Glutathione Conjugation of Alkylating Cytostatic Drugs with a Nitrogen

MARCH 1996 VOLUME 9, NUMBER 2 © Copyright 1996 by the American Chemical Society

Invited Review Glutathione Conjugation of Alkylating Cytostatic Drugs with a Nitrogen Mustard Group and the Role of Glutathione S-Transferases Hubert A. A. M. Dirven,† 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 Received August 14, 1995

Introduction Among the group of cytostatic drugs, the so-called alkylating agents still rank as some of the most valuable drugs available in the treatment of cancer patients. Alkylating agents exert their therapeutic effects through the formation of monoalkylation adducts and interstrand DNA cross-links (review: ref 1). The formation of these types of DNA damage causes an interference in DNA replication and transcription in rapidly proliferating cells. Chemically, alkylating agents can be divided into five groups: nitrogen mustards (e.g., melphalan, chlorambucil, cyclophosphamide, ifosfamide), ethylenimines (e.g., thiotepa), alkyl sulfonates (e.g., busulfan), triazenes (e.g., dacarbazine), and nitrosureas (e.g., carmustine, streptozocin) (Figure 1). Drug resistance is one of the problems frequently encountered with these agents in the clinic (review: refs 2 and 3). Drug resistance toward alkylating agents is only in rare instances related to overexpression of the MDR1 gene (P-glycoprotein) (4). Enhanced nonenzymatic or GST1-catalyzed glutathione conjugation is a mechanism associated with the development of drug resistance toward alkylating agents. Elevation of cellular GST levels and/or an increase in GSH levels may occur in tumor cells in response to anticancer drug selection pressure (5-8). For chlorambucil and * Author for correspondence. † Present address: Nycomed Imaging, Department of Toxicology, Oslo, Norway.

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melphalan an inverse relationship was found between the GSH concentration and/or the GST activity and the number of DNA cross-links formed (9 ,10). General aspects about GSH and GST in the development of drug resistance have been summarized in a number of excellent review articles (4, 11-13). Correlative observations in wild type versus drugresistant tumor cells are not conclusive evidence for a direct participation of GSH/GSTs in the development of drug resistance (14). It was recognized that direct proof for the participation of GSH/GSTs in drug inactivation was lacking. In the last years, a number of papers have been published in which the glutathionyl conjugates of, especially, nitrogen mustard alkylating agents like mechlorethamine, melphalan, chlorambucil, phosphoramide mustard, ifosfamide mustard, and thiotepa have been characterized (15-22). In addition, for a number of these compounds the role of human GSTs in the formation of these glutathione conjugates has been studied (20-25). In this review, we summarize the results of these studies and elaborate their potential applicability in the clinic. Since marked differences in substrate specificities be1Abbreviations: CP, cyclophosphamide; IF, ifosfamide; GSH, glutathione; GST, glutathione S-transferase; 4-OOHIF, 4-hydroperoxyifosfamide; 4-OHCP, 4-hydroxycyclophosphamide; aldoIF, aldoifosfamide; PM, phosphoramide mustard; PMGS, monoglutathionyl phosphoramide mustard; 4-OHIF, 4-hydroxyifosfamide; IM, ifosfamide mustard; FAB-MS, fast atom bombardment mass spectrometry; 4GSCP, 4-glutathionyl cyclophosphamide; IMGS, monoglutathionyl ifosfamide mustard; GSCB, monoglutathionyl chlorambucil.

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352 Chem. Res. Toxicol., Vol. 9, No. 2, 1996

Figure 1. Chemical structures for a number of alkylating cytostatic drugs mentioned in this review.

