[CANCER RESEARCH54, 6215—6220,December 1, 19941

ABSTRACT

Alkylating agents can be detoxified by conjugation with glutathione(GSH). One of the physiological significances ofthis lies in the observationthat cancer cells resistant to the cytotoxic effects of alkylating agents havehigher levels of GSH and high glutathione S-transferase (GST) activity.However, little is known about the GSH-/GST-dependent biotransformalion ofalkylating agents, including cydophosphamide. Cyclophosphamidebecomes cytostatic after the enzymatic formation of 4-hydroxycyclophos

phamide. The ultimate alkylating species formed from cyclophosphamideis phosphoranside mustard. In this paper we describe the involvement of

purified human glutathione S-transferases isoenzymes GST Al-i, A2-2,Mla-la, and Pi-l in the formation of two types of glutathionyl conjugatesof cyclophosphamide, @e.,4-glutathionylcyclophosphamide (4-GSCP) andmonochloromonoglutathionylphosphoramide mustard.

When 0.1 mM 4-hydroxycyclophosphamide and 1 mM GSH was incubated in the presence of 10 pM GST Al-i, A2-2, Mla-la, and P1-i theformation of4-GSCP was 2—4-foldincreased above the spontaneous level.Enzyme kinetic analysis demonstrated the lowest Km (0.35 IflM) for GSTAl-i. Kmvalues for the other GST enzymes ranged from 1.0 to 1.9 mM.

Glutathione S-transferase Al-i (40 pM) also increased the conjugationof phosphoramide mustard and GSH (both 1 mi'@i)2-fold, while the othermajor human Isoenzymes, A2-2, Mia-ia, and P1-i, did not influence the

formation of monechlorumonoglutathionylphosphoramide mustard.These results Indicate that only one enzyme within the class of human

GST a enzymes was able to catalyze the reaction of the aziridinium ion ofphosphoramide mustard with glutathione.

Thus increased levels of GST Al-i in tumor cells can contribute to anenhanced detoxification of phosphoramide mustard and hence to thedevelopment of drug resistance. Since all of the human GSTs tested didcatalyze the formation of 4-GSCP, the role of 4-GSCP either as a transport form of activated cyclophosphamide or as a detoxification product isdascussed.

INTRODUCTION

Resistance to anticancer chemotherapeutic drugs remains a majorobstacle in cancer chemotherapy. Recent studies suggest that GSH,3an intracellular cysteine-containing tripeptide present at high concentrations (up to 5—10mM) in most mammalian cells, is a criticaldeterminant in tumor cell resistance to alkylating cytostatic agents (forreview see Ref. 1). This has been attributed to the ability of GSH tocompete with DNA for drug binding. The actual structures of thesedrug-GSH conjugates have been characterized for a number of theseagents, i.e., melphalan, chlorambucil, and cisplatin (2—4).

In addition to higher intracellular concentrations of GSH a higherGST activity also has been observed in tumor cells resistant to

Received 6/27/94; accepted 9/30/94.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 in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I This study was supported by Dutch Cancer Society Grant TNOV-92-93.

2 To whom requests for reprints should be addressed.

3 The abbreviations used are: GSH, glutathione; NMR, nuclear magnetic resonance;

4-OOHCP (4-hydroperoxycyclophosphamide), 2-[bis(2-chloroethyl)amino]tetrahydro-2-oxide-2H-1,3,2-oxazaphosphorine-4-yl-hydmperoxide; 4-OHCP, 4-hydroxycyclophosphamide; PM, phosphoramide mustard; 4-GSCP, 4-glutathionylcyclophosphamide;PMGS, monochloromonoglutathionylphosphoramide mustard; OST, glutathione S-transferase;CP, cyclophosphamide;PM,phosphoramidemustard;HPLC,high-performanceliquid chromatography; LC-MS, liquid chromatography-mass spectrometry; TFA,trifluoroacetate.

alkylating agents. A concentration of 4—40 @tgGST/mg cytosolicprotein has been reported in tumors or tumor cell lines (5—7).According to our calculations this correponds to a 30—240fLMconcentrationof GST.

