[CANCER RESEARCH 40, 1463-1468, May 1980]0008-5472/80/0000-OOOOS02.00

Mutagenicity, Cytotoxicity, and DMA Binding of Platinum(ll)-chloroammines in Chinese Hamster Ovary Cells1

Neil P. Johnson,2 James D. Hoeschele, Ronald O. Rahn, J. Patrick O'Neill, and Abraham W. Hsie3

University of Tennessee-Oak Ridge Graduate School of Biomédical Sciences [N. P. J.], and Biology [R. O. R.. J. P. O., A. W. H.¡and Health and Safety Research[J. D. H.] Divisions, Oak Ridge National Laboratory. Oak Ridge, Tennessee 37830

ABSTRACT

The cytotoxicity and mutagenicity of the six plat-

inum(ll)chloroammines have been investigated in Chinese hamster ovary cells. For these compounds, the observed slopes ofthe mutation-induction curves (mutants/106 cells/juM) were:

c/s-Pt(NH3)2CI2 (31.5), K[Pt(NH3)CI3] (2.78), [Pt(NH3)3CI]CI(0.11), K2[PtCI4] (0.12), irans-Pt(NH3)2CI2 (0.013), and

[Pt(NH3)4]CI2 (0.0). The relative cytotoxicity of these compounds follows the same order and is of similar magnitude. Theobserved relative mutagenicities of these compounds paralleled their reported potencies in the Ames assay and theirrelative antitumor activities. Results indicate that Chinese hamster ovary cells are useful in quantifying low mutagenic activityof chemicals such as platinum compounds.

Studies with 195mPt-labeled c/s- and frans-Pt(NH3)2CI2

showed that during treatment both compounds enter the celland bind to the DMA with comparable efficiency. Hence, therelative mutagenicities of c/s- and frans-Pt(NH3)2CI2 are not a

consequence of different initial levels of DNA binding.

INTRODUCTION

c-Pt(NH3)2CI2" is an inorganic antitumor agent which is par

ticularly effective in combination chemotherapy against testic-

ular (5) and ovarian cancer (32). Its effectiveness has stimulated the search for other antitumor platinum compounds, ofwhich the 1,2-diamminocyclohexane platinum compounds (27)

appear promising.In addition to its antitumor activity, c-Pt(NH3)2CI2 is known to

enhance prophage induction (21 ), inactivate transforming DNA(17), inactivate viruses (26), induce filamentous growth in bacteria (24), selectively inhibit DNA synthesis (9), cause muta-

genesis (15, 16, 19, 30), produce chromosomal abnormalities(33), and kill cells (20). For these same biological effects, thefrans isomer is either completely inactive or less potent than isthe c/s form. It is generally thought that c- and t-Pt(NH3)2CI2

form different lesions with DNA which have different biologicalactivities (23).

The platinum(ll)chloroammines are square-planar transition

1 Research sponsored jointly by the Environmental Protection Agency (IAG-D5-E81) and the Office of Health and Environmental Research, United StatesDepartment of Energy, under Contract W-7405-eng-26 with the Union CarbideCorporation.

2 Supported by an American Cancer Society Postdoctoral Fellowship and

Carcinogenesis Training Grant CA 05296 from the National Cancer Institute.Present address: Laboratoire de Pharmacologie et de Toxicology Fondamentales. 205 Route de Narbonne, 31078 Toulouse Cedex, France.

3 To whom requests for reprints should be addressed.4 The abbreviations used are: c- and t-Pt(NH3)2CI2. c/s- and frans-dichloro-

diammineplatinum(ll): CHO:HGPRT system. Chinese hamster ovary:hypoxan-thine-guanine phosphoribosyltransferase; F12FCM5. F12 medium supplementedwith 5% extensively dialyzed fetal bovine serum.

Received October 17. 1979; accepted January 23, 1980

metal complexes which have diverse chemical properties. Thecharge on these complexes ranges from +2 {[Pt(NH3)4]'t2} to-2{[PtCI4]~:?}. The chloride, the most labile group (7), is re

placed by a nitrogen when these compounds bind to DNAbases (23); as a result, the number of reactive sites varies from4, for the tetrachloride, to 0, for the tetraamine.

