Metabolism of chlorambucil by rat liver microsomal glutathione S-transferase

Chemico-Biological Interactions 149 (2004) 61–67

Metabolism of chlorambucil by rat liver microsomal glutathione S-transferase Jie Zhang, Zhiwei Ye, Yijia Lou∗ Department of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310031, China Received 10 April 2003; received in revised form 11 July 2003; accepted 11 July 2003

Abstract Clinical efficacy of alkylating anticancer drugs, such as chlorambucil (4-[p-[bis [2-chloroethyl] amino] phenyl]-butanoic acid; CHB), is often limited by the emergence of drug resistant tumor cells. Increased glutathione (␥-glutamylcysteinylglycine; GSH) conjugation (inactivation) of alkylating anticancer drugs due to overexpression of cytosolic glutathione S-transferase (GST) is believed to be an important mechanism in tumor cell resistance to alkylating agents. However, the potential involvement of microsomal GST in the establishment of acquired drug resistance (ADR) to CHB remains uncertain. In our experiments, a combination of lipid chromatography/electrospray ionization mass spectrometry (LC/ESI/MS) was employed for structural characterization of the resulting conjugates between CHB and GSH. The spontaneous reaction of 1 mM CHB with 5 mM GSH at 37 ◦ C in aqueous phosphate buffer for 1 h gave primarily the monoglutathionyl derivative, 4-[p-[N-2-chloroethyl, N-2S-glutathionylethyl] amino]phenyl]-butanoic acid (CHBSG) and the diglutathionyl derivative, 4-[p-[bis[2-S-glutathionylethyl] amino]phenyl]-butanoic acid (CHBSG2 ) with small amounts of the hydroxy-derivative, 4-[p-[N-2-S-glutathionylethyl, N-2hydroxyethyl] amino]phenyl]-butanoic acid (CHBSGOH), 4-[p-[bis[2-hydroxyethyl] amino]phenyl]-butanoic acid (CHBOH2 ), 4-[p-[N-2-chloroethyl, N-2-S-hydroxyethyl]amino]phenyl]-butanoic acid (CHBOH). We demonstrated that rat liver microsomal GST presented a strong catalytic effect on these reactions as determined by the increase of CHBSG2 , CHBSGOH and CHBSG and the decrease of CHB. We showed that microsomal GST was activated by CHB in a concentration and time dependent manner. Microsomal GST which was stimulated approximately two-fold with CHB had a stronger catalytic effect. Thus, microsomal GST may play a potential role in the metabolism of CHB in biological membranes, and in the development of ADR. © 2004 Published by Elsevier Ireland Ltd. Keywords: Chlorambucil; Microsomal glutathione S-transferase; Acquired drug resistance Abbreviations: CHB, chlorambucil (4-[p-[bis [2-chloroethyl] amino] phenyl]-butanoic acid); GSH, glutathione (␥-glutamylcysteinylglycine); CHBSG, 4-[p-[N-2-chloroethyl, N-2-S-glutathionylethyl] amino]phenyl]-butanoic acid; CHBSG2 , 4-[p-[bis[2-S-glutathionylethyl] amino]phenyl]-butanoic acid; CHBSGOH, 4-[p-[N-2-S-glutathionylethyl, N-2-hydroxyethyl] amino]phenyl]-butanoic acid; CHBOH2 , 4-[p-[bis[2-hydroxyethyl] amino]phenyl]-butanoic acid; CHBOH, 4-[p-[N-2-chloroethyl, N-2-S-hydroxyethyl]amino]phenyl]-butanoic acid; GST, glutathione S-transferase; ADR, acquired drug resistance; NEM, N-ethylmaleinimid; LC/ESI/MS, lipid chromatography/electrospray ionization mass spectrometry; CDNB, 1-chloro-2,4-dinitrobenzene. ∗ Corresponding author. Tel.: +86 571 87217206; fax: +86 571 87217206. E-mail address: [email protected] (Y. Lou) 0009-2797/$ – see front matter © 2004 Published by Elsevier Ireland Ltd. doi:10.1016/j.cbi.2003.07.002