GSTs might catalyze the formation of drug-GSH conjugates. However, little is known about the role of GSTs in the formation of theseglutathionyl conjugates. Ciaccio et a!. (8), reported that human GSTaand@Tenzymesenhancedtheformationofmonochloro,monoglutathionylchlorambucil. Meyer et a!. (9) reported that onlyGST a enzymes catalyzed this reaction and not GST@ and ‘zr.Formelphalan the formation of monochloromonoglutathionylmelphalanwas increased by mouse liver GST a isoenzymes but not by GST@and ir (10). Knowledge on the specificity of GSTs for the conjugationreactions of alkylating agents is necessary to provide insight in therole of GSTs in the development of tumor cell resistance since intumor cells mainly GST ii@enzymes are expressed (for review seeRef. 11).

CP, an orally active alkylating agent, has a wide spectrum of clinicaluses and is an essential component of numerous combination chemotherapeutic regimens. Cyclophosphamide becomes cytostatic after oxidationby hepatic microsomal enzymes to form 4-OHCP (Fig. 1). Three specifichuman cytochrome P450 enzymes, i.e., CYP2B6, CYP2C8, andCYP2C9, have been identified as major catalysts of cyclophosphamideactivation (12). 4-OHCP equilibrates with the ring-opened aldophosphamide which undergoes spontaneous decomposition to yield PM andacrolein. It is proposed that 4-OHCP can, while PM cannot, enter cells so4-OHCP might be considered as the carrier molecule of PM across cellmembranes (13). Phosphoramide mustard possesses DNA-alkylating activity and is generally considered to be the therapeutically significant

cytotoxic metabolite of cyclophosphamide. Phosphoramide mustard alkylates DNA through a positively charged reactive intermediate, i.e., anaziridinium ion formed by the loss of a chlorine atom. Phosphoramide

mustard has two chloroethyl groups and is able to form alkylatedproducts at two separate nucleophilic sites.

Two types of glutathionyl conjugates of cyclophosphamide havebeen described, i.e, diglutathionyl-PM and 4-GSCP (14, 15), and thekinetics of the nonenzymatic formation of both conjugates has beenstudied using 31P NMR spectroscopy (16). Four stereoisomeric4-GSCP conjugates were found. The formation of 4-GSCP was foundto be reversible, and at hydrolysis, PM was being formed. 4-GSCP cantherefore be considered as a stabilized reservoir for the production ofPM. Conjugation of PM was found to be preceded by the formation ofPMGS and subsequently diglutathionyl-PM.

Whether these reactions are catalyzed by the isoenzymes of humanGSTs is unknown. We describe in this paper the influence of purifiedhuman GSTs on the rate of formation of 4-GSCP and PMGS in vitro.The results confirm a catalytic effect of all three classes of humanGSTs tested on the formation of 4-GSCP as well as an effect of GSTAl-i on the rate of formation of PMGS. The impact of these findingson the role of GSTs in drug resistance toward CP is discussed.

MATERIALS AND METHODS

Chemicals. 4-OOHCP and phosphoramide mustard [N,N-bis(2-chloroethyl)phosphorodiamidic acidi were kindly provided by Dr. J. PohI from ASTA

6215

Involvement of Human Glutathione STransferase Isoenzymes in the Conjugation ofCyclophosphamide Metabolites with Glutathione'

Hubert A. A. M. Dirven,2 Ben van Ommen, and Peter J. van Bladeren

TNO Nutrition and Food Research Institute, Division of Toxicology, P.O. Box 360, 3700 AJ Zeist. the Netherlands

