODS

Cell Cultures. Primary HEC were prepared by trypsinization of eviscerated and decapitated embryos after 13 to 14days of gestation. Cells were resuspended in MDM (6)supplemented with 10% heat-inactivated FBS and NaHCO3(0.22 g/100 ml). Approximately 5 x 10e cells in 5 ml ofmedium were plated into 60-mm plastic Petri dishes andincubated in a 5% CO2 atmosphere at 37°.Total cell countsafter 3 days ranged from 3.7 to 4.5 x 10@cells/plate.

Continuous lines of monkey kidney cells, Vero and BSC1, were subcultured weekly in 100-mm plastic Petni dishes.Growth medium consisted of Eagle's minimum essentialmedium with 10% heat-inactivated FBS.

Virus. SimianadenovinusSA7was inoculatedinto Veroor BSC-1 cell cultures in 100-mm dishes at an input multiplicity of 2 to 3 plaque-forming units/cell. After the virushad been adsorbed for 2 hr, 5 ml of medium were added toeach plate. Cytopathic effects were usually complete by 72hr, after which the infected cells were harvested and thenfrozen-thawed 4 times, and the virus was separated fromcell debris by low-speed centnifugation. Virus stocks fortransformation assays were stored in 1- or 2-mi aliquots at—65°.

Transformation Assays. Adenovirus transformation procedunes have been presented elsewhere (4, 6). Briefly, theprocedure was as follows. SA7 was added to HEC (3 to 4 x10@plaque-forming units/culture) and adsorbed for 3 hr;the virus-inoculated cells were removed with trypsin (0.25%trypsin in MDM with 0.1 mM CaCI2), centrifuged, and resuspended to 10@cells/mi in MDM with 10% FBS and NaHCO3(0.11 g/100 ml). The cells were vigorously mixed and platedinto 60-mm dishes using 2 x 10@cells/dish; 3 ml of theabove medium were then added to each plate. After incubation for 3 days, the medium was changed to MDM with

3 The abbreviations used are: HEC, hamster embryo cells; MOM, modified

Dulbecco's medium; FBS, fetal bovine serum; TF, transformation frequency.

ABSTRACT

Thirty-eight metal salts were tested for their capacity toenhance transformation of Syrian hamster embryo cells bya simian adenovirus, SA7. All of the metal salts with knowncarcinogenic potential in animals on mutagenic activity inmicrobial on mammalian cells increased the SA7 transfonmation frequency. Metals were classified into three groupsaccording to the concentration necessary to produce significant enhancement. Those showing highest activity (positive at less than 0.05 mM) were the salts of antimony,arsenic, cadmium, chromium, and platinum. The secondgroup (positive from 0.05 to 0.6 mM) included beryllium,cobalt, copper, lead, manganese, mercury, nickel, silver,thallium, and zinc. Iron salts were placed in a third group(only positive at concentrations greater than 0.9 mM). Withthe exception of ZnCI@and ZnSO4, enhancement was demonstrated by both a relative increase in the viral transfonmation frequency and an absolute increase in the numberof transformed foci among treated cells. The latter observation and the demonstration of enhancement in the absence of overt cell killing negate the possibility that enhancement resulted from the selection of transformationsensitive cells.

INTRODUCTION

The development and validation of short-term tests forthe detection of carcinogens and mutagens are urgentlyneeded. These tests should produce no false-negatives,generate few false-positives, be responsive to the variousgroups of chemical and physical agents, and be quantitativeso that reliable risk assessments can be made when datafrom the various test systems are evaluated . Several in vitrosystems are currently under consideration for use in predicting the carcinogenic or mutagenic activity of environmental chemicals, including mutagenesis assays in microbial and mammalian cells, transformation of mammaliancells in vitro , and analysis of DNA damage and repair. Nobioassay can be used alone to categorize all classes ofsuspect carcinogenic or mutagenic agents. However, witha number of selected chemicals of known activity, microbial(31) on mammalian cell (26) mutagenesis, mammalian celltransformation (9, 17, 37, 38), DNA repair (44, 48), or DNAfragmentation (9, 51) assays have been reported to detectapproximately 90% of the chemical carcinogens tested.

ITheworkuponwhichthispublicationIsbasedwasperformedpursuantto Contract NCI-NO1-CP-45615 wIth the National Cancer Institute, Department of Health, Education and welfare.

