(CANCER RESEARCH 48, 5136-5144, September 15, 1988]

Intracellular Biotransformation of Platinum Compounds with the1,2-Diaminocyclohexane Carrier Ligand in the LI 210 Cell Line1

Stanley K. Mauldin,2 Gregory Gibbons, Steven D. Wyrick, and Stephen G. Chancy3

Dépannent of Biochemistry and Nutrition, School of Medicine [S. K. M., G. G., S. G. C,], and Division of Medicinal Chemistry, School of Pharmacy fS. D. WJ,University of North Carolina, Chapel Hill, North Carolina 27599

ABSTRACT

We have previously reported the development of a two-column high

performance liquid chromatography system for separation of platinum! 11)complexes with the 1,2-diaminocyclohexane (DACH) carrier ligand

(Mauldin et al., Anal. Biochem., 157: 129, 1986). Here we report theapplication of this technique to the study of the intracellular biotransformations of (DL)-rrani-l,2-diaminocyclohexanedichloroplatinum(II)|l'i( l.(/ranv-l)A( Il)| and (DL)-frans-l,2-diaminocyclohexanemalonato-platinum(II) |l»i(mal)(min.Y-I>A(ïl)| in the 1,1210 cell line. The two-

column high performance liquid chromatography system allowed separation and identification of both parent drugs and intracellular biotransformation products containing glutathione, methionine, cysteine, arginine,lysine, aspartate or glutamate, and serine or threonine. With the exception of the platinum-glutathione complex, the relative abundance of each

biotransformation product was independent of drug concentration. Therelative abundance of the platinum-glutathione biotransformation product

increased with increasing platinum concentration, suggesting that platinum drugs cause an increase in intracellular glutathione levels in a dose-

dependent manner. This hypothesis was verified by direct measurementof intracellular glutathione levels. In continuous uptake experiments, theintracellular levels of the parent compounds peaked between 2 and 5 hand declined to negligible levels by 24 h. In pulse-chase experiments, thechemical t., for I>K I,(rrunv-1)A( 11) and !'(( mal)(m/n.v-I >A( ÕI) inside thecell at 37°Cwas determined to be 12-15 and 21-28 min, respectively.

This is far shorter than previously determined rates for the displacementof either ligand in vitro. The platinum-amino acid complexes accumulatedgradually throughout the 24-h incubation. The free frans-DACH carrier

ligand also accumulated to a level approaching 20% of filterable countsduring the 24-h incubation, probably due to rraiu-labilization of thecarrier ligand by sulfur-containing nucleophiles. A combination of reverse

phase high performance liquid chromatography and a DNA binding assaywas used to identify and quantitate the reactive biotransformation products. As expected from previous studies (Mauldin et al.. Cancer Res., 46:2876, 1986), the PtCl2(rraiu-DACH)-treated cells had approximately 3

times more reactive platinum biotransformation product at early times,but the levels of reactive biotransformation product fell much morerapidly than in l'i(m:il)(rran.v-l)A<ÕIHrcated cells. In the l't( b(rran.v-

I>AO IHri-aleil cells, the major reactive biotransformation product wasthe aquachloro species at all time points tested. In l'i(mal)(rrun.v-l)A( 11)-

treated cells, however, one or more additional reactive biotransformationproducts were found to accumulate at later times. We believe that thesetechniques are applicable to detailed biotransformation studies of a widerange of platinum compounds.

INTRODUCTION

Platinum(II) and platinum(IV) complexes have come intowidespread use as anticancer agents in recent years (1,2). Basedon in vitro studies it is clear that most platinum drugs must beactivated intracellularly before reacting with DNA, and the

Received 5/20/87; revised 11/3/87, 3/4/88, 5/17/88; accepted 5/26/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This research was supported in part by USPHS Grant CA 34082 awarded bythe National Cancer Institute. S. K. M. was supported by a predoctoral traineeship(National Research Service Award 5T32 ES-07126) from the National Instituteof Environmental Health Sciences.

2 Present address: Molecular and Cell Biology Program, Pennsylvania StateUniversity, State College, PA 16802.

1To whom requests for reprints should be addressed.

reactive species are thought to be the aquated derivatives of thedrug (3, 4). Yet in spite of widespread use of these anticanceragents, very little is known about their biotransformationsinside the cell. Scanlon et al. (5) have tentatively identified m-[Pt(H2O)(Cl)(NH3)2]+4 and c/s-[Pt(H2O)2(NH3)2]2+ as intracel

lular biotransformation products of the LI210 cell line bystrong cation exchange HPLC. More recently, Andrews et al.(6) have reported detection of unchanged cisplatin and peakswhich comigrated with the cisplatin-methionine complex andthe various aquated species in a human carcinoma cell line byion pair-reverse phase HPLC (7). Unfortunately, previous separation systems have suffered from limited resolution and aninadequate number of platinum standards. We have recentlydescribed a two-column HPLC separation system for platinumcompounds and have characterized the retention times of 18potential biotransformation products (8). In this paper we report the use of this method for the characterization and quan-titation of the major intracellular biotransformation productsof two second generation platinum drugs in the LI 210 cell line,with special emphasis placed on the quantitation and kineticsof formation of the platinum-amino acid complexes andaquated species.

We have chosen to study platinum drugs with the DACHcarrier ligand because this class of platinum compounds hasshown considerable potential as second generation anticanceragents (9) and because it is possible to prepare ^I- labeled drugwith high specific activity (10), providing the sensitivity neededfor these intracellular studies. PtCl2(DACH) was chosen because it is the prototype drug of this class and because it is alikely biotransformation product of tetraplatin, a compoundcurrently recommended for preclinical and phase I/II clinicaltrials (11, 12). Pt(mal)(DACH) was chosen because of unresolved questions concerning the activation of platinum drugswith bidentate leaving ligands (13,14). The effects of these twocompounds on LI210 cells in culture (15) and the stability andtransformation products of Pt(mal)(fra/«-DACH) in tissue culture medium (16) have been reported elsewhere. The differentisomers of diaminocyclohexane have slightly different effectiveness and toxicity in the various tumor cell screens (17,18). Themixed trans-DL isomers of the 1,2-diaminocyclohexane carrierligand were used throughout this study.