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GST.MEDIATED GLUTATHIONE CONJUGATION OF CYCLOPHOSPHAMIDE

0

rjicDa

LKB, Uppsala, Sweden). Eluent A was 0.02 MTns buffer (pH 6.8), and eluentB was 0.02 MTris buffer (pH 6.8) with 0.15 MNaC1.Flow was 1 ml/min. Thesolvent program started isocratically with 100% solvent A for 5 mm followed

by a linear gradient to 15% solvent B in 10 mm.Mass Spectrometry. In HPLC-MS experiments with glutathionyl conju

gates ofphosphoramide mustard a Supersher RP select B (200 x 4 mm) columnwas used with 10 mMammonium trifluoroacetate with 0.5% TFA (solvent A)and acetonitrile (solvent B). The gradient applied was 5—30%solvent B in 15mm (flow, 1 mI/min). Mass spectrometric analyses were conducted using ionspray ionization at an API III triple-quadruple mass spectrometer (Sciex,Thornhill, Ontario, Canada).

In HPLC-MS experiments with 4-glutathionylcyclophosphamide a Spherisorb 55 ODS-2 (200x 0.32-mm) column was used. Mobile phase A was 0.1%TFA in water, and mobile phase B was 0.1% TFA in acetonitrile. The solventprogram started with 100% solvent A for 5 mm, followed by a linear gradient

to 20% solvent B in 15 mm. Mass spectrometric analyses were performedusing electrospray ionization (Analytics) on a Finnigan MAT TSQ 700.

Peaks used for quantification of 4-GSCP and mono- and diglutathionylconjugatesof PMwereisolated,lyophilized,andcharacterizedwithfast atombombardment mass spectrometry using a Finnigan-MAT900 instrument asdescribed elsewhere (16).

Analysis of Results. Kinetic parameters (Km and Vm@) were calculatedusing the Michaelis-Menten equation. The curve-fitting program for theanalysis of enzyme kinetic data “EZ-fit―was used for this purpose (22).

RESULTS

Conjugation of 4-Hydroxycydophosphamide and Glutathione.Incubation of 6 mM 4-OOHCP and 60 mist glutathione resulted inliquid chromatography-mass spectroscopy detection of a peak withmass fragments mlz 566 and m/z 568. The mass spectrum of this peak,corresponding to 4-GSCP, is presented in Fig. 2. When this incubationmixture was analyzed with reversed-phase HPLC with radiochemicaldetection one major radioactive peak was found at 26.5 min (Fig. 2).In Fig. 3A the nonenzymatic rate of formation of 4-GSCP at 25°Cand37°Cis shown with time. The conjugate was found to be relativelystable at 25°Cbut less stable at a temperature of 37°C.

All further incubations were performed at a temperature of 25°C.When 0.1 mM 4-OOHCP and 1 m@.iGSH were incubated with increasing concentrations of GST Al-i, an enzyme concentration-dcpendent increase in the formation of 4-GSCP was found (Fig. 3B). InFig. 4 the formation of 4-GSCP in the presence of 10 p.MGST Al-i,Mia-la, and P-i is shown. All 3 isoenzymes tested had enhanced therate of formation of the 4-GSCP conjugate. When these purified GSTenzymes were incubated for 15 min with concentrations of 4-OOHCPup to 2000 p.Ma concentration-dependent increase in the formation of4-GSCP was found. From the data of these experiments apparent Kmand Vm,,,@values were calculated using Lineweaver-Burk plots (Table1). Km and Vmss values were relatively high. The lowest Km value wasfound for GST Al-i (0.35 mM). Km values for the other OST enzymesranged from 1.0 to 1.9 mM.

Conjugation of Phosphoramide Mustard and Glutathione.Upon incubation of 6 missPM with 60 mistGSH two peaks were foundin the chromatogram (Fig. 5). With liquid chromatography-massspectrometry it was shown that both m/z 493 (corresponding tomonochloromonoglutathionylphosphoramide mustard) and m/z 762(corresponding to diglutathionylphosphoramide mustard) were present in the incubation mixture (Fig. 5). Also mass fragment m/z 473(corresponding to monohydroxymonoglutathionylphosphoramide musturd was found.