tween rodent and human GST exist, mainly studies using human GST are discussed.2

Reactions of Nitrogen Mustard Cytostatic Drugs with Glutathione The serendipitous observation that victims of mustard gas used during World War I often had leucopenia in addition to severe damage to the mucous membranes and skin blistering (26) can be considered as one of the first observations on the biological activity of alkylating agents in rapidly proliferating tissues (2). In the Second World War, as part of a program to develop improved chemical warfare agents, nitrogen mustard (mechlorethamine) was developed. From 1942 this compound was used in clinical trials in the treatment of leukemia, and the results were published in 1946 (27). In cytostatic drugs with a nitrogen mustard group, the biological efficacy is closely related to the reactivity of the functional 2-chloroethyl groups. This reactivity is linked to the basicity of the corresponding nitrogen atoms. Substitution of electrophilic groups at the amino nitrogen reduces the basicity and thus reduces the reactivity of the functional groups, thereby increasing the chemical half-life of the drug in blood. Mechlorethamine. Mechlorethamine is a structural analog of chemical warfare agents like sulfur mustard in which the sulfur atom was replaced by an amide to produce a less reactive substance. Mechlorethamine is used intravenously, primarily in the treatment of advanced Hodgkin’s disease, almost exclusively in combination chemotherapy (28). Mechlorethamine exerts its cytotoxic effect through the alkylation of cellular components. This alkylation is believed to proceed via a twostep mechanism (Figure 2). The sequence is initiated by the nucleophilic displacement of chloride by the bis(chloroethyl)amine nitrogen to form the reactive aziridinium ion. 2Human glutathione transferases GSTA1-1 and GSTA2-2 belong to the alpha class, GSTM1a-1a belongs to the mu class, and GSTP1-1 belongs to the pi class (92).

Dirven et al.

Figure 2. Reactions of mechlorethamine with water and glutathione according to Gamcsik et al. (15). 1 ) monohydroxy mechlorethamine, 2 ) monoglutathionyl mechlorethamine, 3 and 4 are aziridinium intermediates, 5 ) dihydroxy mechlorethamine, 6 ) monohydroxy, monoglutathionyl mechlorethamine, and 7 ) diglutathionyl mechlorethamine.

The aziridinium ion is a strong electrophile. It can react with nucleophiles present in proteins and DNA and also with glutathione. In aqueous solution, mechlorethamine is quite reactive. At 30 °C in 0.05 M phosphate buffer (pH 7.0) the half-life time of mechlorethamine is 5.5 min (15). The aziridinium ion formed reacts rapidly with water, leading to the formation of hydrolysis products. In the presence of GSH, both mono- and diglutathionyl conjugates were formed. Gamcsik et al. (15) showed that these conjugation reactions, indeed, proceed through an aziridinium intermediate. No information on the role of GST in these reactions is available. Chlorambucil. A reduction of the reactivity of, for example, mechlorethamine was achieved by coupling of the nitrogen mustard group to a benzene ring. Chlorambucil (Figure 1) is used alone or as a component of various chemotherapeutic regimens in the treatment of mainly chronic lymphocytic leukemia, malignant non-Hodgkin’s lymphomas, and advanced Hodgkin’s disease (28). This compound does not require metabolic (i.e., enzymatic) activation to exert its cytotoxic effect. The major mechanism by which chlorambucil enters and exits cells appears to be passive diffusion (29). The major metabolite of chlorambucil (phenylacetic acid mustard) is also a bifunctional alkylating compound with activity against neoplastic cell lines. Chlorambucil reacts with nucleophiles by the formation of aziridinium compounds in analogy to mechlorethamine. Glutathionyl conjugates of chlorambucil were first characterized by Dulik et al. (16). After incubation of chlorambucil in PBS at 37 °C in the presence of a 45fold excess of glutathione, mainly mono- and diglutathionyl conjugates were found, and no mono- and dihydroxy conjugates (23). With human GST, it was shown that at pH 6.5 only GST alpha and to a lesser extent GST pi catalyzed the formation of monoglutathionyl chlorambucil (GSCB) (24). Km values were 19, 220, and 830 µM for GSTA1-1, A2-2, and P1-1, respectively (24). Meyer et al. (23) performed

Invited Review

Figure 3. Reactions of melphalan with glutathione (32, 53).