MATERIALS AND METHODS

PtCl2(frans-DACH) and Pt(mal)(fra/w-DACH) were prepared withhigh specific activity, nonexchangeable tritium in the 4,5 positions of

4The abbreviations and trivial names used are: cisplatin or cis-PtCl2(NH3)2,n'í-diamminedichloroplatinum(II); [c/.s-Pt(H2O)(Cl)(NH3)2]*, c/j-diamminea-quachloroplatinum(II); |c/.v IM(il..<>b(Nlli)j|-'. m-diamminediaquaplatinum(II);

HPLC, high performance liquid chromatography; TLC, thin layer chromatography; DACH, 1,2-diaminocyclohexane; irons-DACH, (DL)-trans- 1,2-diaminocyclohexane; PtCl2(íraní-DACH),(DL)-rrani-1,2-diaminocyclohexanedichloro-platinum(II); tetraplatin, (i>i ) iran.v 1,2-diaminocyclohexanetetrachloro-platinum(IV); Pt(mal)(franí-DACH), (DL)-frani-l,2-diaminocyclohexanemalon-atoplatinum(II); [Pt(H2O)(Cl)(rrans-DACH)]*, (DL)-irans-1,2-diaminocyclohex-aneaquachloroplatinum(II); [Pt(H2O)2(irans-DACH)]!*, (DL)-rrans-1,2-diamino-

cyclohexanediaquaplatinum(II). Platinum(II) complexes with various amino acidsare indicated as follows: Pt(amino acid)(irans-DACH); in most cases the exactstoichiometry or charge of the complex is not known.

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INTRACELLULAR BIOTRANSFORMATION OF PLATINUM COMPOUNDS

the 1,2-diaminocyclohexane moiety. All syntheses of the labeled andunlabeled platinum compounds as well as the starting material, (DI )fra/M-1,2-diaminocyclohexane, are described elsewhere (10). Since ourHPLC separation procedure does not resolve the CMand trims isomersof DACH we have confirmed the conformation of the radiolabeledDACH by nuclear magnetic resonance analysis of the A4-l,2-diamino-

cyclohexene intermediate and by thin layer Chromatographie separation(19) of the cis and trans-(oL) isomers of the 3H-labeled 1,2-diaminocy-clohexane. Two previous paper (8, 15) referred to the cw-DACHisomers of these platinum compounds, based on the procedure ofCraven (20) for the preparation of A4-cw-l,2-diaminocyclohexene.

However, in a recent réévaluationof this synthesis, we have found thatthe step involving reflux in hydrazine hydrate allows the conversion ofthe cis isomer of l,2-dicarbethoxycyclohex-4-ene to the trans isomer ofcyclohex-4-ene-l,2-dihydrazide. Stock solutions (200 Mg/ml) of [3H]-PtClj(rrans-DACH) and [3H]Pt(mal)(fra/w-DACH) were prepared in

0.15 M NaCl and water, respectively. Stock solutions were usuallyprepared the day before the experiment and were kept in the dark at4"C until used.

The LI210 cell line was obtained from Dr. Alan Eastman (EppleyInstitute for Cancer Research, Omaha, NE). Growth medium consistedof RPMI 1640 supplemented with 15% fetal bovine serum and penicillin/streptomycin. The cells were subcultured twice a week and weregrown in a humidified incubator with an atmosphere of 95% air-5%CO.. The original cell line was stored in liquid nitrogen. Fresh cellswere removed and cultured from the original stock every 3-6 months.

Isolation of Intracellular Platinum Biotransformation Products. Intra-cellular platinum biotransformation products were isolated from theultrafilterable fraction of LI 210 cells. LI 210 cells at 10" cells/ml (100ml/75-cm2 flask) were incubated with either [3H]PtCl2(fra/w-DACH) or[3H]Pt(mal)(frani-DACH) for various lengths of time at 37°C.The cells

were centrifuged and washed twice with 13 ml of cold phosphate-buffered saline, and the cell pellets stored at —80°Cuntil needed for

HPLC analysis. Control experiments have shown that the cell pelletscan be stored at —80°Cfor approximately 1 month without any detect

able change in the amounts or distribution of platinum biotransforma-tion products.

The ultrafilterable fractions for HPLC analysis were prepared byresuspending the cell pellets in 1 ml of water and sonicating the cellson ice. The final sonicate was then filtered at 4°Cthrough an Amicon

YMT membrane with a M, 30,000 cutoff (Amicon Corp., Danvers,MA) to remove protein-bound platinum. (Our control experimentshave shown that the low molecular weight platinum complexes do notbind to the Amicon YMT membrane.) The ultrafiltrates were collectedand immediately separated by the two-column HPLC system. Analiquot was counted before and after filtration in order to determinethe total and filterable quantities of intracellular platinum.

HPLC Separation. The two-column HPLC system used has beendescribed previously (8). The elution profiles were analyzed and plottedwith the Spectrodata software package (Spectrofuge Corp., Carrboro,NC). The individual biotransformation products were identified bycomparing their retention times on reverse phase and cation exchangeHPLC with the retention times of in v//ro-prepared standards (8).Quantitation was performed by integrating the area under the peaksand expressing the data as a percentage of the total filterable counts.In some cases the data were converted to pmol platinum/106 cells based

on the known specific activity of the two drugs.Pulse-Chase Experiments. For the pulse-chase experiments, 1,1210

cells at 10' cells/ml (100 ml/75-cm2 flask) were pulse-labeled with 6Mg/ml [3H]PtCI2((frai«-DACH) or 100 Mg/ml [3H]Pt(mal)(frani-DACH) for 1 and 2 h, respectively at 37°C.After pulse-labeling, thecells were cooled rapidly to 4"C, centrifuged, washed with 60 ml of cold

phosphate-buffered saline, and resuspended in the original volume (100ml) of prewarmed medium. The cells were further incubated at 37*C

for 15 min to 2 h (chase) and then centrifuged and washed twice with13 ml of cold phosphate-buffered saline. A zero time control wasprepared in the same fashion immediately after the 1- or 2-h pulselabel. The final cell pellets were frozen at -80°Cuntil needed for HPLC

analysis. The rate of disappearance of the PtCl2(frans-DACH) andPt(mal)(irarts-DACH) in the ultrafilterable fraction was measured using

only the ion pair-reverse phase HPLC column since no other intracellular biotransformation products appear to comigrate with either compound on that column. The peaks containing the parent drugs werequantitated, and the data at each time point were expressed as apercentage of the zero time control. For efflux measurements, an aliquot(0.5 ml) of medium was removed from the 0 time control and at eachchase time point just after the first centrifugaron. The initial rate ofdrug efflux was estimated from plots of radiolabeled diaminocycloh-exane-platinum released versus time.

Quantitation of Reactive Platinum Biotransformation Products. Quantitation of the reactive platinum species in the uitrafilterable fractionswas determined directly after filtration by a modification of the DNA-binding assay of Johnson et al. (3). Samples (400 M')were incubated at25°Cfor 60 min with 100 n\ of 1 mg/ml salmon sperm DNA. The final

salt concentration was adjusted to 5 mM NaOO4, pH 5.5. The sampleswere precipitated on ice for 15 min with 600 n\ of cold 10% TCA and100 n\ of calf thymus DNA (1 mg/ml) as carrier. DNA pellets werecollected by centrifugation for 20 min at 1200 x g, redissolved in 200n\ of 0.1 M NaOH, and reprecipitated with 200 M!of cold 10% TCA,followed by addition of 2 ml of cold 5% TCA. The precipitated DNAwas again collected by centrifugation and dissolved in 0.5 ml NCSTissue Solubilizer (Amersham, Arlington Heights, IL). Solubilizationof the DNA took approximately 1-2 h at 37'C or overnight at room

temperature. After complete solubilization, 5 ml of Neutralizer Cocktail(Research Products International, Elk Grove Village, IL) were addedand the samples were counted for 'II incorporation. Measurement of

background incorporation into DNA was performed as above exceptthat the samples were precipitated with 10% TCA immediately afteradding the salmon sperm DNA.