2 To whom requests for reprints should be addressed, at Blolabs, Inc.,

2910 MacArthur Boulevard, Northbrook, III. 60062.Received July 14, 1978; accepted October 10, 1978.

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Enhancementof virus transformationofEC by metalsaltsCorn

undP0SA7

transformationInfidelityof DNAsynthe

SjSCCarcino

genic/muta

genic@'Enhance-(mM) Concen

ment ratio°tration@@AICI30.9

4.100AI@(SO4)@1.20.600Sb(C2H@O2)33.30.003AsCI32.2

0.003+BaCI@0.94.800BeSO42.60.56++Cd(C2H@O2)@2.20.001++CdCI@2.10.002++CaCI@0.86.800CaCrO42.00.006++Co(C2H@O2)@7.20.2++CoMoO46.10.25++Cu2S16.2

0.38+CuSO42.20.08+FeCI@2.72.500+FeSO42.20.900+PbO3.80.05++PbCrO43.20.04++LiCI0.523.60Mg(C@H@O2)@0.94.700MnCI@2.20.13++HgCl@5.60.05+NiSO43.40.38++PtCI41.80.03+K@CrO42.40.01++AgNO32.70.06++NaAsO22.40.10+Na@SO41.27.000NaOH0.82.500SrCI20.93.800Tl(C@H@O2)2.20.1TICI2.40.1WSbO42.20.08WCI61.10.5TiO20.812.50ZnCI@8.70.180+ZnCrO44.1

0.01+ZnSO41.80.2+

B. C. Casto et a!.

0.1 mM CaCI (4, 21), 10% FBS, and NaHCO3(0.22 g/100 ml).After 6 days, transformation assay plates were overlaid with3 ml of the above medium containing Bacto-agar (0.3 g/100ml). At intervals of 4, 5, and 6 days, 3 ml of additional agarmedium were added. Final focus counts were made 25 to30 days from the beginning of the experiment.

Survival Assays. After resuspension of the HEC to 10@cells/mI (see “TransformationAssays―),the cells were diluted 1:300 in medium to give 333 cells/0.1 ml. Two-tenthsml (666 cells) was then added to each of 5 plastic dishes,followed by 3 ml of MDM with 10% FBS and NaHCO3 (0.11g/100 ml). Five to 6 days later, 3 ml of medium with doublethe amount of NaHCO3were added to each plate, and after8 to 9 days of total incubation the cell colonies were fixed in10% buffered fonmalin and stained with 0.02% crystal violet.The cloning efficiency of virus-inoculated cells under theseconditions was usually 8 to 12%.

Chemical Treatment. Fresh stock solutions of metal saltswere prepared for each experiment by dissolving the saltsin acetone: water (1:1). Appropriate dilutions were thenmade in the complete medium to give the desired finalconcentration. In each experiment, 2 plates of HEC weretreated with metal salts for 18 hr prior to inoculation , on 5 to10 plates were treated at 5 hr after virus inoculation. Afterpretreatment, HEC were rinsed with complete medium andinoculated with SA7. Transformation and clonal assayswere then performed with each treatment group as descnibed above. When treated after virus inoculation, themetal salts remained in the medium for 48 hr.

Determinationof Enhancement.The fraction of cellssurviving treatment was determined from assay plateswhich were seeded with 666 cells/plate. The number ofcolonies in 5 plates seeded with treated cells was dividedby the number of colonies in 5 plates with control cells togive the surviving fraction of treated cells.

The total number of SA7 foci in 5 control plates, eachreceiving 200,000 cells, was used as the frequency oftransformation per 106 virus-inoculated control cells. Thefrequency of transformation of treated cells (number of SA7foci per 10@surviving cells) was calculated by multiplyingthe number of SA7 foci from 5 plates by the reciprocal ofthe surviving fraction of treated cells. Detailed methods fordetermining the frequency of transformation and enhancement ratios using data from actual enhancement expeniments have been presented (11, 12). Similar methods forcalculation have been used for the determination of mutation frequency (14) and for the demonstration of enhance@ientof SV4O transformation by DNA base analogs (52) in

mammalian cells.Enhancement was expressed as the ratio between the TF

of treated, surviving cells and the TF of control cells.Statistical significance was determined using a table ofratios (10) derived from the Lorenz table (29), which isbased upon the Fkisson distribution. The increased TF wasconsidered statistically significant at the 5 or 1% confidencelevel if the enhancement ratio exceeded the appropriatevalue obtained from the Lonenz table.