The formation of glutathionyl conjugates with time was studiedusing 6 mM PM and 60 m@tGSH at 25°Cand 37°C,respectively (Fig.6A). Within 60 mm, peaks characterized as monochloromonoglutathionyl-PM and diglutathionylphosphoramide mustard were found inthe HPLC chromatogram. The pattern of formation of these two

6216

HO@S NH

@@L.:-'L@HO HO

CI@ OS

as

0 GSHGSH

GSTA1-1@

—(PM(Gs,()

@oramidsmu@au

(PM)

@ndH@mwls@msdi@s

m.nothuro.

— muu@d‘PMr,s,

Fig. 1. Metabolism of cyclophosphamide (simplified) and possible conjugation reactions with glutathione.

Medica, Frankfurt am Main, Germany. 4-OOHCP is used as a precursor of4-OHCP because of its higher stability in a crystalline state and its easiersynthesis. Glutathione was from Boehringer Mannheim GmbH (Mannheim,Germany); glutathione reductase and NADPH were from Sigma Chemical Co.(St. Louis, MO). [3551G5Hwas obtainedfrom DuPont de Nemours, NENDivision, Den Bosch, the Netherlands. All other chemicals used were of thehighest purity obtainable.

GST Purification and Assay. GST isoenzymeswere purifiedfromhumanliver and placenta using affinity chromatography (S-hexylglutathione-Sepharose 6B) and chromatofocusing as described previously (17). Purity wasconfirmed with HPLC analysis as described elsewhere (18); protein wasdetermined according to the method of Lowry et al. (19) with bovine serumalbumin as standard. GST activity was assayed using 1-chloro-2,4-dinitrobenzene as a substrate (20). Specific activities (p@mol/min/mgprotein) were31—45,10—23,67—150,and 54, respectively, for GST Al-i, GST A2-2, GSTMia-la, and GST P1-i. All enzyme concentrations were expressed as theconcentration of the subunit (Mr 25, 900, 25, 900, 26, 700 and 24, 800,respectively, for subunits Al, A2, Mia, and P1).

Incubation of 4-OOHCP and PM with Glutathione. Incubations werecarried out in 0.07 M phosphate buffer (pH 7.0) with 5 mM EDTA, 3 mMNADPH, 0.8 unit/ml glutathione reductase, and 1 or 60 mM GSH/35SGSH,with or without GST. Incubations were performed at a pH 7 since the median

tumor pH is 7.1 (21).Reactions were started with the addition of 4-OOHCP or PM, which was

dissolved immediately before use. Incubation temperature was 25°Cor 37°C,respectively.

Incubation mixtures containing 4-hydroxycyclophosphamide were terminated by the addition of acetic acid until a final concentration of 0.05 Mwasachieved and stored at —20°Cuntil analysis. Incubations containing phosphoramide mustard were terminated by the addition of N-ethylmaleimide until a

final concentration of 10 msi was attained, and they were also stored at —20°Cuntil analysis.

Analysis of the Reaction Products. The HPLC system that was usedconsisted of a Pharmacia LKB HPLC pump 2248 (Uppsala, Sweden) equippedwith a flow through radioactivity detector (Radiomatic Ho-one A500; Men

den, England.) A 2-ml flow cell was used. As scintillation cocktail Flo-ScintA (Packard Instrument, Groningen, the Netherlands) was used with a flow of2 mllmin.

Formation of 4-glutathionylcyclophosphamide was assayed on a 25th

4.6-mm Zorbax ODS column (Rockland Technologies, Chadds Ford, PA).Eluent A was water with 0.2% TFA and eluent B was acetonitrile with 0.2%TFA (flow, I mI/mm). The solvent program started isocratically with 100%eluent A for 5 mm, followed by a linear gradient to 10% eluent B in 15 mm,

followed by a linear gradient to 100%eluent B in 5 mm (flow, 1 mI/mm)whichwas held for 10 mm.