Under these assay conditions, the reaction of [Pt(H2O)(Cl)(fran.s-DACH)]+ and [Pt(H2O)2(mj/w-DACH)]2+ towards DNA was essentially

complete at 60 min, with final reactivities of 43.6 ±5.9% (mean ±SD,n = 10) and 73.8 ±4.4% (n = 8), respectively. The reason for incompletereaction with DNA is not known, but the values obtained were reproducible and were characteristic of the compounds tested. l't(m:il)(r/-un.s-DACH) and most platinum-amino acid complexes tested showed 2%or less reactivity towards DNA, except for the platinum-lysine andplatinum-arginine complexes with 10% reactivity. PtCl2(frans-DACH)had 5% reactivity towards DNA under these assay conditions. Forboth [Pt(H2O)(CI)(irani-DACH)]+ and [Pt(H2O)2(frfl/w-DACH)]2+, the

amount of platinum bound to DNA was directly proportional to theamount of platinum added to the assay (data not shown).

Recovery of the aquated platinum complexes from an LI 210 sonicateunder our assay conditions was determined as follows. An LI210sonicate prepared from 10s cells in 1 ml 11_>()was spiked with a knownamount of 3H-labeled [Pt(H2O)(Cl)(fraiw-DACH)]+ or [Pt(H2O)2(fra;w-DACH)]2*. The sonicates were immediately filtered through an Amicon

YMT membrane and diluted 1:25 in 5 mM NaClO4, pH 5.5, forthe DNA-binding assay. (We found significant inhibition of DNA-binding activity at dilutions of 1:10 or less, but no effect of dilutionon the percentage of DNA binding at dilutions of 1:25 or greater).Under these conditions, we obtained 50.2 ±6.7% (n = 8) recovery of [Pt(H2O)(Cl)(/rans-DACH)r and 9.4 ±0.8% (n = 3) recovery of [Pt(H2O)2(mj/i.s-DACH)2+. Clearly, quantitation of the[Pt(H2O)(CI)(frani-DACH)]* species is possible under our assay con

ditions with corrections for both recovery of the aquachloro species inthe cell sonicate and incomplete reaction with DNA. While the quantitation of the [Pt(H2O)2(frans-DACH)]2+ species would be difficult, the

data of Lim and Martin (21) for the ethylenediamine-platinum complexes indicate that the intracellular concentration of the (H2O)2 speciesshould comprise only 3-4% of the total aquated species present in thecell. Assuming comparable pKa values for the coordinated aqua ligands,the intracellular concentrations of [Pt(H2O)2(fra/w-DACH)]2+ should

be similarly low.Identification of the reactive biotransformation products was based

on a reactivity profile determined by measuring the DNA-bindingactivity of each peak fraction obtained from the reverse phase HPLCseparation step described previously. Fig. 1, A and B, shows the elutionand reactivity profiles of [Pt(H2O)(CI)(fra/w-DACH)]+ in water (8).When [Pt(H2O)(Cl)(rra/tj-DACH)]+ was added to a cell sonicate and

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INTRACELLULAR BIOTRANSFORMATION OF PLATINUM COMPOUNDS

Io

oLU_Lum

402060300,6030O—

l 1—_A.b/I

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RETENTION TIME (min)e l g

PEAKFig. 1. Distribution of DNA-binding activity following reverse phase Ill'l ( .

Platinum complexes were separated by reverse phase I ll'l < as described previously (8) i.l. C, and / ). The peak fractions from each 1ll'l ( run were pooledand assayed without dilution for DNA-binding activity as described in the text.Since the retention time of these peak fractions varies slightly from column tocolumn, each column was calibrated with in vitro prepared standards (8) and theidentification of individual peaks was based on the retention times of thosestandards on the same column [PtCI2(frani-DACH) = peak b; [Pt(H2O)(Cl)(fra/u-DACH)]* = peak f, Pt(methionine)(franj-DACH) = peak g; and [Pt(H2O)2(irani-DACH)]2* = peak g). The observed DNA-binding activity was corrected for

inhibition of the reaction by HPLC buffer (IS to 35% depending on the composition of buffer in the fraction assayed). Only fractions e through g had appreciableDNA-binding activity and their reactivity profile is shown in B, D, and /•'.(I and

B) [Pt(H2OXClXfra/ts-DACH)r prepared by incubating PtCl2(frani-DACH) inwater (1). Peak b = PtCI2(froni-DACH), peak f = [Pt(H2O)(Cl)(fra/u-DACH)r,and peak g = |Pt(H2O)2(rranj-DACH)l:*. Recovery of [Pt(H2O)(Cl)(frans-DACH)1* from the reverse phase column was 85.4 ±0.8% (n = 4); (C and D)[l'iliI.())(( 'I) (rmm 1>A( 11)| after addition to an I 1210 sonicate and nitrationthrough an Amicon YMT membrane filler (see text); (/, and /•')10' 1.1210 cellsin 100 ml of RPMI 1640 were incubated for 5 h with 12 wg/ml of 3H-labeledPtCI2(/ra/ts-DACH). The filterable fraction was prepared for injection onto HPLCas described in "Materials and Methods."

processed as described above, one sees the expected formation ofother platinum biotransformation products (Fig. 1C). However, thereactivity profile is essentially unchanged (Fig. ID), suggesting that[Pt(H2O)(Cl)(íraní-DACH)]*is still the only reactive biotransformation

product present. Fig. 1, E and F, shows the profiles obtained from cellslabeled with PtCl2(fran.s-DACH) in culture. Again the reactivity profileis similar, but not identical, to that obtained with |l'i(H..())(C'l)(ira;i.v-DACH)]+. [Pt(H2O)(Cl)(fra/M-DACH)]+ is the only reactive biotrans-

formation product we have identified which élûteswith peak f.PtCI2(irans-DACH) élûteswith peak b, [Pt(H2O)2(fra;w-DACH)]2*élûteswith peak g, and the platinum-arginine and platinum-lysinecomplexes elute with peak h. Thus, these data allow tentative identification of [Pt(H2O)(Cl)(iro/ii-DACH)]* as the major reactive biotrans

formation product present in that sample.