experiments using approximately physiological amounts of human glutathione S-transferases at pH 7. Based on the disappearance of chlorambucil, a catalysis of 5-10% of the spontaneous rate was found with 52 µM GSTA11, but not with 24 µM GSTA2-2, 30 µM GSTP1-1, and 25 µM GSTM1a-1a. However, based on the formation of GSCB, a more pronounced catalytic effect of GSTA1-1 was found. It was proposed that noncovalent binding of GSTA1-1 to GSCB protects this intermediate from further reactions. Meyer et al. (23) showed that, indeed, monoglutathionyl chlorambucil was a competitive inhibitor of the activity of GSTA1-1/A2-2 (Ki’s were 2.2 and 1.2 µM, respectively) but not of GSTP1-1 and M1a-1a (Ki’s of 10 µM). Melphalan (Phenylalanine Mustard). Melphalan is the L-isomer of the phenylalanine derivative of mechlorethamine. It was designed in the hope that its resemblance to phenylalanine, a precursor of melanin, would facilitate its uptake in melanoma cells. Melphalan is used alone or as a component of various chemotherapeutic regimens in the treatment of mainly multiple myeloma and ovarian carcinoma (28). The entry of melphalan into cells is mediated by two energy-dependent transport systems, both of which are normally engaged in the transport of amino acids (30). Thus, melphalan is actively concentrated in cells, resulting in much higher intracellular concentrations than found for chlorambucil. The formation of the aziridinium ion intermediate from melphalan is the rate-determining step for the formation of monohydroxy melphalan and monoglutathionyl melphalan (25) (Figure 3). Glutathionyl conjugates of melphalan were first characterized by Dulik et al. (17). The reaction of the aziridinium ion of melphalan in the presence of 2.5-5 mM glutathione (pH 7.4) was found to proceed at a 5.5-11-fold greater rate, respectively, than the reaction of the aziridinium ion with water (25). At pH 6.5 the contribution of GSH to the detoxification of melphalan was only as high as that of water (25). The addition of 40 µM GST alpha increased the formation of monoglutathionyl melphalan, 9-fold at pH 6.5 and 2.5-fold at pH 7.4 (25). Evidence was presented that the alpha GST-monoglutathionyl melphalan complex slowly dissociates (25). In a Chinese hamster ovary cell line with a 20-fold acquired resistance to melphalan, an increased expression of an alpha form of GST was found. This GST was

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purified and shown to accelerate the formation of glutathione-melphalan adducts (31). In addition to mono- and diglutathionyl melphalan, the formation of (4-glutathionyl)phenylalanine has been described. The formation of this conjugate is apparently catalyzed by microsomal GST (32). Cyclophosphamide. In an attempt to target nitrogen mustard compounds to tumor cells, cyclophosphamide was developed. It was hoped that cyclophosphamide would be specifically activated by tumor enzymes (review: ref 33). Later, it was shown that cyclophosphamide is activated by hydroxylation to 4-hydroxycyclophosphamide (4-OHCP), a reaction mainly catalyzed by hepatic cytochrome P450s. Cyclophosphamide is used alone and as a component of various chemotherapeutic regimens in the treatment of Hodgkin’s disease and malignant lymphomas, multiple myeloma, leukemia, neuroblastoma, ovarian and breast carcinoma, and retinoblastoma (28). Cytochrome P450s belonging to the 2B and 2C family were identified as major catalysts of cyclophosphamide activation (34). 4-OHCP equilibrates with the ringopened aldophosphamide, which spontaneously decomposes to phosphoramide mustard (PM) and acrolein (Figure 4). It is proposed that 4-OHCP can enter cells, while PM cannot, so 4-OHCP might be considered as the carrier molecule of PM across cell membranes (35). Phosphoramide mustard possesses DNA-alkylating activity and is generally considered to be the therapeutically significant cytotoxic metabolite of cyclophosphamide. Phosphoramide mustard reacts with nucleophiles through the formation of an aziridinium ion formed by the loss of a chlorine atom (18). The pKa value of phosphoramide mustard is 4.9, and theoretical calculations indicated that the aziridinium ion formed from PM is an unstable species, unlikely to be detected in solution (36). Two types of glutathionyl conjugates of cyclophosphamide have been described, i.e., mono- and diglutathionyl PM, and 4-glutathionyl cyclophosphamide (4-GSCP) (1820). Furthermore, the kinetics of the nonenzymatic formation of both conjugates has been studied using 31P NMR spectrometry (37). Four stereoisomeric 4-GSCP conjugates were found. The formation of 4-GSCP was found to be reversible, and upon hydrolysis PM was being formed. 4-GSCP might therefore be considered to be a stabilized reservoir of activated cyclophosphamide for the production of PM (37-39). All GSTs tested enhanced the formation of 4-glutathionyl cyclophosphamide 2-4 times above the nonenzymatic rate (pH 7.0). Km values were in the range of 1.0-1.9 mM for GSTA2-2, M1a-1a, and P1-1, and 0.3 mM for GSTA1-1 (20). Conjugation of PM was found to proceed by the formation of monoglutathionyl phosphoramide mustard (PMGS) and subsequently diglutathionyl PM. The formation of the aziridinium ion was the rate-limiting step in the reaction with nucleophiles (37). Formation of monoglutathionyl PM was only catalyzed by GSTA1-1, but not by GSTA2-2, M1a-1a, or P1-1. At pH 7.0, in the presence of 40 µM A1-1 the rate of formation of the monoglutathionyl phosphoramide conjugate was 2-fold increased above the spontaneous level (pH 7.0) (20). Ifosfamide. Ifosfamide is a compound that differs in only one aspect from cyclophosphamide. In cyclophosphamide both 2-chloroethyl groups are linked to the extracyclic nitrogen, while in ifosfamide only one 2-chloroethyl group is linked to the extracyclic nitrogen, while