The formation of mono- and diglutathione conjugates of phosphoramidemustard was assayed on a Mono 0 HR 5/5 anion exchange column (Pharmacia

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0.

@ 1.80

0

EI.20

GST-MEDIATED GLUTATHIONE CONJUOA11ON OF CYCLOPHOSPHAMIDE

,@ 0100

SO

_ cc—'cf@r-(@@j@0

J40

20307.1 43@.0

243.0 I

LL.:zi..@;@@@0

0

Time(mm)Fig. 2. Separation of 4-glutathionylcyclophosphamide from glutathione by reverse phase liquid chromatography. 4-Hydroxycyclophosphamide (6 mM)and GSH (60 mM)were

incubatedfor5 h at 37°C;40 @dof thereactionmixturewereanalyzedas describedin the text.Thepeakwitha retentiontimeof 6 mmis glutathione,andthepeakwitha retentiontime of 25.6 mm is 4-glutathionylcyclophosphamide. inset, IC-MS spectrum of 4-glutathionylcyclophosphamide.

B

3.00 14

2.40@ 8

A

0.60

0.00

0 200 400 600 800 1000 1200 0 10 20 30 40 50

time (mm) concentration GST Al-I (pM)

Fig.3.A,timecourseof formationof 4-glutathionylcyclophosphamidein an incubationmixtureof 6 ms 4-hydroxycyclophosphamideand60 ms GSHat 25°(O)and37°C(•).B, formationof4-glutathionylcyclophosphamide in the presenceof varying concentrationsof GST Al-i. The nonenzymaticformationof 4-glutathionylcyclophosphamideis subtractedfromall values

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0 6 12 18 24 30

Km (mM)VmlIx(11m01/11th1/

mgprotein)GSTAl-i0.35 ±0.09iO.0 ±0.8GSTA2-21.5 ±0.4i5.i ±2.2GSTMia-lai.04 ±0.310.4 ±1.6GSTP1-I1.9 ±0.435.1 ±4.8

GST.MEDIAThD GLUTATHIONE CONJUGA11ONOF CYCLOPHOSPHAMIDE

products as found in this experiment was comparable with that described in an earlier study in which these reactions were monitored by31P NMR spectroscopy (16). Diglutathionyl-PM appeared to be arelatively stable conjugate, at both 25°and 37°C(Fig. 6A).

When 1 mM PM and 1 mist GSH were incubated at 37°Cwithvarying concentrations of GST Al-i, an enzyme concentration- dependent increase in the formation of monochloromonoglutathionylphosphoramide mustard was found (Fig. 6B). Using the sameexperimental conditions no increase in the formation of monochloromonoglutathionylphosphoramide mustard was found using GSTA2-2 (results not shown).

When 40 p.MOST Al-i, A2-2, Mia-la, and P1-i was incubated for24 min with 1 misi PM and 1 miss GSH the formation of the monoglutathionyl conjugate was 2-fold increased above the spontaneousrate in incubations with GST Al-i but not with GST Mia-la, A2-2,orP-i(Fig.7).

DISCUSSION

If tumor cells have the capacity to inactivate alkylating agents, theamountofDNAdamagecausedbytheseagentsmaybelessthanincellsin which these mechanisms are not present Inactivation by both nonenzymatic and GST-catalyzed GSH conjugation has been postulated to berelated to the development of such drug resistance in tumor cells.

The question addressed here is whether cyclophosphamide metabolites are substrates for human GST isoenzymes. Previous studies onthe spontaneous reactions of activated cyclophosphamide with glutsthione showed that mainly two types of conjugates were formed, i.e.,4-GSCP and mono- and diglutathionylphosphoramide mustard (16).