RESULTS

Quantitation of Intracellular Biotransformation Products. Theinitial goal of these experiments was to test the applicability ofour two-column HPLC procedure (8) to the quantitation ofintracellular biotransformation products in cultured L1210 cellsincubated with equitoxic doses of PtChi/raws-DACH) orPt(mal)(fra/Js-DACH). Since the biotransformation productswhich accumulated in the first few hours were of the greatestinterest, the drug concentrations were based on the 3-h 50%inhibitory doses determined previously (15). Drug levels 5-10times the 50% inhibitory doses were generally used to provideadequate sensitivity. Initially, the cells were incubated with eachdrug for 12 h at 37°C.The HPLC elution profiles are shown

for PtCl2(fra/tt-DACH) (Fig. 2) and Pt(mal)(ira«s-DACH) (Fig.

10 20 30 4O 5O 6O

RETENTION TIME (min)

Fig. 2. HPLC separation of the intracellular biotransformation products ofPtCI2(rra/u-DACH). L1210 cells (10') in 100 ml of RPMI 1640 were incubatedfor 12 h with 12 „g/mlof 3H-labeled PtCl2(rranj-DACH) (1.31 Ci/mmol). The

filterable fraction was prepared and analyzed on reverse phase HPLC as describedin "Materials and Methods." Peak fractions from the reverse phase column werecollected and concentrated 10-fold before separation by cation exchange HPLCat pH 4 (8). The elution profiles were plotted as described in "Materials andMethods." ( I ) reverse phase elution profile; (B) cation exchange elution profile

of peak b; (O cation exchange elution profile of peak e; (D) cation exchangeelution profile of peak f; (/ ) cation exchange elution profile of peak g; (F) cationexchange elution profile of peak h.

10 20 30 40 50 O 10 20

RETENTION TIME (min)

30 40 50 60

Fig. 3. HPLC separation of the intracellular biotransformation products ofPt(mal)(frani-DACH). This experiment was carried out as described in Fig. 2except that 100 >ig/ml of 'H-labeled Pt(mal)(/rani-DACH) (0.15 Ci/mmol) was

used. B, cation exchange elution profile of peak c.

3). The reverse phase elution profiles showed 5-6 peaks ofintracellular biotransformation products for each drug (Fig. 2Aand 3/1). Each major peak from the reverse phase column waspooled, concentrated 10-fold, and separated by strong cationexchange HPLC at both pH 2.3 (data not shown) and pH 4(Fig. 2, B-F, and 3, B-F). Obviously, the two-column HPLCsystem allowed the resolution of a large number of intracellularbiotransformation products. The patterns obtained were extremely reproducible and were, for the most part, qualitativelysimilar for both drugs. The only significant differences in theelution profiles were the presence of Pt(mal)(frans-DACH)(peak c) in the Pt(mal)(frfl/is-DACH)-treated culture and thepresence of an unknown biotransformation product (peak h9)in the PtCl2(fra«s-DACH)-treated culture.

The identification of the major biotransformation products5138

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INTRACELLULAR BIOTRANSFORMATION OF PLATINUM COMPOUNDS

was carried out by comparing their retention times under threeHPLC conditions (reverse phase, cation exchange at pH 4, andcation exchange at pH 2.3) with those of a series of 18 standardsprepared in vitro (8) (Table 1). Six species could be identifiedand quantitated unambiguously. The lysine and arginine complexes were readily identified, but quantitation was somewhatmore difficult because they were not as well resolved from otherbiotransformation products on cation exchange HPLC (Figs.2F and 3F). The identification of the aspartate/glutamate andthe serine/threonine complexes was somewhat more tentativebecause [Pt(H2O)(Cl)(frans-DACH)]+ and several minor plati-num-amino acid complexes would be expected to make smallcontributions to the peaks containing those compounds (8).The biotransformation products identified in this manner represent 64 and 77% of the ultrafilterable platinum in cells treatedwith PtCl2(fra/is-DACH) and Pt(mal)(fra«s-DACH), respectively. With the exception of peak h9, most of the remainingbiotransformation products each represented 2% or less of theultrafilterable platinum. The aquated platinum species were notquantitated by this method because they were not adequatelyresolved from the platinum-amino acid complexes (8).

Reproducibility and Concentration Dependence. To test forreproducibility, two separate cultures of L1210 cells were incubated with each drug for 12 h and the major intracellularbiotransformation products were quantitated as a percentage ofthe total filterable counts (Table 2). With a few exceptions, thequantitation of individual biotransformation products was reproducible considering the very small amount of material. Therange was only ±10-20% for most samples. In comparing thetwo drugs some interesting differences were observed for levelsof the peak containing the serine and threonine complexeswhich was significantly higher in cells treated with PtC\2(trans-DACH) and for the glutathione complex which was significantly higher in cells treated with Pt(mal)(ira/«-DACH).

The effect of different drug concentrations on the relativelevels of the major biotransformation products was also testedfor both drugs (Table 3). With the exception of the glutathionecomplex, the intracellular biotransformation products showedessentially the same proportions over a wide range of drugconcentrations. The differences observed were well within the±20%margin of error seen in Table 2. The 2.5-fold increase inthe relative abundance of the glutathione complex at higher

concentrations of PtCl2(fra/ii-DACH) suggested that incubation of LI210 cells with platinum might actually cause anincrease in glutathione levels as part of the protective responseof the cell. Therefore, total glutathione levels (reduced andoxidized) were measured in the 1,1210 cells after a 24-h exposure to various concentrations of unlabeled PtCl2(fra/ii-DACH)(Table 4). There was a 50% increase above basal glutathionelevels at 6 ¿tg/mlPtCl2(/rans-DACH) and a further 2-foldincrease between 6 and 50 Mg/ml. This correlated well withthe increase seen in the relative abundance of thePt(glutathione)(/ra/w-DACH) complex.

Time Course for Accumulation of Intracellular Biotransformation Products. Having established the reproducibility of theassay and the validity of using high drug concentrations tomaximize sensitivity, we examined the kinetics of formationof the intracellular biotransformation products of bothPtCl2(fr<z/is-DACH) (Fig. 4) and Pt(mal)(fra/is-DACH) (Fig. 5)at 6 and 100 Mg/ml, respectively. The kinetics of total andfilterable platinum uptake (Figs. 4A and 5A) has been describedpreviously (15). The plateauing phenomenon observed for theuptake of PtCl2(frans-DACH) is primarily due to a more rapidconversion of PtCl2(fra/is-DACH) to inactive transformationproducts in RPMI 1640 (15). In cells treated with either drug,the major intracellular complex product observed at early timeswas unchanged drug (Fig. 4B and 5B). Due to the slower uptake of the malonato species (15), intracellular levels ofPt(mal)(fra/is-DACH) accumulated and decayed somewhatmore slowly than PtCl2(fra/w-DACH). The Pt(mal)(fra/w-DACH)-treated cells also accumulated significant levels ofPtCl2(ira«i-DACH) (Fig. SB), presumably due to the displacement of malonate by chloride in the extracellular medium andthe rapid uptake of the resulting dichloro complex (15). Thetime course for the formation of the other biotransformationproducts was very similar for the two drugs. Those biotransformation products which did accumulate in LI210 cells appearedto be primarily dead-end products. With the exception of thelysine and arginine complexes, the amino acid complexes whichaccumulated have negligible reactivity toward DNA (16).