Dirven et al.

Figure 4. Reactions of cyclophosphamide with glutathione (20).

Figure 5. Reactions of ifosfamide with glutathione (21).

the other is linked to the endocyclic nitrogen (Figure 1). The metabolism of IF is quite similar to the metabolism of CP (Figure 5). IF is activated to 4-hydroxyifosfamide (mediated by enzymes from the cytochrome P450 3A family (26, 40)). The ultimate alkylating metabolite formed is ifosfamide mustard. N-Dechloroethylation reactions play a more important role in the metabolism of IF compared to CP (review: ref 41). It has been proposed that 4-OHIP can enter cells, while IM cannot enter cells due to its polar nature (42).

No evidence for the formation of 4-glutathionyl ifosfamide was found (21). Kwon et al. (43) described two mechanisms for the formation of 4-thioCP conjugates. One mechanism (a base-catalyzed elimination of a proton from the endocyclic nitrogen leading to the formation of iminocyclophosphamide) is not possible with IP, since no proton is present on the endocyclic nitrogen (Figure 5). The other mechanism involves the formation of a hemithioacetal from aldoifosfamide that subsequently cyclizes to 4-thio conjugates. It was proposed that the glutathio-

Invited Review

Figure 6. Reactions of thiotepa with glutathione (22).

nyl aldoifosfamide conjugate can be formed (21), but that ring closure is prevented because of steric hindrance by the chloroethyl group at the endocyclic nitrogen atom. The in vitro half-life time for IM is larger than that for PM (21, 37, 44), but the intracellular half-life time for IM was the same as that for PM (45). Ifosfamide mustard reacts with nucleophiles via the formation of an aziridinium intermediate (21). In contrast to the aziridinium ion formed from PM, the aziridinium ion formed from IM can deprotonate upon formation, leading to the formation of a (noncharged) aziridine species. This intermediate (N-(2-chloroethyl),N′-aziridinylphosphorodiamidic acid) is relatively stable and was recently characterized (21). The formation of this intermediate provides evidence that the alkylating reactions of ifosfamide indeed proceed via the formation of aziridinium ions. At pH 7.0, the formation of monoglutathionyl ifosfamide mustard was 3-5-fold catalyzed by GSTP1-1, but not by the other major human GST isoenzymes tested (A1-1, A2-2, and M1a-1a) (21). In the pH range 5.5-7 the formation of monoglutathionyl ifosfamide mustard was up to 2-10-fold increased above spontaneous levels by GSTP1-1 (21). Thiotepa. Thiotepa was first synthesized in 1952 and underwent clinical trial in the 1960s which indicated it was active against a wide variety of tumors. Recently, renewed interest in this drug in combination with bone marrow transplantation was raised (46). Thiotepa is an alkylating agent containing a fourcoordinated phosphorus atom and three aziridine moieties (Figure 1). Cell death is probably the result of formation of cross-links within DNA (47, 48). The reactions of thiotepa have been studied by 31P NMR (22). Thiotepa is quite a stable compound in aqueous solution at pH 7.4 (t1/2 ) 3300 min). In the presence of glutathione, the rate of disappearance of thiotepa increased greatly (t1/2 ) 282 min). Both mono- and diglutathionyl conjugates of thiotepa were characterized (Figure 6) (22). GSTA1-1 and P1-1, but not GSTM1a-1a and A2-2, increased the rate of formation of monoglutathionyl thiotepa (22). For both GSTA1-1 and P1-1 the Km for the formation of monoglutathionyl thiotepa was relatively high (5-7 mM). However, this is not unusual for hydrophillic substrates like thiotepa. GSTA1-1 did not increase the rate of formation of diglutathionyl thiotepa, suggesting that only thiotepa and not its monoglutathionyl conjugate is a substrate for GSTs (22). Also, the