RESULTS

The enhancement of viral transformation with 38 compounds (salts of 25 different metals), the known cancino

genicity-mutagenicity of these salts, and their reportedactivity in the infidelity of DNA synthesis assay are summanized in Table 1 (46, 47). The metal salts showing highestactivity (Group 1), based upon the concentration requiredto significantly enhance viral transformation, were those ofantimony, arsenic, cadmium, platinum, and chromium(<0.05 mM). A second group of metal salts, including thoseof beryllium, cobalt, copper, lead, manganese, mercury,nickel, silver, thallium, and zinc, increased the viral TFwhen HEC were treated for 18 hr with 0.05 to 0.6 mM. Thethird group, consisting of ferrous chloride on sulfate, hadthe lowest activity, since concentrations of 0.9 mM ongreatenwere requiredforenhancement.

Of the metal salts causing enhancement, only those ofzinc yielded inconclusive results. With ZnCl.@and ZnSO4,enhancement was observed in only 3 of 6 and 3 of 7 trials,

Table1

a Determined by dividing the TF of treated cells by that obtainedfrom control cells. Numbers in italics are statistically significant atp <0.01.

b Lowest concentration tested that was positive for enhancement (italicized values) or the highest concentration that failed toenhance.

C Sirover and Loeb (46, 47).

d Determined from Refs. 22, 47, 49, and 50.

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Enhancementof viraltransformati‘onby zincsaltsConcentra

SurvivingEnhanceChemicaltion

(mM)afrac tion@'SA7foci@'ment ra

tio'ZnCI@0.34

0.170.080.0400.01

0.031.021.091.000

10394941NA@

8.70.91.2

1.0ZnSO40.20

0.100.050.02500.44

0.730.600.781.0050

534361601.8

1.21.21.3

1.0ZnCrO40.02

0.010.0050.002500.33

0.731.130.901.0085

1106863376.9

4.11.61.91.0

ChemicalConcentration(mM)aSurviving

fractionbSA7

focicEnhancement ratio@FeCl@5.0

2.51.20.600.56

0.620.630.671.0061

513634303.7

2.71.9e1.7

1.0CuSO40.64

0.320.160.0800.30

0.430.660.911.0023

558756282.7

4.54.62.2

1.0K@CrO40.025

0.0120.0060.00300.20

0.810.891.181.0058

756656319.5

3.02.41.51.0

Enhancement of Viral Transformation by Metals

Table 2 Table 3Enhancementof viral transformationby salts of iron, copper, and

chromium

a Chemical dilutions were added to mass cultures of HEC 18 hrbeforeor 5 hr after SA7.Virus was absorbedfor 3 hr, and the cellswere transferred for survival (500to 700 cells/dish) and for transformation assays (200,000 to 300,000 cells/dish).

b Determined from plates receiving 500 to 700 cells. The numberof colonies from virus- and chemical-treated cells was divided bythe number of colonies from virus-inoculated control cells to givethe surviving fraction. Cloning efficiency of control cells was from10to 15%.

C Number of foci from 10 plated cells.

d Determined by dividing the TF of treated cells (TF = SA7 focix reciprocal of the surviving fraction) by that obtained from controlcells. Numbersin italics, arestatisticallysignificant at the 1% level.

e NA, not applicable.

respectively. In each instance, the enhancement was basedonly upon a relative increase in TF when the proportion ofcells surviving treatment was used for the enhancementcalculation. When zinc was combined with an active metalsuch as chromium (ZnCrO4), both a relative and an absoluteincrease in foci occurred (Table 2); these increases werequantitatively greater than those observed with CaCrO4 orK@CrO4.With the exception of zinc and iron salts, all metalsalts found positive for enhancement in Table 1 were alsopositive in the infidelity of DNA synthesis assays (47).

The type of response observed after treatment of HECwith FeCI2is shown in Table 3; similar toxicity and enhancement patterns were seen after treatment with FeSO4. Incontrast to the concentration required and/on the degree ofenhancement resulting from treatment with iron salts, exposune of HEC to metal salts of the first or second group(active at less than 0.05 mM or at 0.05 to 0.6 mM, nespectively) usually resulted in a greater increase in absolutenumbers of foci, and enhancement was generally observedover a relatively wide dose range. Representative enhancement patterns observed with the latter 2 groups are shownin Table 3 using CuSO4 and K@CrO4 as examples.