All GSTs tested enhanced the formation of 4-glutathionylcyclo

phosphamide 2—4 times above the nonenzymatic rate. Km values

I

I

U00

tim. (mm)

Fig. 4. Time course of formation of 4-glutathionylcyclophosphamide from 0.1 inst4-hydroxycyclophosphamide and 1 inst GSH at 25°Cwithout enzyme (•)and in thepresence of 10 gLMGSTA1-l (A), GST Mla-ia(O), and GST P-i (+). By means of linearregression analysis it was found that the slope ofthe lines ofGST Al-i, Mla-la, and P-iwere statistically significant different (P < 0.05) from the slope of the line withoutenzyme.

Table 1 Apparent V@ and Km VO.lU@Sfor human glutathione S-transferase catalyzedformation of 4-glutathionylcyclophosphamide

—I

HOCç@@p-.NHa

0

Ge

-a

Ea-V

psdi2

HOOS @NH,

peak 1 peak 2

0 nVz

Fig. 5. Separation of monochiommonoglutathionylphosphoramidr mustard and diglutathionylphospborainide mustard from glutathione with anion exchange chromatography.Phosphoramide mustard 6 maoand GSH 60 inst were incubated for 3 h at 25°C;100 p1 of the incubation mixture were analyzed as described in the text. Inset, LC-MS spectrum ofmonochloromono@lutathionvlohosohoranndemustard and dialutathionylahosphoramide mustard.

6218

Time (mm)

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GST-MEDIAThDGLUTAThIONECONJUGATIONOF CYCLOPHOSPHAMIDE

A B

30

20Cl)0

a-

10

c,J00

00

0@

E

Fig.6.A, timecourseof formationof monochloromonoglutathionylmustardanddiglutathionylphosphoramidemustardin an incubationmixtureof 6 instphosphoramidemustardand60 mstOSHat 25°Cand37°C.0, monochloromonoglutathionylphosphoramidemustard,25°C;•,diglutathionylphosphoramidemustard,25°C;U monochloromonoglutathionylpho.phoramidemustard,37°C;, diglutathionylphosphoramidemustard,37°C.B, formationof monochioromonoglutathionylphosphoramidemustardin the presenceof varyingconcentrations of GST Al-i at 37°C.The nonenzymatic formation of monochloromonoglutathionylphosphoramide mustard is subtracted from all values.

0 10 20 30 40 500 240 480 720 960 1200

found were in the range of 1.0—1.9m@tfor GST A2-2, Mia-la, andP1-i and 0.3 mM for GST Al-i. Km values in the mitt range are alsoobservedfor othersubstratesfor GSTs,e.g.,l-chloro-2,4-dinitrobenzene,a substrateusedin the quantificationof GST activity (20, 23).

In contrast to the formation of 4-glutathionylcyclophosphamide theformation of monocfflommonoglutathionyl-PM was catalyzed only byGST Al-i but not by GSTA2-2, Mia-la, or Pi-i. In the presence of 40@AMGST Al-i the rate of formation of the monoglutathionylphosphor

amide @ugatewas2-foldincreasedabovethespontaneouslevel.The formationof 4-thio derivativesof cyclophosphamideas a

“stabilizedreservoir of alkylating activity―was suggested by Draegeret al. (24). This study and a previous study (16) showed that formation

of 4-glutathionylcyclophosphamide is reversible and therefore that4-GscP @i@iitbe consideredas a stabilizedformof 4-OHCP.

Kwon et cil. (25) described two mechanisms for the formation of4-thiocyclophosphamide conjugates. One mechanism involved theformation of iminocyclophosphamide and, the other mechanism involved the formationof a hemithioacetalfrom aldophosphamidethatsubsequently cyclized to 4-thiocyclophosphamide.

Conjugation of PM with OSH was shown to proceed through theformation of aziridinium ions and not by direct replacement of chlo

50

40

rime (i4). In previous 31P NMR experiments we found that the PMsignal in incubations with glutathione disappeared at the same rate asin incubations without glutathione. These findings indicate that therate-limiting step in the conjugation of PM with GSH is the formationof the aziridinium ion. Also the reaction of the nitrogen mustardagents melphalan and chlorambucil with glutathione is believed toproceed via the aziridinium intermediate (26).