Figure 7. Influence of the pH on the GSTP1-1 and A1-1 dependent formation of monoglutathionyl thiotepa. Thiotepa (0.2 mM) was incubated with 1 mM GSH without or with 10 µM GSTP1-1/A1-1 for 45 min. O min GST, b GSTP1-1, and 9 GSTA1-1.

formation of monoglutathionyl conjugates of tepa, the major (oxidized) metabolite of thiotepa, was strongly enhanced by GSTA1-1 and P1-1 (22). The nonenzymatic reaction of the aziridinium moieties of thiotepa with glutathione is strongly dependent on the pH. In the pH range 5.7-7.0 no nonenzymatic formation of monoglutathionyl thiotepa was observed. GSTA1-1 and P1-1 increased the rate of formation of monoglutathionyl thiotepa in the pH range 5.5-8.5 (Figure 7) (22).

Role of GST Theta Enzymes in the Glutathione Conjugation Reactions with Alkylating Agents In addition to GST alpha, mu, and pi, a fourth cytosolic GST family has been described recently, i.e., theta. Two catalytically rather distinct isoenzymes of rat GST theta have been purified, i.e., Yrs-Yrs and GST5-5 (49, 50). The human counterpart of GST5-5 is GSTT1-1 (51). The role of GST5-5 in the conjugation reactions of alkylating cytostatic agents has been studied (Table 1).3 Compared to incubations without GSTs, the formation of monoglutathionyl thiotepa was 80-fold increased by 10 µM GST55. The formation of monoglutathionyl ifosfamide mustard was slightly but significantly (1.2-fold) enhanced by GST5-5. In contrast, the formation of monoglutathionyl conjugates of either phosphoramide mustard or melphalan was not catalyzed by GST5-5. These results show that the aziridine groups present in thiotepa are good substrates for GST theta and the aziridine group or the aziridinium group in ifosfamide mustard is a weak substrate for GST theta enzymes. At present, nothing is known about the presence of GST theta in tumors. It is therefore difficult to predict the role of GST theta enzymes in the development of drug resistance.

Human GST Isoenzyme Specificity for Nitrogen Mustard Compounds The rate-limiting event in the conjugation reactions of chlorambucil, melphalan, IM, and PM is the formation of the aziridinium intermediate (49, 25, 37, 21). The aziridinium/aziridine intermediate of IM is a substrate for GSTP1-1 (21), and the aziridine/aziridinium intermediate of thiotepa is a substrate for both GSTP1-1 and GSTA1-1 (22), while the aziridinium intermediates of 3Dirven

et al., unpublished results.


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Table 1. Role of GST Alpha, Pi, and Theta (10 µM) in the Formation of Monoglutathionyl Conjugates of Phosphoramide Mustard, Ifosfamide Mustard, Melphalan, and Thiotepaa,b phosphoramide mustard ifosfamide mustard melphalan thiotepa

min GST

GSTA1-1

GSTP1-1

GST5-5

29.0 ( 1.8 20.9 ( 0.6 11.4 ( 0.5 0.3 ( 0.2

36.9 ( 1.2 (P ) 0.007) ND 14.1 ( 0.3 (P ) 0.04) ND

ND 29.0 ( 0.6 (P ) 0.006) ND 29.9 ( 4.9 (P ) 0.008)

31.0 ( 0.6 (P ) 0.25) 24.6 ( 0.37 (P ) 0.0319) 13.8 ( 0.8 (P ) 0.13) 24.7 ( 1.0 (P ) 0.0001)

a All activities are expressed as µM monoglutathionyl product formed. The GST-catalyzed product formation compared to the spontaneous product formation was tested for statistical significance using the paired t-test. P values of 0.05 are considered to be of statistical significance. ND ) not determined. b 1 mM PM was incubated for 24 min with 1 mM GSH and analyzed as described before (20). 2 mM IM was incubated for 60 min with 1 mM GSH and analyzed as described before (21). 0.2 mM thiotepa was incubated for 60 min with 1 mM GSH and analyzed as described before (22). 0.1 mM melphalan and 1 mM GSH were incubated for 60 min and analyzed as described by Bolton et al. (53).