Salts of metals such as antimony, platinum, and thallium,that have not been extensively tested in animals on in vitro,also increased the viral TF (Table 4). Antimony acetate wasparticularly effective, causing an increase in transformationat 0.003 mM when added 5 hr after virus inoculation.However, when tested as WSbO4, 0.08 mM was the minimum concentration required for enhancement. Platinumchloride (Table 4) as well as the chemothenapeutic agent,cis-dichlonodiammineplatinum , enhanced transformation

a Chemical dilutions were added to mass cultures of HEC 18 hrbeforeor 5 hr after SA7.Viruswas absorbedfor 3 hr, and the cellswere transferred for survival (500to 700 cells/dish) and for transformation assays (200,000 to 300,000 cells/dish).

b Determined from plates receiving 500 to 700 cells. The numberof colonies from virus- and chemical-treated cells was divided bythe number of colonies from virus-inoculated control cells to givethe surviving fraction. Cloning efficiency of control cells was fromlOto 15%.

C Number of foci from 10 plated cells.

d Enhancement ratio was determined by dividing the TF oftreated cells (TF = SA7foci x reciprocal of the surviving fraction)by that obtained from control cells. Numbers in italics are statistically significant at the 1%level.

e Significant at the 5% level.

when HEC were treated for 18 hr prior to virus addition. A2.4-fold increase in TF and an absolute 2-fold increase inthe number of SA7 foci resulted from treatment with lessthan 0.001 mM cis-PtCL@(NH@, whereas 0.03 mM PtCI4wasrequired to increase the TF and SA7 foci by 1.8-fold.

Ten compounds were negative for enhancement, including the chloride or sulfate salts of aluminum, barium,calcium, lithium, magnesium, sodium, and strontium; titanium dioxide was also negative when tested at up to 12.5mM(Table5).

DISCUSSION

Treatment of primary HEC with different classes of chemical carcinogens and mutagens leads to the enhancementof transformation by viruses, notably by the simian adenovirus, SA7, and many carcinogens and mutagens have beenshown to transform Syrian hamster cells when added alone(13).@The virus-transformed foci were easily distinguishedfrom chemically transformed foci since the cellular andclonal morphology was characteristic for 5A7 transfonmation (4, 5) and was unlike published descriptions of chemically transformed cells. Furthermore, formation of chemically transformed foci has not been observed with tech

4 J. A. DiPaolo and B. C. Casto. Quantitative studies of In vitro morpho

logic transformation of Syrian hamster cells by inorganic metal salts.Submitted for publication to Cancer Research.

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ChemicalConcentration (mM)@'Surviving

fraction@@SA7

fociCEnhancement ratio'@@Sb@H@O2)@0.015

0.00750.00320.001600.13

0.240.460.611.003

403934260.9

6.53.3

2.11.0PtCI40.12

0.060.030.01500.42

0.751.001.091.0040

465637313.1

1.91.81.1

1.0TI(C@H@O2)0.2

0.10.050.02500.49

0.840.991.171.0034

301515164.3

2.20.90.91.0

Failure to enhance viral transformation after treatment ofhamstercellswith saltsof aluminum, strontium, andtitaniumConcentra-

Surviving EnhanceChemical frac- SA7 foci@' ment ra

tion (mM)° tion@@tio@AL@(SO4)@

0.6 0.74 181.20.3

0.83 161.00.151.02 160.80.071.04 150.801.00 191.0SrCI2

4.0 1.25 270.92.01.07 381.41.01.03 250.90.51.12 301.101.00 241.0Ti02

12.5 1.03 190.86.250.87 130.73.121.00 281.31.560.80 201.201.00 22 1.0

B. C. Casto et a!.

Table5. Table 4

Enhancement of viral transformation by salts of antimony,platinum, and thallium

a Chemical dilutions were added to mass cultures of HEC 18 hrbeforeor 5 hr after SA7.Viruswas absorbedfor 3 hr, and the cellswere transferred for survival (500to 700cells/dish) and for transformation assays (200,000 to 300,000 cells/dish).

b Determined from plates receiving 500 to 700 cells. The numberof colonies from virus- and chemical-treatedcells was divided bythe number of colonies from virus-inoculatedcontrol cells to givethe surviving fraction. Cloning efficiency of control cells was fromlOto 15%.