Most of the platinum(ll)chloroammines have been tested fortheir mutagenic activity in the Ames assay (15) and for theirability to induce prophage (21); in these experiments, theplatinum compounds exhibited different levels of biologicaleffectiveness which paralleled their relative antitumor activityin mice (3). Consequently, it seemed likely that they mightshow a similar range of mutation induction in mammalian cells.We have extended a previous study of the mutagenicity andcytotoxicity of c-Pt(NH3)2CI2 (19) to include all 6 plat-

inum(ll)chloroammines in order to investigate the ability of theCHO:HGPRT system (18) to quantify mutagenicity of chemicalssuch as platinum compounds. In addition, we have investigatedthe correlation between mutation induction and DNA bindingfor c- and t-Pt(NH3)2CI2 by means of 195mPt-labeledcompounds.

MATERIALS AND METHODS

Platinum Compounds. Platinum(ll)chloroammines were synthesized according to published procedures (6, 13, 14, 25,29). For the cellular uptake and DNA binding studies, 195mpt(1

mCi/mg platinum; f, ,2 = 4.02 days), prepared by neutronbombardment of 194Ptin the High Flux Isotope Reactor at Oak

Ridge National Laboratory, was used as the starting material(10). Radioactive compounds were dissolved in 0.9% NaCIsolution and stored refrigerated in the dark for as long as 3days prior to treatment. Under these conditions, 97% of the c/sand 99% of the frans isomers are in the dichloro form (1). Theamount of platinum either present in cells or bound to isolatedDNA was determined from radioactivity measured with a Hewlett-Packard automatic gamma counter. Sample radioactivity

was compared with standards of known concentration in orderto determine the amount of platinum present. For the mutationassays, nonradioactive compounds were dissolved in sterileSaline G (8) immediately prior to treatment, and their concentrations were determined by absorbance (2). Saline G (8)contains (in g/liter) glucose (1.1), NaCI (8.0), KCI (0.4),Na2HPO4•¿�7H2O (0.29), KH2PO4 (0.1 5), MgSO4 •¿�7H2O (0.1 54),and CaCI2 (0.016), and it was adjusted to pH 7.5. We preparedconcentrations of [Pt(NH3)4]CI2 >1 mM by dissolving the compound directly in medium.

Mutagen Treatment and Selection for 6-Thioguanine Resistance. Mutagen treatment and determination of mutationinduction have been described in detail previously (18). Briefly,5 X 105 cells were plated in 25-sq cm Falcon flasks containing

5 ml of F12FCM5 and incubated overnight to allow cell growth

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N. P. Johnson et al.

to 106 cells/flask. No more than 200 ¡itiof the freshly dissolved

platinum compounds were then added for various periods oftime. For short treatment times, medium was removed, cellswere washed 3 times with Saline G (8), and new medium wasadded. The cultures were grown for 16 to 24 hr after additionof mutagen and were then dissociated from the flask by gentletrypsinization; the cell number was determined by means of aCoulter Model B counter. Approximately 106 cells were re-

plated in F12FCM5 for expression of mutant phenotype, and200 to 1000 cells (depending on the expected cell survival)were plated in triplicate for determination of the effect oftreatment with platinum compounds on the cellular cloningefficiency. Untreated control cultures exhibited cloning efficiencies of 60 to 100%. To obtain maximum stable phenotypicexpression, we subcultured the treated cells every 2 days andthen selected for 6-thioguanine resistance on the eighth or

tenth day after treatment (18, 19). For mutant selection, 2 x106 cells were plated in 100-mm dishes (5 plates = 106 cells)

in hypoxanthine-free F12FCM5 containing 10 ¡J.M6-thioguan

ine. For determination of cloning efficiency of the culture at thetime of mutant selection, 200 cells were plated in triplicate inthe hypoxanthine-free F12FCM5 without thioguanine. After

incubation for 7 days in selective medium, mutant colonieswere fixed, stained, and counted. Mutation frequency, expressed as mutants/ 106 clonable cells, was calculated by

division of the total number of mutant colonies by the numberof cells plated (1 06), which were corrected for cloning efficiency

of the culture at the time of mutant selection. For these experiments, the spontaneous mutation frequency of untreated con

trols ranged from less than 1 to 15, with an average of 3.3mutants/106 cells.