Figure 8. Model of the role of glutathionyl conjugates of cyclophosphamide in the metabolism of this drug (37, 21, 39).

chlorambucil, melphalan, and phosphoramide mustard are substrates for only GSTA1-1 (24, 23, 25, 20, 53). The aziridinium intermediate of IM might deprotonate upon formation, leading to the formation of an aziridine group (21). The aziridinium intermediates of melphalan, chlorambucil, and PM cannot undergo these deprotonation reactions. It is thus tempting to speculate that the isoenzyme selectivity is determined by the aziridine/ aziridinium equilibrium and thus in the case of IM and thiotepa on the extent of protonation. However, quantum chemical calculations performed thus far, on the aziridinium intermediates of PM and IM as well as on the noncharged aziridine intermediate of IM, indicate that net atomic charges are unlikely to influence the isoenzyme specificity of the GST-catalyzed conjugation reactions (Table 2). Since in ifosfamide mustard the two chloroethyl groups are not linked to the same nitrogen atom as in melphalan, chlorambucil, and phosphoramide mustard, steric differences might also explain the observed differences in isoenzyme specificity. Further studies on the differential selectively are clearly warranted.

Role of GSTs in the Biotransformation of Cytostatics: An Example Knowledge on the formation of glutathione conjugates of nitrogen mustard cytostatic drugs in vivo is limited.

Table 2. Net Atomic Charges for Selected Atoms in the Aziridinium Forms of Phosphoramide Mustard (A) and Ifosfamide Mustard (B) and in N-(2-Chloroethyl), N′-aziridinyl Ifosfamide Mustard (C)a PdO PdO PsOH PsOH N-azirid. C-azirid. N-amido Cl q(aziridine)b q(phosphoryl)b P-N bond order

A

B

C

+2.67 -1.05 -0.81 +0.32 -0.52 -0.10 -0.99 -0.011 +0.31 +0.69 0.442

+2.66 -1.03 -0.81 +0.33 -0.56 -0.11 -0.93 -0.112 +0.29 +0.71 0.442

+2.80 -1.13 -0.83 +0.27 -0.94 -0.04 -0.92 -0.11 -0.56 +0.56 0.612

a The charge distribution was computed using the semiempirical AM1-Hamiltonian with limited configuration interaction over four frontier molecular orbitals using the AMPAC 2.1 (QCPE) program. b These values were computed by adding all net atomic charges for those atoms bonded to the aziridine moiety and the phosphoryl moiety, respectively.

However, we can speculate about the role of these glutathione conjugations in vivo, for example, for cyclophosphamide (Figure 8). Upon hydroxylation of CP by cytochrome P450 in the liver, 4-OHCP is formed. 4-OHCP can conjugate with GSH, leading to the formation of 4-GSCP. This conjugation might occur nonenzymatically, but is also catalyzed by all major classes of cytosolic GSTs, of which the alpha and mu class enzymes are

Invited Review

abundantly present in human liver (54). We postulate that 4-GSCP acts as a transport 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 reconjugate with GSH. This reaction occurs nonenzymatically but is also catalyzed by GST alpha, mu, and pi enzymes (20). 4-GSCP is possibly transported out of the cell by an ATP-dependent glutathione S-conjugate export pump (55, 56). 4-GSCP also equilibrates with 4-OHCP/aldophosphamide, giving rise to PM and acrolein. PM can conjugate with glutathione either nonenzymatically or catalyzed by GSTA1-1. Monoglutathionyl PM cannot form DNA crosslinks, while diglutathionyl PM cannot interact with DNA at all. Monoglutathionyl PM is again possibly transported out of the cell by the glutathione S-conjugate export pump. The proposed model shows that both glutathione levels and the presence of GST, and especially GSTA1-1, can influence the concentration of aziridinium intermediates formed from PM and hence the cytotoxicity of cyclophosphamide. Recent studies in a human breast cancer cell line support this hypothesis (57).