The results of the studies with melphalan, chlorambudil (8—10,26),and phosphoramide mustard (present study) all indicate that mainlyOST a enzymes can catalyze the reaction of the aziridinium intermediatewith glutathione.Increasedlevelsof GST a enzymesin tumorsmight therefore be related to an enhanced formation of drug-GSHconjugates and hence to the development of drug resistance.

A number of findings suggest a role of GST a enzymes in thedevelopment of drug resistance toward alkylating agents. Severalalkylator-resistant sublines have been shown to overexpress GST aenzymes (27—30).The introduction of rat or human GST a genes intolines of cultured cells conferred resistance toward alkylating agents(31—33).In addition, a number of studies showed that GSH protectsagainst the cytotoxicity of CP (34, 35). Peters et al. (7) showed a25-fold increase in the concentration of GST a enzymes in esophagealtumors compared to normal esophageal tissue.

Thus, we have identified two types of glutathione conjugates ofcyclophosphamide. GSTs were able to catalyze both conjugationreactions. It is tempting to speculate about the role of the formation ofthese products in the biotransformation of cyclophosphamide andtheir role in the development of drug resistance. Our findings have ledto the following model. Upon hydroxylation of CP by cytochromeP450 in the liver, 4-OHCP is formed, which can conjugate with GSH,leading to the formation of 4-GSCP. This conjugation might occurnonenzymatically, but it is also catalyzed by all three major classes ofcytosolic GSTs, of which the a- and p.-class enzymes are abundantlypresent in human liver (36). We postulate that 4-GSCP acts as atransport form of 4-OHCP in blood. Uptake of activated cyclophosphamide in cells is likely to occur by conversion of 4-GSCP to

4-OHCP by a number of trans-mercaptalization reactions. Intracellularly, 4-OHCP can conjugate with GSH. This reaction might occurnonenzymatically but is also catalyzed by GST a, p., and it- enzymes.4-GSCP is possibly transported out of the cell by an AlT-dependentglutathione S-conjugate export pump (4). 4-GSCP would also

-GST GSTAI-1 GSTA2-2 GSTNIa-la GST P1-I

0300

a-

@ 20

10

0

Fig. 7. Formation of monochloromonoglutathionylphosphorainide mustard without orwith 40 @LMenzyme. Phosphoramide mustard (1 msi)was incubated with OSH (i mst) for1 h at 37°C.The formationof the conjugatewith enzyme was comparedto the spontaneous formation by means of the paired t test. P values were 0.006, 0.02, 0.06, and 0.04,respectively,forGSTAi-1, A2-2,Mia-la, andP-i.

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14. Yuan, Z., Smith, P. B., Bnmdrett, R. B., Calvin, M., and Fenselau, C. Glutathioneconjugation with phosphoramide mustard and cyclophosphamide. A mechanisticstudy using tandem mass spectrometry. Drug Metab. Dispos., 19: 625—629,1991.

15. Pallante, S. L, Lisek, C. A., DU1ik, D. M., and Fenselau, C. Glutathione conjugates,immobilized enzyme synthesis and characterization by fast atom bombardment-massspectrometry. Drug Metab. Dispos., 14: 313—318,1986.

16. Dirven, H. A. A. M., Venekamp, J. C., van Ommen, B., and van Bladeren, P. J. Theinteractionofglutathione with 4-hydroxycyclophosphamideand phosphoramide mustard, studied by 31P nuclear magnetic resonance spectroscopy. Chem.-Biol. Interact.,93: 185—196,1994.

17. Vos, R. M. E., Snoek, M. C., van Berkel, W. J. H., Muller, F., and van Bladeren, P. J.Differential induction of rat hepatic glutathione S-transferase isoenzymes by hexachlorobenzene and benzyl isothiocyanate: comparison with induction of phenobarbital and 3-methylcholanthrene. Biochem. Pharmacol., 37: 1077—1082,1988.