We calculated the slopes of the concentration-dependent

mutation induction curves and used the f test to determine thestatistical significance of nonzero slopes. As a correction forspontaneous mutation, 15 mutants/106 cells were subtracted

from each data point. For calculation of the i parameter, theslope was divided by its S.E., and confidence levels weredetermined from statistical tables (31). The variability of themutation induction within an experiment was as large as thatfor the variability between experiments (Chart 1); therefore, thenumber of degrees of freedom was calculated as the numberof data points minus 2.

Determination of the Uptake of Platinum Compounds intoCells and Their Binding to DNA. For these studies, 20 to 40x 106 cells were treated with 195mPt-labeled compounds for

various time intervals. The treated cells were then washed 3times with 5 ml Saline G prior to trypsinization, and the cellnumber and radioactivity of this sample were measured todetermine the uptake of the platinum compound into the cell.Cells were then lysed in 1.5 ml of 0.1 % sodium dodecyl sulfatein 0.1 M Tris (pH 8) containing 1 rnw EDTA and were incubatedat least 2 hr at 50°with proteinase K (200 fig/ml; EM Biochem-

icals). This incubated mixture was made 0.5 M in NaOH,incubated for an additional 2 hr at 50°, and neutralized. For

DNA extraction, the aqueous phase was washed repeatedlywith distilled phenol saturated with 0.1 M Tris (pH 7.5), followedby chlorofornrisoamyl alcohol (24:1). After the interphase haddisappeared, the aqueous phase was chilled on ice, 70%

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Chart 1. Effects on percentage of survival (•*. •¿�,A) and mutation induction (D, O, O, A) of 16- to 24-hr treatment with the indicated concentrations of c-PKNH3)2CI2 (a), K[Pt(NH3)CI3] (b), [Pt(NH3)3CI]CI (c), MPtCU) (<J),t-Pt(NH3)2CI2 (e), and [PKNH3)<CI2] <M. Different symbols refer to data from separate experiments.The spontaneous mutation frequency ranged from fewer than 1 to 15 mutants/10" clonable cells and was not subtracted from the data. The slopes of straight line fits

of the mutation-induction data from the linear regression analysis are given in Table 1.

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Mutagenesis of Pt(ll) Compounds

perchloric acid was added to make a 7% solution, and theprecipitated DNA was centrifuged at 1000 rpm for 15 min in anInternational centrifuge. The pellet was then washed with cold7% perchloric acid and resuspended in 0.5 M NaOH. Theconcentration of DNA in this solution was determined spectro-

photometrically with an extinction coefficient corresponding tothe denatured DNA [emax= 8645 (mol/liter cm)"1]. Radioactiv

ity of the same sample was measured to determine the platinumconcentration, and the results are reported as platinum molecules per DNA nucleotide. Control experiments with knownamounts of radioactive platinum bound to calf thymus DNA(Sigma Chemical Co., St. Louis, Mo.) showed that the platinum:DNA product was stable during the isolation procedures.

RESULTS

Three platinum(ll)chloroammines exhibited mutagenic activity in the CHO:HGPRT system; among them, c-Pt(NH3)2CI2 was

the most potent (Chart 1a). Treatment with a 3 /IM concentrationof this compound increased the number of mutants to about140/106 cells; there was a shoulder in the survival curve.