Nonenzymatic vs GST-Catalyzed Glutathione Conjugation A number of studies showed that GSH protects against the cytotoxicity of alkylating agents (58-65). An increased level of GSH in tumor cells is probably the result of an increased intracellular GSH synthesis due to increased expression of γ-glutamylcysteine synthetase and/or γ-glutamyl transpeptidase (63, 66). The aziridinium ion is highly polarized, carrying a high positive charge density at the electrophilic center, and is defined as a hard electrophile. Glutathione is a soft nucleophile because the larger atomic volume of sulfur gives rise to a diffuse electron density which is polarizable. Electrophiles will tend to react most rapidly with nucleophiles of similar hardness (67). The aziridinium ion will therefore more favorably react with hard nucleophiles like the oxygen of phosphates and amino groups of proteins than with thiol groups in, for example, GSH (67). GST catalyzes the conjugation of aziridinium/ aziridine groups with GSH by bringing the two into close proximity, creating a conductive hydrophobic environment and reducing the apparent Ka of the thiol group in the cysteine group of GSH to a more neutral value (4). These factors contribute to an increase in rate and extent of the enzyme-catalyzed conjugation compared to the spontaneous reaction. Because the interior of tumors has often a poor vascularization, leading to decreased availability of oxygen and a high glycolysis activity, the pH in parts of the tumor might be lower than that of normal cells (68). The GST-catalyzed formation of IMGS, PMGS, monoglutathionyl thiotepa, and monoglutathionyl melphalan in the pH range 6-7.4 is independent of the pH (25, 2022), while the spontaneous formation is strongly reduced at these pH’s, suggesting that expression of GSTs in tumors might result in an enhanced detoxification.

GST Expression in Cell Lines and Tumors A concentration of 30-240 µM GST has been reported in tumors and tumor cell lines (69-71). In most human


tumors and cell lines, GST pi is the major isoenzyme expressed. GST pi is often increased in colon, stomach, esophagus, uterine, cervix and lung tumors. In liver and kidney tumors, GST alpha is often decreased. In breast in lymphocytic leukemia the relative levels of GSTs alpha, mu, and pi are not altered (review: ref 72). However, in tumors from lung, ovarian, and liver a substantial expression of GST alpha was found (73). In ovarian carcinoma samples an alpha class GST concentration of 5-10 µM was found (74), but the concentration of GST alpha in ovarian tumors was lower than in normal ovarian tissue (75, 71). In esophageal tumors an increase in GST alpha was found (70). Several alkylating cytostatic drug-resistant cell lines have been shown to overexpress GST alpha enzymes (7, 8, 76-79). It is often considered that enhanced resistance to a particular drug must be shown in a transfected cell line in order for the gene product to be causatively linked to the resistance phenotype. Tew (4) gave a number of factors why GST transfection in some cell lines does not (always) produce significantly enhanced drug resistance, for example, substrate specificity of the transfected GST for the alkylating agent tested, presence and/or efficiency of a GSH conjugate membrane efflux pump, effective compartmentalization of GST/GST, and intrinsic GSH/ GST levels. The introduction of rat or human GST alpha genes into lines of cultured cells conferred resistance toward alkylating agents (80-82). Transfection of GST P1-1 into NIH 3T3 cells failed to alter their sensitivity to chlorambucil and melphalan (83). Leyand-Jones et al. (84) were unable to detect an alteration in chlorambucil sensitivity in a human breast tumor cell line stably transfected with a human alpha class GST. Thus, there is conclusive evidence that GST pi is expressed in tumors. In addition, a number of tumors express GST alpha enzymes. Overexpression of both GST alpha and pi enzymes is found in cell lines which are resistant to alkylating agents. In vitro studies have shown that both GST alpha and pi enzymes play a role in the formation of glutathionyl conjugates of alkylating cytostatic drugs. This suggests that the presence of GST alpha and pi in tumors contribute to an enhanced detoxification of alkylating cytostatic drugs and hence to the development of drug resistance.