Intracellular Half-Lives of the Parent Drugs. Time courseexperiments such as the ones described above tell us little aboutthe relative displacement rate of the chloro and malonatoligands in the cell. Thus, we measured the rate of disappearance

Table 1 Identification of the major intracellular biotransformation products ofPtCI2 (trans-DACH) and Pt(mal)(trans-DACH) in Li 210 cells'

Peaks fromHPLCClear

identificationPeakbPeak

cPeakelPeake2Peakg4Peakg5Peakg6Peak

hSTentative

identificationPeakOPeak

f4Peak

h6Peakh7Retention

time onreverse phase*(min)10(8-12)15(13-17)27

(24-27)27(24-27)33(31-34)33(31-34)33(31-34)35(35-40)29

(28-30)29

(28-30)35

(35-40)35(35-40)Fractional

retention nine''

on cationexchangepH40.110.140.110.190.420.670.760.670.240.310.780.88pH2.30.130.200.840.650.890.650.290.840.89IdentificationPtCb

(fra/ii-DACH)Pt(mal)(/ranj-DACH)Pt(cysteine)(rra«i-DACH)Pt(cysteine)(»ra/ts-DACH)Pt(glutathionc)(rranj-DACH)Free

/rani-DACHPt(methionine)(fra/u-DACH)Overlap

of peakg5Pt(aspartate)(ira/u-DACH)

andPt(glutamate)(frani-DACH)Pt(serine)(ira/tf-DACH)

andPt(threonine)(rra/ts-DACH)Pt(lysine)(frani-DACH)Pt(arginineX<rans-DACH)

" Identification of the peaks in Figs. 2 and 3 was based on comparison of retention times on both the reverse phase and cation exchange HPLC columns to the

retention times of 18 standards on the same columns. In the case of the cation exchange column, the identification was based on separations at both pH 4 and pH2.3. The preparation of standards and the HPLC separations have been described previously (8).

* The retention times of the peak fraction are indicated on the left. The fractions actually pooled for further analysis on cation exchange are shown in parentheses.' Retention time of the unknown peak divided by the retention time of the |I'KH:(»:(rrunv I)A('II)|-'' standard on the same column (8).

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INTRACELLULAR BIOTRANSFORMATION OF PLATINUM COMPOUNDS

Table 2 Quanlitation of the major biotransformation products ofPtCh (trans-DACH) and Pt(mal)(trans-DACHf

% of total Filterable platinumat 12 h*

Platinum biotransformationproductsPtCW/ranj-DACH)Pt(mal)(/ranj-DACH)

Free trans-DACHPt(serine/threonineXfrani-DACH)Pt(methionine)(»ra/«-DACH)Pt(glutathione)(>ra/u-DACH)Pt(aspartate/glutamate)('ran5-DACH)Pt(cysteine)(ironi-DACH)Pl(arginine)(rrans-DACH)Pt(lysine)(frani-DACH)PtClj

(trans-DACH)5.4,

5.415.0,

17.017.6, 14.9

5.3, 3.96.8, 2.85.5,5.1

(7.4)(2.8)(1.6)Pt(mal)(frani-

DACH)10.2,

8.89.2, 8.5

15.5, 16.48.1,8.86.4, 6.3

14.5, 12.94.8, 3.97.5, 7.21.3,0.91.3, 1.5

•L1210cells were incubated for 12 h at 37'C with either 12 »ig/mlPtCl2(trans-DACH) or 100 »ig/mlPt(mal)(rranj-DACH). Intracellular biotransforma-tion products of PtCI2(fra/u-DACH) and Pt(mal)(rra;ts-DACH) were separatedby the two-column I Il'l.( ' procedure described in "Materials and Methods" (seeFigs. 2 and 3). Quantitation was done as described in "Materials and Methods."

All values were corrected for column recovery. The values reported represent twoseparate experiments (except where indicated in parentheses).

*Total filterable platinum at 12 h was 24.2 pmol/10* cells for PtCI2 (trans-DACH) at 12 >ig/ml and 60.8 pmol/106 cells for Pt(malXfrani-DACH) at 100

Mg/ml.

Table 3 Effect of platinum drug concentration on the amounts of the majorbiotransformation products of PtCliftrans-DACH) and Pt(mal)(trans-DACHf

Major biotransformation products at 12 h as a % oftotal filterable platinum*

Concentration of Mal- Serine/ Methi- Gluta- Freedrug (Mg/ml) C12 onate Threonine onine thione DACH

PtCl2(fra/u-DACH)6122050Pt(mal)(»ra/ii-DACH)201005.45.44.14.19.4

10.09.58.816.416.215.713.87.18.46.94.64.713.45.16.43.94.87.910.510.213.712.816.014.512.418.116.0

" 10* L1210 cells in 100 ml of RPMI 1640 were incubated for 12 h with either'H-labeled PtCI2(fra/ts-DACH) or Pt(mal)(fraits-DACH) at the concentrations

indicated. Separation and quantitation of the intracellular biotransformationproducts were carried out as described in "Materials and Methods." The valuesreported for PtCl2(fra/u-DACH) at 12 ^g/rnl and Pt(mal)(irans-DACH) at 100i.u mI represent the average of two separate experiments (Table 2).

* Total filterable platinum for cells treated with PtCl2(/ra/«-DACH) for 12 hrwas 9.7 pmol/10* cells at 6 «ig/ml,24.2 pmol/10' cells at 12 pg/ml (average oftwo experiments), 31 pumi nr cells at 20 fig/ml, and 79.3 pnml 10" cells at 50»ig/ml.For cells treated with Pt(mal)(rrani-DACH) the values were 8.9 pmol/10*cells at 20 wg/ml and 60.8 pmol/106 cells at 100 >ig/ml (average of two experi

ments).