K[Pt(NH3)CI3] (Chart 1b) was less mutagenic than was c-

Pt(NH3)2CI2. Treatment with 50 ¡MK[Pt(NH3)CI3] increased thenumber of mutants to 130/106 cells and decreased the survival

exponentially. [Pt(NH3)3CI]CI was less mutagenic than wasK[Pt(NH3)CI3] (Chart 1c). With increasing concentration of[Pt(NH3)3CI]CI to 360 /¿M,the number of mutants increased toa maximum value of about 70/106 cells, and the cell survival

decreased exponentially. Based on the slope of the linearportion of the mutation induction curve, the approximaterelative mutagenic activity of c-Pt(NH3)2CI2:K[Pt(NH3)CI3]:

[Pt(NH3)3CI]CI is 100:8:0.3.The mutagenic potential of K2[PtCI4] (Chart 1d) and t-

Pt(NH3)2CI2 (Chart 1e) was less clear. For both compounds, thenumber of mutants increased with treatment concentration, butthe maximum number of induced mutants was not much greaterthan was the maximum number of spontaneous mutants. Eachcompound exhibited a shoulder in its survival curve. The max-

260-

imum concentration of t-Pt(NH3)2CI2 tested (34 /IM) was determined by the solubility of the compound (see "Materials andMethods") rather than by its toxicity.

[Pt(NH3)4CI2] did not appear to be mutagenic (Chart 1f).Treatment with concentrations of this compound up to 6600fiM induced fewer mutants than did the highest spontaneousmutation frequency; the cell survival decreased to about 30%over this concentration range.

The mutagenicity and cytotoxicity of c-Pt(NH3)2CI2 were investigated in more detail. Examination of results of 7 experiments (Chart 2) showed that the experimental uncertaintyincreased with mutation frequency. Consequently, slopes ofthe concentration-response curves were calculated by

weighted linear regression (31). Within experimental uncertainty, the mutagenicity increased with increasing treatmentconcentration for all of the platinum(ll)chloroammines (Chart1). We noted that for c-Pt(NH3)2CI2 (Chart 2), in addition to the

linear fit, the quadratic fit in the concentration response wasalso significant at a >99% confidence level. The quadratic andstraight-line fits of the data in Chart 2 differ by fewer than 20mutants/106 cells from 0 to 2.5 /IM c-Pt(NH3)2CI2. The S.E. of

the curve was 3.47 for the linear fit and 3.24 for the quadraticfit. Hence, the straight-line fit of the concentration-dependentmutagenicity of c-Pt(NH3)2CI2 can be used for purposes ofcomparing the concentration-dependent mutation induction of

the platinum(ll)chloroammines.We have quantified the mutagenic potential of the

platinum(ll)chloroammines by 2 methods. First, we simply compared the maximum number of mutants induced by each compound. The relative mutagenicity was c-Pt(NH3)2CI2 >K[Pt(NH3)CI3] > [Pt(NH3)3CI]CI > K2[PtCI„],t-Pt(NH3)2CI2, and[Pt(NH3)4]CI2 (Table 1). One application of this method is todetermine whether or not a compound is mutagenic, a positiveresult being a significantly larger number of mutant coloniesthan the number observed for the untreated control. The spontaneous mutation induction for the present experiments rangedfrom 0 to 15 mutants/106 cells. If 30 mutants/106 cells, twice

the maximum spontaneous mutation frequency, are chosen asan arbitrary cutoff, K2[PtCI4], t-Pt(NH3)2CI2, and [Pt(NH3)4]CI2

are not mutagenic. The main weakness of this approach is thatit in no way considers the concentration of compound tested.

Second, we measured the slopes of the mutation-inductioncurves (Table 1); this method indicated the same relative mutagenicity as the first method except that K2[PtCI4] and

Table 1

Mutagenicity of the platinum(ll)chloroammines

Compoundc-Pt(NH3)2CI2K[Pt(NH3)CI3][Pt(NH,)3CI]CIK2[PtCU]t-Pt(NH3)2CI2[Pt(NH3)4]CI2Concen

trationrange(ilM)0-30-500-3600-650-340-6600Maxi

mumno.ofmu

tants/106cells314013070251217Slope6mutants/106cells//iM

concentration31.5±0.9C2.78

±0.070.11±0.0070.12

±0.020.013±0.007(2.5±2.3) X 10~3No.

ofdatapoints46717233521%of

confidencethatslope>0999999999550

Chart 2. Effects on mutation induction (M) of 16- to 24-hr treatment with c-Pt(NH3)2CI2as a function of concentration (C). Poisson-weighted data have beenfit by the best straight line, M = 39.9C + 1.33 (S.E. = 3.47). and the bestquadratic. M = 9.69C2 + 19.1C + 1.79 (S.E. = 3.24). The quadratic term is

significant at the 99% confidence level. Spontaneous mutation frequency wasnot subtracted.