Conclusions and Future Directions The critical event in the development of resistance to alkylating cytostatic drugs is the ability of tumor cells to lower the concentrations of aziridinium ions present in the cytoplasm. This can be achieved by lowering the concentration of the precursor of the aziridinium ions (like, for example, phosphoramide mustard, ifosfamide mustard, melphalan, chlorambucil, and thiotepa) or by effective scavenging of the aziridinium ions formed. The intracellular chloride concentrations of 8 mM predict that aziridinium formation is essentially irreversible (4). The concentration of alkylating cytostatic drugs in tumor cells is likely to be very low; the concentration of the aziridinium ions derived from these drugs is even lower. It can be assumed that the intracellular concentration of both GSH and GST is much higher than the concentration of aziridinium ions, favoring both enzymatic and nonenzymatic glutathione-dependent detoxification of these drugs. The relative importance of the GST-


catalyzed formation of glutathionyl conjugates is dependent on the class of GST present in the tumor cells, the concentration of GSTs, and the drug used in the treatment. Expression of GST alpha enzymes might contribute to the development of drug resistance toward melphalan, chlorambucil, phosphoramide mustard, and thiotepa. The presence of GST pi might be an important factor in the development of drug resistance toward ifosfamide mustard and thiotepa. An important factor to be considered is the fate of the GSH conjugates in cells. Not much is known about the alkylating activities of monoglutathionyl conjugates of alkylating agents. In principle, the monoglutathionyl conjugates of the nitrogen mustards and thiotepa could still alkylate cell macromolecules, while 4-GSCP can reform 4-OHCP and thus serve as a transport form for the alkylating agent. In any event, the organ and cell selectivity of the glutathione conjugates are likely to be quite different from that of the parent compounds (review: ref 85). Meyer et al. (23) showed that CHBSG did not alkylate plasmid DNA, but did alkylate other cell macromolecules. To prevent protein/DNA alkylation, the monoglutatyhionyl conjugates of nitrogen mustard alkylating cytostatic drugs should be removed from the tumor cells. An ATPdependent transport mechanism for cisplatin glutathionyl conjugates has been described by Ishikawa and AliOsman in leukemia cells (55). Active accumulation of glutathionyl conjugates into membrane vesicles from the endocytic-secretory pathway and exocytose are probably involved in this transport (86). Recently, the multidrug resistance-associated protein (MRP) was identified as a glutathione S-conjugate carrier in leukemia and in lung carcinoma cells (56, 87). Increased amounts of MRP have been detected in several drug-selected cell lines that do not overexpress P-glycoprotein (88). It is likely that both increases in the GSH and/or GST concentration in cells and expression of MRP can be considered as part of a mechanism in which tumor cells become resistant toward alkylating cytostatic drugs. If the intracellular concentration of monoconjugates is low due to an effective export of conjugates by a plasma membrane ATP-dependent system, cellular damage resulting from protein alkylation is expected to be of minor importance. If the export of monoconjugates is slow, the intracellular concentration of monoconjugates will be high and protein alkylation and probably inhibition of GST alpha enzymes (as found by Meyer et al. (23) for GSCB) are likely to occur. More work on the distribution and kinetics of glutathionyl conjugates in cells is needed. In this respect, it is also important to obtain data on the fate of the monoglutathionyl conjugates in cellular compartments, including the nucleus. Cells which have increased levels of GSH/GSTs are often cross-resistant to a number of alkylating agents (10, 89, 90), suggesting that this resistance is determined by common biological mechanisms. If GSTs do indeed play a role in the development of drug resistance toward alkylating drugs, the findings that phosphoramide mustard is detoxified by GST alpha enzymes and ifosfamide mustard is detoxified by GST pi enzymes suggest that tumor cells which are resistant to phosphoramide mustard should still respond to a treatment with ifosfamide mustard. This might be a determinant in the observed lack of cross-resistance between CP and IP in experimental tumors (41). A better understanding of GST

Dirven et al.

substrate specificities with anticancer alkylating agents can explain alkylating agent cross-resistance patterns and may eventually be useful in detecting cross-resistant phenotypes in individual patients from GST assays on tumor biopsy specimens. More information is needed on the isoenzyme specificity for these glutathione conjugation reactions. Molecular modeling studies using the published crystal structures of GSTA1-1 and P1-1 (review: ref 91) can give clues about groups present in the cytostatic drugs that are important for enzymatic detoxification. This knowledge might lead to the development of cytostatic drugs that are not substrates (or are poor substrates) for GSTs.

Acknowledgment. This study was supported by Grant TNOV-92-93 of the Dutch Cancer Society. We would like to thank Dr. R. A. van Gurp of the Prins Maurits Laboratory of TNO in Rijswijk, The Netherlands, for performing the atomic charge calculations.

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TX950143C

Glutathione Conjugation of Alkylating Cytostatic Drugs with a Nitrogen

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