Table 4 Effect of platinum drug concentration on intracellular glutathioneconcentrations"

Concentration ofPtCI2(rrans-DACH)

Oig/ml)06

122050Intracellular

glutathioneconcentrations (nmol/

mg protein)16.9

26.034.043.451.5

" I I : Hi cells (10") in 100 ml of RPMI 1640 were incubated for 12 h withI'll l.u/.im ])A( Hi at the concentrations indicated. Intracellular glutathione

levels were determined by the method of Tietz (30). Determinations were induplicate with a precision of ±5%or better. Control experiments demonstratedthat the platinum drug itself caused no enhancement of the assay.

of intracellular PtCl2(mz«s-DACH) and Pt(mal)(fra/is-DACH)after a short pulse label [l h for PtCl2(franj-DACH) and 2 hfor Pt(mal)(frans-DACH)], as described in "Materials andMethods." The pulse times and drug concentrations were cho

sen to give comparable intracellular levels of both parent drugsat the beginning of the chase period. The rate of disappearance

TIME (hrs)

Fig. 4. Time course for formation of intracellular biotransformation productsin LI210 cells treated with PtCl2(fra/u-DACH). L1210 cells (10»)in 100 ml ofRPMI 1640 were treated with 6 »ig/ml'H-labeled PtCl2(rranj-DACH) for the

times indicated. Preparation of the filterable fraction and separation by HPLCwas carried out as described in Fig. 2. Individual biotransformation products werequantitated as described in Table 2. Conversion to pmol/10* cells was based onthe total 'II counts in the filterable fraction and the specific activity of the

drug (1.31 Ci/mmol). (I) uptake of platinum into I 121(1cells; (B) formationof individual biotransformation products in the filterable fraction: •,PtCI2-(trans-DACH); •. free frans-DACH; A, Pt(cysteine) (fram-DACH); O,Pt(methionine)(/ranj-DACH); D, Pt(glutathione)(irofts-DACH).

Fig. 5. Time course for formation of intracellular biotransformation productsin LI210 cells treated with Pt(mal)(fraas-DACH). This experiment was carriedout as described in Fig. 4 except that 100 ng/ml of 3H-labeled Pt(mal)(fra/ts-DACH) (0.15 Ci/mmol) were used. O, Pt(mal)(frani-DACH). The rest of thesymbols are described in Fig. 4.

of either drug from the filterable fraction could be due to severalfactors: (a) binding to intracellular macromolecules; (h) bio-transformation to other low molecular weight compounds; and(c) movement of unchanged drug out of the cell (efflux). Whilewe were primarily interested in measuring the half-lives due tointracellular biotransformation only, the correction for effluxwas difficult to estimate since one may not be able to assumethat all of the platinum which diffuses out of the cell is in theform of the parent drug (6). By simply measuring the amountof the original platinum complex remaining at each time point,we obtained a minimum estimate of stability (since some of theparent drug was lost by efflux). By correcting these values forthe total amount of platinum retained by the cell at each timepoint, we obtained a maximum estimate of stability (since someintracellular biotransformation products may have diffused outof the cell). Fortunately, the rates of efflux were slow relative

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INTRACELLULAR BIOTRANSFORMATION OF PLATINUM COMPOUNDS

to the intracellular displacement reactions, so the maximumand minimum estimates of half-life were not significantly different. The half-lives of PtCl2(ira/ji-DACH) and Pt(mal)(fra/is-DACH) inside the cell were found to be in the range of 12-15and 21-28 min, respectively. These values are approximately6- to 10-fold faster than any rate of biotransformation observed

extracellularly (13, 14, 22).The approximate rates of efflux of radiolabeled platinum

from PtCl2(rra/is-DACH) and Pt(mal)(fra/rs-DACH)-treatedLI210 cells were also determined in the same experiments, asdescribed in "Materials and Methods." The estimated effluxrates were 0.1 pmol/min/106 cells for PtCl2(frans-DACH)-treated cells and 0.05 pmol/min/106 cells for Pt(mal)(trans-DACH)-treated cells.

Reactive Biotransformation Products in the Cell. Finally, wewished to determine the levels and identities of the reactivebiotransformation products in the cells. Our previous experiments had suggested that the relative abundance of reactivebiotransformation products must be 2-3 times greater forPtCl2(frans-DACH) than for Pt(mal)(fra//s-DACH) at earlytimes (15). The same study also suggested that the reactivebiotransformation products were more persistent in Pt(mal)-(frans-DACH)-treated cells. Thus, it was of particular interestto quantitate the reactive biotransformation products at varioustimes to see if the patterns obtained matched those predicted.Time course experiments were carried out as described previously (Figs. 4 and 5) and total levels of reactive biotransformation products in the cell were determined by the DNA-binding assay described in "Materials and Methods." The re

active species as a percentage of total filterable platinum areshown in Table 5 (column 1). As predicted, about 3 times moreof the filterable platinum from PtCl2(fra«s-DACH)-treated cellswas in the form of reactive biotransformation products at earlytimes. By 5 h the percentage of reactive platinum species inPtCl2(/rans-DACH)-treated cells had started to decrease rapidly, and the reactive species became 2-3 times more abundantin Pt(mal)(fra/is-DACH)-treated cells between 8 and 24 h. Thus,the relative abundance of reactive biotransformation productsas determined by the DNA-binding assay followed exactly thepattern predicted for both platinum drugs.

While the DNA binding assay allowed quantitation of totalreactive biotransformation products, it did not allow identifi

cation of those compounds. We used a combination of thereverse phase HPLC separation and the DNA-binding assayfor identification of reactive biotransformation products (see"Materials and Methods"). LI 2 10 cells were incubated with

PtCl2(franj-DACH) or Pt(mal)(fra/u-DACH) for various periods of time, and ultrafiltrates were prepared as describedpreviously. Following reverse phase separation of the filtrates,the peak fractions were pooled and assayed for reactivity by theDNA-binding assay. Peaks a, b, c, and d showed essentially noreactivity under the assay conditions used. The reactivity profiles for peaks e through h are shown in Figs. 6 [PtCl2(fra/js-DACH)] and 7 [Pt(maI)(fra/ii-DACH)]. By comparing thesereactivity profiles with that obtained for a [Pt(H2O)(Cl)(fra/w-DACH)]* standard in a L1210 sonicate (Fig. ID), it was

possible to estimate how much of the reactive platinum in thecell was actually the aquachloro complex. These estimates aresummarized in Table 5, column 2. The reactivity profiles andestimates of percentage of [Pt(H2O)(Cl)(/ra/is-DACH)]+ may

not be accurate at early times due to the very low total countsavailable for the DNA-binding assay. However, from 5 h onward a very interesting pattern emerged. For PtC\2(trans-DACH)-treated cells, the aquachloro complex comprised 75-80% of the total reactive platinum at all time points. ForPt(mal)(fra/is-DACH)-treated cells, however, the relative abundance of the aquachloro complex declined to less than 50% by24 h, suggesting significant accumulation of one or more reactive biotransformation products other than the aquachloro complex. The additional reactive biotransformation product(s) cannot be identified by this assay but appear to be more tightlybound to the HPLC column than the aquachloro complex underthe ion pair-reverse phase conditions used (Fig. 7). Based onthe data shown in Table 5 and the specific activity of eachplatinum drug, it was also possible to estimate the actual levelsof [Pt(H2O)(Cl)(fra/w-DACH)]+ at each time point (Table 5,

column 3). As for most other intracellular biotransformationproducts, the relative abundance of aquachloro complex wasindependent of drug concentration over the concentration rangeof 12 to 100 Mg/ml (data not shown).