18. Bogaards, 1. J. P., van Ommen, B., and van Bladeren, P. 1. An improved method forthe separation and quantification of glutathione S-transferases subunits in rat tissueusing high-performanceliquid chromatography. J. Chromatogr., 474: 435—440,1989.

19. Habig, W. H., Pabst, M. J., and Jakoby, W. B. Glutathione S-transferase: the first stepin mercapturic acid formation. J. Biol. Chem., 249: 7130—7139, 1974.

20. Lowry, 0. H., Rosebrough, N. J., Farr, A. L, and Randall, R. 1. Protein measurementwith the Folin phenol reagent. 1. Biol. Chem., 193: 265-275, 1951.

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23. Meyer, D. L Significance of an unusually low Km for giutathione in glutathionetransferases of the a, g.@and ir classes. Xenobiotica, 23: 823—834,1993.

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26. Bolton, M. 0., Hilton, J., Robertson, K. D., Streeper, R. T., Colvin, 0. M., and Noe,D. A. Kinetic analysis of the reaction of melphalan with water, phosphate, andglutathione. Drug Metab. Dispos., 21: 986—996,1993.

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29. Buller, A. L, Clapper, M. L, and Tew, K. D. Glutathione S-transferases in nitrogenmustard-resistant and -sensitive cell lines. Mol. Pharmacol., 31: 575—578.

30. Yang, W. Z., Begleiter, A., Johnston, J. B., Israels, L G., and Mowat, M. R. A. Roleof glutathione and glutathione S-transferase in chioranibudil resistance. Mol. Pharmacol., 41: 625—630,1992.

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33. Schecter, R. L, Alaoui-Jamali, M. A., Woo, A., FaIn, W. E., and Batist, G. Expression of a rat glutathione S-transferase complementary DNA in rat mammary carcinoma cells: impact upon alkylator-induced toxicity. Cancer Res., 53: 4900—4906,1993.

34. Peters, R. H., Ballard, K., Oates, J. E., Jollow, D. J., and Stuart, R. K. Cellularglutathione as a protective agent against 4-hydroperoxycyclophosphamide cytotoxicity in K-562 cells. Cancer Chemother. Pharmacol., 26: 397—402,1990.

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36. van Ommen, B., Bogaards, J. J. P., Peters, W. H. M., Blaauboer, B., and vanBladeren, P. J. Quantification of human hepatic glutathione S-transferases. Biochem.J., 269: 609-613, 1990.

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equilibrate with 4-OHCP/aldophosphamide giving rise to PM andacrolein. PM can conjugate with glutathione either nonenzymaticallyor catalyzed by GST Al-i. Monoglutathionyl PM cannot form DNAcross-links, while diglutathionyl PM cannot interact with DNA at all.The proposed model indicates that both glutathione levels and thepresence of GSTs, including GST P1-i, can influence the number ofDNA alkylations and hence the cytotoxicity of cyclophosphamide.Therefore it is likely that a relationship exists between increasedlevels of GSH and GSTs in tumor cells and the development of drugresistance. In future studies we will study the formation of theseglutathione conjugates in vivo.

ACKNOWLEDGMENTS

We wish to thank L. G. Gramberg (TNO-SIA) for his assistance with thefast atom bombardment measurements, Dr. E. R. Verheij (TNO-SIA) for hisassistance with the micro LC-MS experiments, and Dr. J. Blanz (Universität

Tubingen, Tubingen, Germany) for the initial LC-MS analysis of glutathionylconjugates of phosphoramide mustard.

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on March 21, 2020. © 1994 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

1994;54:6215-6220. Cancer Res   Hubert A. A. M. Dirven, Ben van Ommen and Peter J. van Bladeren  Glutathionethe Conjugation of Cyclophosphamide Metabolites with

-Transferase Isoenzymes inSInvolvement of Human Glutathione

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