* Chart 1 spontaneous mutation frequency not subtracted.

Least-squares straight line for data in Chart 1 by use of Poisson weights.Spontaneous mutation frequency (15 mutants/106 cells) has been subtracted

from the data, and mutation frequencies <15 have been set equal to 0.5 mutant/106cell.

c Mean ±S.E.

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N. P. Johnson et al.

[Pt(NH3)3CI]CI are indistinguishable. Using this second method,we consider a compound to be mutagenic if the slope of themutation induction as a function of concentration is significantlygreater than zero. Results reported in Table 1 indicate that, forall compounds except t-Pt(NH3)2CI2 and [Pt(NH3)4]CI2, there

was >99% probability that the slope of the mutation inductionas a function of concentration was significantly greater than 0,and there was <1 % probability that the observed positiveslopes arose from random fluctuations in the mutation induction. Since [Pt(NH3)4]CI2 cannot react with DMA (7, 23) andtherefore could be considered as a negative control, resultswith this compound show that this method can distinguish weakmutagens from a nonmutagen. The slopes of the mutation-induction curves were computed after 15 mutants/106 cells

were subtracted from the data to account for spontaneousmutants (see "Materials and Methods"). If the subtraction

is not performed, then all of the compounds, including[Pt(NH3)4]CI2, show a positive slope which is significant with>99% confidence according to the t test. We are developinga more rigorous method than subtraction, which will take intoaccount the spontaneous mutation induction and which wehope will define the sensitivity limits of our mutation assay.

The mutagenicity of t-Pt(NH3)2CI2 as measured by the slopeof the mutation-induction curve (Table 1) appears to lie on the

lower limit of mutagenic response, and the mutation inductionper /¿Mconcentration of c-Pt(NH3)2CI2 was at least 2400 timesgreater than that for t-Pt(NH3)3CI2.

The log percentage of survival per /ÃŒMconcentration on theexponential portion of the survival curve (Chart 1) is —¿�0.876± 0.079 for c-Pt(NH3)2CI2 and -0.005 ± 0.001 for t-

Pt(NH3)2CI2. Thus, the eis isomer is approximately 16 timesmore cytotoxic than is the trans isomer.

The numbers of c- and t-Pt(NH3)2CI2 molecules bound perDMA nucleotide after a 16-hr incubation with 195mPt-labeled

compounds are shown in Chart 3. The slopes of the DMAbinding curves are 2.8 x 10~5 and 8.7 x 10~6 [(platinum/DNA

nucleotide)/juM], respectively. Although both isomers bound toDNA during treatment, the level of binding was 3.2 times largerper UMconcentration for the c/'s than it was for the trans isomer.

After 4 x 107 cells were treated for 16 hr with 40 ml of 2

UM c-Pt(NH3)2CI2 inF12FCM5, 0.2% of the total concentration

had entered the cells, and 0.015% was bound to DNA. After

6-

4-

2-

the same treatment with 2 P.M t-Pt(NH3)2CI2, 0.04% of the

compound was in the cell, and 0.005% had bound to DNA.Hence, the larger DNA binding of c- than t-Pt(NH3)2CI2 was

primarily a consequence of greater uptake of the c/s isomerinto the cell.

The relative DNA binding of the 2 isomers as a function oftime was markedly different. c-Pt(NH3)2CI2 concentrations in

the cell increased during the experiment, and DNA binding ofthis compound was linear with treatment time (Chart 4). For t-

Pt(NH3)2CI2, however, the platinum per cell increased for about5 hr and thereafter decreased (Chart Ab). DNA binding of thiscompound as a function of treatment time showed a similarprofile, which suggests that the decreased platinum per cellwas responsible for the decreased platinum per nucleotide.Maximum cellular uptake and DNA binding occurred after thesame treatment time (~5 hr) regardless of concentration (Chart

4b).One possible explanation of the t-Pt(NH3)2CI2 data in Chart

4 is that no additional compound enters the cell after a few hr,and, therefore, that additional growth in these cultures dilutesthe overall platinum per cell. Chart 5 shows a graph of the totalplatinum in the washed cells as a function of time. After 5 hr,no additional t-Pt(NH3)2CI2 accumulated in the cells, while cellnumber increased. In contrast, c-Pt(NH3)2CI2 entered the cells

during the entire treatment.