DISCUSSION

We have developed a method to determine the intracellularhalf-lives of PtCl2(fra//s-DACH) and Pt(mal)(fra/w-DACH) and

Table 5 Quantitation of the intracellular reactive biotransformation products derived from PtCliftrans-DACH) and Pt(mal)(trans-DACH¡f

Drug and time ofincubation(h)PtCM/ra/u-DACH)12581224Pt(mal)(rra/ii-DACH)12581224Reactive

species (% oftotal filterablePt)31.0

±5.3 (n =8)22.0±5.2 (n =6)6.6±2.4 (n =6)4.6±1.4 (n =5)0.65±0.10(n

=5)0.68±0.20 (n =5)11.2

±5.4 (n =6)7.0±2.5 (n =6)8.0±2.5 (n =6)9.3±2.6 (n =5)2.5±0.1(n =5)1.0±0.1 (n = 5)%

of reactive speciesas

[Pt(H2OXCI)(rra/u-DACH)|**44668177827542617346S342[Pt(H2O)(ClXfranj-DACH)r(pmol/106cells)0.42

±0.070.84±0.200.77±0.280.75±0.230.13

±0.020.11±0.030.39

±0.200.80±0.29l.66±

1.032.03±0.580.80±0.040.31±0.05

°L1210 cells (10') in 100 ml RPMI-1640 medium were incubated with either PtCl2(/ra/is-DACH) at 12 ng/ml or Pt(mal)(íraní-DACH)at 100 pg/ml. All values

are reported as the mean ±SEM. At the indicated times, the cells were harvested, processed, and assayed for their reactivity towards salmon sperm DNA as describedin "Materials and Methods."

* Calculation of the percentage of the reactive species as 11'u11.•'>)(('l )(r;w;v I>A( 11)| was based on a comparison of the distribution of the DNA-binding activityin each sample following HPLC separation (Figs. 6 and 7) with the comparable distribution of a [Pt(H2O)(ClX»rani-DACH)]* standard added to an LI210 cell

sonicate (Fig. ID).

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INTRACELLULAR BIOTRANSFORMATION OF PLATINUM COMPOUNDS

80£

40P

*•*00

80

5m

40_i0

0

fe80a«

40ftIhr^

5hr21612

hr

-,2,0585606*"2772hr8hr15624

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ö453535525147II»•¡«.ilo255

Ufa -

E F G H

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Fig. 6. Distribution of the reactive platinum biotransformation products ofPtCMfrani-DACH) following reverse phase HPLC. L1210 cells (10") in 100 ml

of RPMI 1640 medium were incubated for the indicated times with 12 »mml of3H-labeled l't< Ur/um 1>A( 11). The filterable fraction was prepared and sepa

rated by reverse phase HPLC as described in Fig. 2. The peak fractions (see Fig.I, E and F) were combined and tested for reactivity by the DNA-binding assaydescribed in "Materials and Methods." Peaks a through d had negligible DN A

binding activity under the assay conditions used and are not shown above. Theamount of binding observed in the remaining peaks was expressed as a percentageof the total DNA-binding activity observed for peaks e through h.

80

t 40

§ 0

O 80

5zS 40

¡ofe 80

5«

40

Ihr

5hr

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Hik12hr

2hr

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24 hr

E F G H E F G H

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to propose a model for the activation of Pt(mal)(fra/w-DACH)in cultured 1,1210 cells (Fig. 8). In RPMI 1640 supplementedwith 15% fetal calf serum, the malonate ligand is displaced atapproximately equivalent rates by protein, amino acids (predominantly metliionine), bicarbonate, and chloride (16). Theplatinum-protein and platinum-ammo acid complexes are essentially inactive and are not taken up by the cell, but both thechloride and bicarbonate displacement reactions can be considered extracellular activation pathways. The chloride displacement reaction can be considered an activation pathway becausePtCl2(/ranj-DACH) is taken up 8 times more rapidly by L1210cells than Pt(mal)(/rans-DACH) (15). Once inside the cell,PtC\2(trans-D\CH) should dissociate to form reactive aquatedspecies (14, 21). In fact, it appears likely that most of theintracellular PtCl2(fra/is-DACH) arises from the extracellulardisplacement reaction. This conclusion is based on two observations: (a) displacement of the malonate ligand by chlorideis exceedingly slow at intracellular chloride concentrations(<5% displacement in 24 h at 37°C);(b) the time course for

PtC\2(trans-DACti) accumulation and disappearance inside thecell (Fig. 5) closely parallels that seen in RPMI 1640 (16). Theextracellular bicarbonate displacement reaction can also beconsidered an activation pathway because the bicarbonato complex is unstable and readily decomposes to give various aquatedspecies (16). At the chloride concentrations which prevail inthe medium, at least some of these aquated complexes wouldbe converted to PtCl2(/ranj-DACH) for entry into the cell.

Once Pt(maI)(fra/ii-DACH) enters the cell, the malonateligand is very rapidly displaced (fv, 21-28 min). The rate ofPt(mal)(fra/ts-DACH) breakdown is fully consistent with thedisplacement rates of the malonate by glutathione and variousamino acids (16) and previous estimates of intracellular aminoacid concentrations in eukaryotic cells (23). Thus, the majorportion of the observed intracellular rate of malonate displacement can probably be accounted for by the reaction of Pt-

Pt Protein(inactive)

sProteins

Pt AA (inactive!l V

Fig. 7. Distribution of the reactive platinum biotransformation products ofPt(malXfranj-DACH) following reverse phase HPLC. This experiment was carried out as described in Fig. 6 except that 100 Mg/ml ot 'I I labeled l't(nul)(rran.v

DACH) (0.15 Ci/mmol) was used.

to identify and quantitate 6-8 different intracellular biotransformation products from the filterable fraction of LI210 cells(Tables 1 and 2). In addition, we have been able to quantitatethe intracellular levels of the monoaquated biotransformationproduct and show that there were significant levels of at leastone other reactive biotransformation product(s) in L1210 cellstreated with Pt(mal)(fra/is-DACH). The quantitation of individual biotransformation products was reproducible (Tables 2and 5) despite the multiple steps required to obtain completeseparation. Furthermore, with the exception of the Pt-(glutathione)(fra/is-DACH) complex, the relative abundance ofthe various platinum biotransformation products was independent of the drug concentration (Table 3), allowing high concentrations of platinum drugs to be used for increased sensitivity.

Previous data, plus the data presented in this paper, allow us5142

/H20'Pt (reactive)

H20

Pt (reactive)l \H20

/ciPI (reocttv»!

Mostly inactive, out )(some reactive species/

Fig. 8. Model for activation of Pt(malXíraní-DACH)in cultured LI210 cells.II. amino acid.