C Number of foci from 10 plated cells.

d Enhancement ratio was determined by dividing the TF oftreated cells (TF = SA7foci x reciprocal of the surviving fraction)by that obtained from control cells. Numbersin italics are statistically significant at the 1% level.

niques used in the adenovinus enhancement assay (8). Inprevious publications (6, 7, 10—12,17), it was shown thatcarcinogenic chemicals increase not only the frequency ofadenovinus transformation but also the absolute number ofSA7 foci pendish.

Both the positive and negative metal salts from the viralenhancement assay were in agreement with their reportedmutagenic or carcinogenic activity and could also be conrelated with the infidelity of DNA synthesis assay reportedby Siroven and Loeb (47), in which perturbations in DNAsynthesized in vitro were introduced by an increase in basesubstitutions. The latter assay was used as a second basisfor comparison since their test procedures accurately predicted the cancinogenic-mutagenic potential of 29 of 31metal compounds and utilized many of the same metal saltsused in the viral enhancement assays. Further, one of thesuggested roles of chemical carcinogens or mutagens inenhancing viral transformation has been the substitution ofviral nucleic acids under the same conditions where basesubstitutions would be expected to occur (6, 12, 15).

Metal compounds that enhanced viral transformation butwere negative in the infidelity of DNA synthesis assay werethe chloride and sulfate salts of iron and zinc. FeCI2,whichcaused enhancement of viral transformation, was negativefor introducing copy error. Both FeCI2and FeSO4at 0.9 to5.0 mM enhanced SA7 transformation, causing a 2- to 3-foldincrease in the absolute number of SA7 foci pen dish.Although metallic iron has not been shown to be carcinogenic (27), iron dextnan will induce tumors in rats, mice,and hamsters (40). Also, ferrous chloride on fennic chloride

a Chemical dilutions were added to mass cultures of HEC 18 hrbeforeor 5 hr after SA7.Viruswas absorbedfor 3 hr, and the cellswere transferred for survival (500to 700cells/dish) and for transformation assays (200,000 to 300,000 cells/dish).

b Determined from plates receiving 500 to 700 cells. The numberof colonies from virus- and chemical-treated cells was divided bythe number of colonies from virus-inoculatedcontrol cells to givethe surviving fraction. Cloning efficiency of control cells was fromlOto 15%.

C Number of foci from 10' plated cells.

d Enhancement ratio was determined by dividing the TF oftreated cells (TF = SA7 foci x reciprocal of the surviving fraction)by that obtained from control cells. Numbers in italics are statistically significant at the 1% level.

and sulfate were earlier found to induce point mutations inEscherichia co!i (16).

Zinc salts have been reported to induce testicular tumorsin chickens (1, 19, 32) and embryonal tumors in hamsters(24). However, zinc chloride gave a negative response inthe infidelity of DNA synthesis assay and was weakly positive in the enhancement of viral transformation expeniments; zinc sulfate gave similar results. When zinc wastested in combination with chromium as ZnCrO4, a synergistic effect was demonstrated; enhancement was inducedat a lower dose, and the increase in the absolute number ofSA7 foci was greater than that observed with CaCnO4onK2CnO4.The oncogenicity of the chromates for mammaliancells (20) and the mutagenicity for yeast (2) on bacteria (54),as well as the reported carcinogenicity in animals (28, 41),agree with the viral enhancement assays.

Results with lead salts in various assay systems havebeen inconsistent. Muno and Goyen (35) found that 1% leadacetate, in the diet of mice, induced chromosome damagein leukocytes. Sirover and Loeb (47) reported lead chlorideto be positive in the fidelity of DNA synthesis assay, butHesslen (25) could not demonstrate mutagenic activity inthe algae P!atymonas subcordiformis ; viral enhancementassays in HEC were positive with lead acetate (weak), leadoxide (strong), and tetraethyl lead (weak) (B. Casto, unpublished observations). Lead acetate in the diet of rats hasbeen reported to cause tumors (3, 30, 53), but lead administered in the drinking water of mice (45) reportedly did notresult in tumor induction. Subsequently, transformation ofSyrian hamster cells by lead acetate was accomplished by

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Enhancement of Viral Transformation by Metals

10. Casto, B. C., Pleczynski, W. J., and DiPaolo, J. A. Enhancement ofAdenovirus Transformation by Pretreatment of Hamster Cells with Carcinogenic Polycyclic Hydrocarbons. Cancer Res., 33: 819-824, 1978.