1.5-

/¿M

Chart 3. Platinum molecules bound per nucleotide of CHO DNA immediatelyafter 16-hr treatment with c-Pt(NH3)2CI2 (O, A) to t-Pt(NH3)2CI2 (•,A). Differentsymbols refer to data from separate experiments.

Chart 4. Platinum molecules (mol) bound per CHO cell and per DNA nucleotide for various treatment times with c-Pt(NH3)2CI2 (a) (2 ^M; O and D are separateexperiments) and t-Pt(NH3)2Cl2 (D, 2 /ÃŒM;A, 5.5 /ÃŒM;O, 12 pu) (b). DNA bindingafter a 6-hr treatment with 12 /*M t-Pt(NH3)2CI2 was not measured. The values forO have been graphed at one-tenth value to allow use of the smaller scale for Dand A.

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Mutagenesis of Pt(ll) Compounds

"o6-

I«

10-

o"b

16TIME (hr)

24

Chart 5. Cell number (a) and total platinum (b) in all harvested cells for varioustreatment times with c-Pt(NH3)2CI2 (O, 2 JIM) and t-Pt(NH3)2CI2 (D. 2 JIM; A, 5.5UM; O, 12 JUM).The values for O have been graphed at one-tenth value to allowuse of the smaller scale for D and A.

DISCUSSION

A comparison of the mutagenicity and cytotoxicity per DMAlesion for c- and t-Pt(NH3)2CI2 shows that these 2 biological

effects differ in their sensitivity to DMA binding. The mutationinduction by the eis isomer as a function of concentration wasat least 2400 times that of the trans isomer (Table 1). However,the relative binding efficiency of the eis isomer was only 3.2times larger (Chart 3). Hence, the mutagenicity per moleculebound to DMA immediately after 16 to 24 hr of treatment is atleast 750 times larger for the cis than for the trans isomer. Onthe other hand, 50% killing occurs at 1.3 and 35 fiM for c- andt-Pt(NH3)2CI2 (Chart 1), which corresponds to 4 x 10~5 and 36x 10~5 platinum molecules per DMA nucleotide, respectively

(Chart 3, frans extrapolated). Hence, cytotoxicity per DMAlesion is a factor of 9 greater for the cis than for the fransisomer. This figure correlates well with the relative cytotoxicityof these compounds in HeLa cells (33). Furthermore, the cisisomer inhibits T7 DMA replication 5 times more effectively perbound molecule than does the frans isomer (11 ), thus indicatingthat cytotoxicity and inhibition of replication show similar relative sensitivities to these compounds.

The relative uptake of c- and t-Pt(NH3)2CI2 into CHO cells

can in part be understood in terms of their basic chemistry.Both compounds replace a chloride with water before reacting.Aquation is 4 times more rapid for the frans than for the cisisomer (1). However, aquated t-Pt(NH3)2CI2 is 20 times morereactive than is c-Pt(NH3)2CI2 (4) and should be preferentiallysequestered by side reactions with macromolecules in themedium. In this way, the greater reactivity of the frans isomerwith extracellular material may account for its lower uptake andsubsequent lower DMA binding as compared with the cis isomer.

The mutagenicity and cytotoxicity of c-Pt(NH3)2CI2 could be

curvilinear with treatment concentration (Charts 1a and 2). TheDNA binding, however, increased linearly with the administereddose (Chart 3). These data indicate that the mutagenicity andcytotoxicity per DNA lesion are slightly less for low amounts ofc-Pt(NH3)2CI2 bound to DNA than for higher levels of binding.