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INTRACELLULAR BIOTRANSFORMATION OF PLATINUM COMPOUNDS

(mal)(fra/is-DACH) with protein, glutathione, and amino acidswithout the necessity of invoking enzymatic activation (13).Most of the platinum-amino acid complexes which form in thismanner appear to be inert. However, at intracellular concentrations, both bicarbonate and phosphates can effectively displacethe malonate ligand as well, and both form unstable complexeswhich dissociate to give rise to aquated species (16). Thus, thesereactions can be considered as intracellular activation pathways.

The relative contribution of the various activation pathwaysdescribed to the formation of aquated platinum complexesin the cell will depend both on the rate of formation ofthe "activated" intermediates [e.g., PtCl2(fra;ií-DACH), Pt-

(HCO3)2(rra/is-DACH), or Pt(H2PO4)2(ira/is-DACH)] and onthe relative rates of ligand displacement from these activatedintermediates by water and intracellular nucleophiles. We donot yet know the relative rates of the ligand displacementreactions for these compounds, but it is apparent that intracellular concentrations of PtQ2(fra/Js-DACH) are comparable toPt(mal)(fra/M-DACH) at most time points (Fig. 5), suggestingthat the extracellular activation pathway may predominate.

We believe that the present approach allows more accurateestimation of intracellular [Pt(H2O)(Cl)(/rans-DACH)r levelsthan previously available methods. However, as with any newmethod, some comments on the possible limitations are appropriate. First, some change in the levels of aquated species duringpreparation (at 4°C)and storage (at -80°C) of the cell pellet is

possible. However, since both the formation and subsequentreactions of the aquated platinum complexes appear to benonenzymatic processes, it is not clear a priori that significantchanges in the steady-state levels of the aquated species wouldbe expected until the cells were lysed. Secondly, it remainspossible that some unknown reactive biotransformation product^) could comigrate with the aquachloro complex in peak f,leading to an overestimation of [Pt(H2O)(Cl)(fra/ji-DACH)]+

levels. We consider this unlikely at present since we havecharacterized 18 of the most likely biotransformation productsand find none with reactivity towards DNA which migrates inpeak f. Obviously, as we continue to identify and characterizeother possible biotransformation products, the accuracy of thisapproach for quantitation of [Pt(H2O)(Cl)(fru/is-DACH)]+ will

improve.The current method does allow unambiguous quantitation of

those reactive biotransformation products which are not[Pt(H2O)(Cl)(fra/ii-DACH)]+ since essentially all of the reactiv

ity of the aquachloro complex towards DNA is found in peak f(Fig. 10). We have previously shown that Pt-lysine and Pt-arginine complexes form very slowly and have significant reactivity towards DNA (16). At least some of the slowly accumulating reactive platinum complexes seen in Fig. 7 haveelution positions on reverse phase HPLC which are similar tothe lysine and arginine complexes (8), but it is not clear thatthose platinum-amino acid complexes are the only reactivespecies which accumulate. We favor a model in which weaknucleophiles in general would tend to react more slowly withPt(mal)(irani-DACH) and, once they did react, would tend toform unstable platinum complexes. However, it is also possiblethat the lysine and arginine complexes possess special reactivitytowards DNA by virtue of their positively charged side chainswhich could interact with the DNA phosphodiester backbone.

The presence of the Pt(glutathione)(franj-DACH) complexis significant since glutathione is involved in the detoxificationof a wide range of xenobiotics (24). Our data show that intracellular glutathione levels increase in response to incubation ofLI210 cells with PtCl2(fra/is-DACH) in a dose-dependent man

ner (Table 4) and that this is reflected in the relative abundanceof the glutathione complex at these drug concentrations (Table3). We believe this is the first demonstration that platinumcompounds cause a rise in glutathione levels in a sensitive cellline. Presumably, this increase in intracellular glutathione levelsis part of the normal cellular defense mechanism against thistype of compound.

The accumulation of a significant amount (~20% of totalfilterable) of free trans-DACH at 24 h is worth noting. Sinceatomic absorption was not used in these studies due to theextremely low amounts of platinum available, approximately20% of the potential biotransformation products could not befollowed due to the loss of 3H label. We do not think that this

significantly alters the conclusions drawn in these experiments,especially at early times when the accumulation of free trans-DACH is small. However, these data do suggest that it may bevaluable to use '95mPtcompounds in some future experiments.The displacement of the trans-D\CH carrier ligand was probably due to the fra/is-labilization effect of glutathione and othersulfur-containing nucleophiles (25) which are present at relatively high concentrations within the cell. For example, Ismailand Sadler (26) have observed displacement of the ethylenedi-amine(en) ligand from PtCl2(en) in reactions with RNase A andW-acetyl-L-methionine. In contrast, when PtCl2(frani-DACH)or Pt(mal)(/ra/tt-DACH) were incubated for 24 h in RPMI1640 (which contains significantly lower levels of glutathione,cysteine, and me thionine) there was only around 2% labi li/.ationof the trans-D\CH carrier ligand (16).

Our techniques have not allowed the identification of all ofthe intracellular biotransformation products resolved by thetwo-column HPLC system. One solution to this problem wouldbe the in vitro preparation of more standards corresponding topotential intracellular biotransformation products. The compounds we used for standardizing our HPLC columns, primarily platinum-amino acid complexes, were chosen on the basisof the high intracellular concentrations and known reactivity ofamino acids towards platinum(II). Nuclear magnetic resonancestudies suggest that other intracellular metabolites such asacetate, pyruvate, citrate, and succinate could he present in highenough concentrations to displace the chloride or malonateligands from the drugs used (27, 28). Finally, even though theidentification of intracellular biotransformation products isbased on their retention times in three different HPLC systems(reverse phase, cation exchange at pH 4, and cation exchangeat pH 2.3), these identifications cannot be considered conclusivein the absence of positive verification of the compounds isolatedfrom the cell by these HPLC techniques. Unfortunately, mostmethods of structural verification are simply not sensitiveenough to be useful for identification of platinum compoundsin the ng range. We are currently working to develop massspectrometric techniques with sufficient sensitivity to verify thestructures of these platinum biotransformation products.

In summary, the techniques described in this paper allow theidentification and quantitation of intracellular platinum bio-transformation products, the determination of their reactivitytowards DNA, and the half-life of the parent drugs in the cell.This, in turn, has allowed us to postulate activation pathwaysfor one of the platinum compounds used in this study. Finally,these techniques make possible a number of potentially usefulstudies. For example, it should now be possible to compare thebiotransformation of platinum compounds in different celltypes and identify differences in metabolism which might leadto the organ-specific toxicity of certain platinum drugs (29).

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INTRACELLULAR BIOTRANSFORMATION OF PLATINUM COMPOUNDS

ACKNOWLEDGMENTS

The assistance of Dennis Chapman in the quantitation of intracel-lular glutathione levels is gratefully acknowledged. The authors alsogratefully acknowledge the scholarly discussions and helpful suggestions from Dr. Aziz Sanear.

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