11. Casto, B. C., Pieczynski, W. J., and DiPaolo, J. A. Enhancement ofAdenovirus Transformation by Treatment of Hamster Embryo Cells withDiverse Chemical Carcinogens. Cancer Res., 34: 72-78, 1974.

12. Casto, B. C., Pieczynski, W. J., Janosko, N., and DlPaolo, J. A. Significance of Treatment Interval and DNA Repair in the Enhancement of ViralTransformation by Chemical Carcinogens and Mutagens. Chem.-BioI.Interactions, 13: 105—125,1976.

13. Casto, B.C., Pieczynski,W.J., Nelson, A. L., and DlPaoIo,J.A.ln VitroTransformation and Enhancement of Viral Transformation with Metals.Proc.Am.Assoc.CancerRes.,17:12,1976.

14. Chu, E. H. Y. Induction and Analysis of Gene Mutations in MammalianCells in Culture. In: A. Hollaender (ed), Chemical Mutagens, pp. 411-444. New York: Plenum Publishing Corp., 1971.

15. Cleaver, J. E. Repair of Damaged DNA in Human and Other EukaryoticCells. In: 0. W. Ribbons, J. F. Woessner, and J. Schultz (ads.), NucleicAcid-Protein Interactions—Nucleic Acid Synthesis in Viral Infections, pp.87-112.Amsterdam:NorthHollandPublishingCo., 1971.

16. Demerec, M., Bertani, G., and Flint, H. A Survey of Chemicals forMutagenicActionon E. coli. Am.Naturalist,85: 119-136,1951.

17. DiPaolo, J. A., and Casto, B. C. Chemical Carcinogenesis. In: R. C.Gallo(ad.),RecentAdvancesin CancerResearch,pp. 17-47.Cleveland:CRCPress,Inc., 1977.

18. DiPaolo, J. A., Nelson, R. L., and Casto, B. C. In Vitro NeoplasticTransformation of Syrian Hamster Cells by Lead Acetate and Its Relevance to Environmental Carcinogenesis. Brit. J. Cancer, 38: 452-455,1978.

19. Falin, L. I. Experimental Teratoma Testis in Fowl. Am. J. Cancer, 38:199-211, 1940.

20. Fradkin, A., Janoft, A., Lane, B. P., and Kuschner, M. In Vitro Transformation of BHK21 Cells Grown in the Presence of Calcium Chromate.CancerRes.,35: 1058-1063,1975.

21. Freeman, A. E., Black, P. H., Wolford, R., and Huebner, A. J. AdenovirusType 12-Rat Embryo Transformation System.J. Virol., 1: 362-367, 1967.

22. Furst, A., and Haro, A. T. A Survey of Metal Carcinogenesis. Progr.Exptl.TumorRes.,12:102-133,1969.

23. Gunn, 5. A., Gould, T. C., and Anderson, W. A. Cadmium-InducedInterstitial Cell Tumors in Rats and Mice and Their Prevention by Zinc. J.Natl.CancerInst.,31: 745—759,1963.

24. Guthrle, J., and Guthrie, 0. A. Embryonal Carcinomas in Syrian Hamsters after Intratesticular Inoculation of Zinc Chloride during SeasonalTesticular Growth. Cancer Res., 34: 2612-2614, 1974.

25. Hessler, A. The Effects of Lead on Algae. II. Mutagenesis Experimentson Platymonas subcordiformis (Chlorophyta: Valvocales). MutationRes.,31:43-.47,1975.

26. Hsie, A. W., Couch, D. B., San Sebastian, J. A., Sun, W. N. C., Riddle, J.C., Brimer, P. A., Machanoff, R., Forber, N. L., Kenney, F. T., and Hsie,M. H. Mutagenicity of Carcinogens as Determined in a QuantitativeMammalian Cell Mutation System, CHO/HGPRT: Study of 80 Agents.Proc.Am.Assoc.CancerRes.,19:93, 1978.

27. Hueper, w. C., and Payne, w. W. Experimental Studies in MetalCarcinogenesis. Chromium, Nickel, Iron, Arsenic. Arch. Environ. Health,5: 445—462, 1962.