In Table 2, the mutagenicity per DNA lesion in the CHO:HGPRT system is compared for c- and t-Pt(NH3)2CI2, A/-methyl-A/-nitrosourea, A/-ethyl-/V-nitrosourea (28), and UV (22). Themutation induction per JUMconcentration of c-Pt(NH3)2CI2 is

much larger than that for the frans isomer or alkylating agents.However, alkylating agents produce smaller numbers of DNAlesions for a given concentration. The number of mutants perDNA lesion varies over 5 orders of magnitude; the alkylatingagents are the most potent, the platinum compounds are leasteffective, and the UV shows an intermediate effect. Thesenumbers most likely reflect different types of DNA lesions, notall of them mutagenic to the same extent, which may berepaired during expression.

The mutagenicity of the platinum(ll)chloroammines is compared with 3 other biological effects of these compounds inTable 3. A compound is considered to have antitumor activityif treated versus control <0.5 (3); by this criterion, c-

Pt(NH3)2CI2 and K[Pt(NH3)CI3] showed an effect. Prophageinduction is reported as the minimum concentration at whichlysis was observed (21). Reversion to histidine prototropy inSalmonella typhimurium is quantified by the linear part of theconcentration-response curve (15). The correlation amongthese 4 phenomena is very good. In all cases, c-Pt(NH3)2CI2 is

Table 2

Mutagenicity per DNA lesioninCompoundc-Pt(NH3)2CI2

t-Pt(NH3)2CI2N-Methyl-W-nitrosoureaN-Ethyl-N-nitrosourea6UV*Lesions/nu-

cleotide/fiMconcentration2.8

x 10~50.87 x 1CT55.7 x 10"e

0.38 X 1CT81.43 X 10sthe

CHO.HGPRTsystemMutants/

10e cells/

fiMconcentration31.5

0.0132.80.84

400 mutants/ 106

cellsMutants/le

sion89.4

x 10"1.3 x 10~64.1 x 10 21.8 x 10 '2.3 X 10'2

a Mutants/lesion in the HGPRT locus = [(mutants/cell//iM) + (lesions/nu-

cleotide/fiM)] + 1200 nucleotides/HGPRT locus/cell.b Ref. 28.c For treatment with 5 J/sq m (22).

Table 3

Relative mutagenic activities of the platinum(ll)chloroammines

Compoundc-Pt(NH3)2CI2K[Pt(NH3)CI3][Pt(NH3)3CI]CIMPtCU)t-Pt(NH3)2CI2[Pt(NH3)«]CI2Ames

assay (re

venants/nw)con

centration31006.04.52.0No

data0.2CHO/HGPRT

system0Induction

ofprophage+

(12. 5UM)+(50/JM)No

dataNodata-(100/lM)-dOOflM)Antitumoractivity0(T/C)

'1002.9eNo

data0.011.20.01Mutants/106

cells//IM

concentration31.52.780.110.120.0130.0025Maxi

mumno.ofmu

tants/106cells14013070251217

"Ref. 15.6 Minimum concentration at which lysis was observed (21).c T, = weight of Sarcoma 180 tumors from mice treated at the therapeutic

dose; C, weight of Sarcoma 180 tumors from untreated controls (3).Data taken from Table 1.

e[Pt(NH,)4IPt(NH3)CI2]2.

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N. P. Johnson et al.

most biologically active, followed by K[Pt(NH3)CI3] and then by[Pt(NH3)3CI]CI; [Pt(NH3)3]CI2 is inactive.

The CHO:HGPRT system appears to be useful to determine3 weak metal mutagens. The relative mutagenicity correlateswell with the effectiveness of these compounds in the Amesassay, their ability to induce prophage, and their potencies asantitumor agents. Both c- and t-Pt(NH3)2CI2 enter CHO cellsduring treatment and bind to the DMA to a similar extent, butthe eis isomer is considerably more mutagenic than is the transisomer. The subsequent expression and/or repair of theselesions must account for the different biological potencies ofthe 2 isomers.

ACKNOWLEDGMENTS

We thank D. G. Gosslee for statistical analysis.

REFERENCES

1. Aprile. F.. and Martin. D. S., Jr. Chlorotriammineplatinum(ll) ion acid hydrolysis and isotopie exchange of chloride ligand. Inorg. Chem.. ). 551-557.1962.

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