28. Laskin, S., Kuschner, M., and Drew, R. J. Studies in Pulmonary Cardnogenesis. In: M. G. Hanna, P. Nettesheim, and J. A. Gilbert (ads.),Inhalation Carcinogenesis, pp. 321-352. U. 5. Atomic Energy Commission, 1970.

29. Lorenz, A. J. Zur Statistik des Plaque-Testes. Arch. Ges. Virusforsch.,12: 108—137,1962.

30. Mao, P., and Molnar, J. J. Fine Structure of Lead Induced Renal Tumors.Am. J. Pathol., 48: 9a, 1966.

31. McCann, J., ChOi, E., Yamasaki, E., and Ames, B. N. Detection ofCarcinogensas Mutagensin the Salmonella/MicrosomeTest:Assayof300Chemicals.Proc.NatI.Acad.Sci.U.S.,72:5135-5139,1975.

32. Michalowsky, I. Die Experimentelle Erzeugung Elner Tera Toiden Neubildung der Hoden Bein Hahn Mitteilung. Zentr. AlIgem. Pathol. Pathol.Anat., 38: 385—387,1926.

33. Milo, G. E., Schaller, J. P., and Yohn, D. S. Hormonal Modification ofAdenovirus Transformation of Hamster Cells in Vitro. Cancer Res., 32:2338—2347,1972.

34. Monti-Bragadin, C., Tomaro, M., and Banfi, E. Mutagenic Activity ofPlatinum and Ruthenium Complexes. Chem.-BioI. Interactions, 11: 469-472.1975.

35. Muro, L. A. , and Goyer, R. A. Chromosome Damage in ExperimentalLead Poisoning. Arch. Pathol., 87: 660-663, 1969.

36. Nishioka, H. Mutagenic Activities of Metal Compounds in Bacteria.MutationRes.,31: 185-189,1975.

37. Pienta, R. J., Poiley, J. A., Lebherz, w. B., Ill. Morphological Transformation of Early Passage Golden Syrian Hamster Embryo Cells Derivedfrom Cryopreserved Primary Cultures as a Reliable in Vitro Bioassay forIdentifying Diverse Carcinogens. Intern. J. Cancer, 19: 642-655, 1977.

38. Purchase, I. F. H., Longstaff, E., Ashby, J., Styles, J. A., Anderson, D.,

DiPaolo et a!. (18). Cells derived from the transformedcolonies produced fibrosarcomas when inoculated intonude mice.

The enhancement of viral transformation by cadmiumsalts is consistent with their reported cancinogenicity inanimals (23, 39, 42) and the demonstration of chromatidaberrations in Chinese hamster cells (43).

There is little experimental evidence in animals for thecancinogenicity of salts of arsenic, antimony, platinum, orthallium, although arsenic salts have been implicated inhuman cancer (22), are mutagenic in bacteria (36), andtransform Syrian HEC in vitro (13). The enhancement ofviral transformation by platinum compounds agrees withthe data showing mutagenicity of platinum in certain strainsof Sa!mone!la (34) when tested as cis-d ichlonodiammineplatinum.

Much of the enhancement data with metal compoundsalso agrees with data obtained using Rec assays withBacillus subtilis (36). In these studies of 56 metal salts,those of arsenic, cadmium, chromium, mercury, manganese, and molybdenum were considered positive. Saltsof 3 of the strongly positive metals, arsenic, chromium, andmolybdenum, were also mutagenic in E. coli (36). The Recassays, however, failed to detect beryllium, copper, iron,lead, nickel, antimony, on zinc salts.

There are several other reports on the carcinogenic andmutagenic activity of the metal salts shown to be positive inthe viral enhancement assay. These investigations havebeen reviewed by Sunderman (49, 50) and Furst and Hano(22) on were cited by Sirover and Loeb (47).

Because of the use of metals in various industrial applications and the increasing evidence that many are involvedin human cancinogenesis, sensitive and reliable assays forpotential mutagenic on carcinogenic activity are necessary.The positive correlation between results with the viral enhancement assay in HEC and the mutagenic or carcinogenic activity of the metal salts in other systems justifies theuse of the SA7 transformation assay as one of the tests tobe included in screening for potential environmental mutagenic on oncogenic agents.

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1979;39:193-198. Cancer Res   Bruce C. Casto, Judy Meyers and Joseph A. DiPaolo  Carcinogenic or Mutagenic Potential of Inorganic Metal SaltsEnhancement of Viral Transformation for Evaluation of the

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