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1. Introduction Alkylating agents, such as chlorambucil (4-[p-[bis [2-chloroethyl] amino] phenyl]-butanoic acid (CHB), are extensively used in the treatment of neoplastic diseases, such as chronic lymphocytic leukemia, ovarian and head/neck carcinomas, but their effectiveness is often limited by the emergence of acquired drug resistance (ADR), in which repeated dosing of the agent results in a lack of target cell cytotoxicity and therefore, reduced clinical effectiveness. The development of ADR has been associated with several possible factors: altered membrane functions that result in decreased cellular drug uptake or enhanced transport of the drug out of the cell, altered nuclear phenomena such as enhanced DNA repair or decreased drug binding and cytoplasmic phenomena such as decreased activation of a prodrug or increased detoxification of an active metabolite. In the case of the alkylating agents, inducible enzymatic systems for detoxification of the drugs are a major mechanism whereby cells may acquire resistance [1,2]. Both elevated ␥-glutamylcysteinylglycine (GSH) levels and increased activity of the glutathione Stransferase (GST) isozymes have been associated with the resistance of cells to alkylating agents. GSH is the most abundant intracellular nucleophile in mammalian cells, present at concentrations of approximately 5 mM. Several studies have suggested a relationship between GSH and the development of ADR [3–6]. Among these studies is the inactivation of the alkylating agents by conjugation with glutathione. This conjugation can be catalyzed by GST isozymes. The sulfur atom of GSH provides electrons for nucleophilic attack on an electrophilic substrate, with the formation of a thioether. The dichloroethylamino nitrogen mustards appear to alkylate through an aziridinium ion intermediate [7,8], which has strong electrophilic properties and is a likely substrate for GSH conjugation. Multiple reports have confirmed the existence of glutathionyl adducts of the nitrogen mustards [9–12]. The GST isozymes play a central role in the protection of cells from cytotoxic chemicals and have a putative role in the acquired resistance of tumors to alkylating agents. They are found largely in the cytosol as homodimers or heterodimers, and in the rat over 20 different GST subunits have been identified [13]. However, a membrane-bound GST that accounts for up to 3% of microsomal protein also has

been identified [14,15]. This GST isoform bears no obvious structural resemblance (amino acid sequence, molecular weight, or immunological properties) to the cytosolic GSTs. The microsomal GST exists as trimer of identical 17.2 kDa subunits [16–19]. Unlike the cytosolic GSTs, microsomal GST activity is increased by partial proteolysis [20] or by sulfhydryl reagents, such as NEM, that bind covalently to the sole cysteine residue (Cys49) in each polypeptide [21,22]. It is believed that GST catalyzed GSH conjugation of alkylating agents played a pivotal role in the development of acquired drug resistance [2]. Increased GSH conjugation in the presence of cytosolic GSTs in vitro has been demonstrated for several important chemotherapeutic drugs, especially alkylating agents such as melphalan [9] and CHB [12]. Numerous studies suggest that enhanced expression of cytosolic GSTs may contribute to the development of cellular resistance. It has been assumed that the increased expression of ␣-class cytosolic GSTs are primarily responsible for the acquired drug resistance [23–26]. Other studies have suggested that cytosolic GST-␲ and -␮ are probably the cause of resistance [27–30]. However, the relationship between activation of microsomal GST and the development of ADR is still uncertain. The objective of this study is to examine the potential involvement of microsomal GST in the establishment of ADR to CHB. We investigated the ability of CHB to activate rat liver microsomal GST activity, and synthesized and characterized the reaction products between CHB and GSH. Conjugates of CHB were prepared under non-enzymatic and enzymatically conditions using non-activated and activated rat liver microsomal GST with CHB. LC/ESI/MS was employed for structural characterization of the resulting conjugates.

2. Materials and methods 2.1. Materials CHB was purchased from the Sigma (St. Louis, MO, USA); reduced GSH and 1-chloro-2,4-dinitrobenzene (CDNB) were obtained from the Sigma (St. Louis, MO, USA); NEM was purchased from Merck (Darmstadt, Germany); other chemicals were obtained from various sources at the highest quality.

J. Zhang et al. / Chemico-Biological Interactions 149 (2004) 61–67

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2.2. Isolation of microsomal membranes

2.5. HPLC assay

Male Sprague–Dawley rats weighing 180–220 g were starved overnight and liver microsomes were prepared as described previously [31]. The microsomes were washed twice with 0.15 M Tris–Cl, pH 8.0, in order to remove cytosolic contamination [32]. Washed and unwashed microsomes were resuspended in 0.1 M phosphate buffer, pH 7.4.

CHB hydrolysis products and GSH adducts were separated by HPLC as described previously [11], using a Shimadzu LC-10ATvp gradient-controlled HPLC system with a Shimadzu SPD-10AVvp UV–vis detector (254 nm), a Shimadzu CTO-10ASvp column oven (25 ◦ C), a C18 Dikma Diamonsil ODS reverse-phase column (5 ␮m; 250 mm × 4.6 mm), a gradient mobile phase (0.1 mM ammonium acetate:methanol, 10 to 100% in 30 min) at a flow rate of 1.0 ml/min. The sample injection volume was 10 ␮l.

2.3. Assay of microsomal GST activity Microsomal GST activity was determined spectrophotometrically according to the method of Habig et al. [33]. The assay was performed in 0.1 M potassium phosphate buffer, pH 6.5, at 25 ◦ C using 1.0 mM GSH and 1.0 mM CDNB as substrates. The assay buffer also contained 0.5% Triton X-100 to obtain full activity of the solubilized microsomal GST. Protein concentration was measured using the Bio-Rad protein assay, with bovine serum albumin as standard [34]. Microsomes (1 mg/ml) treated with 0.243 mM NEM were incubated for 1 min at room temperature. Treatment with CHB was performed by pre-incubating microsomes (1 mg/ml) at 0–5 mM CHB for 1 min or at 0.156 mM CHB for 0–5min at room temperature. Km and Vmax values were calculated from Lineweaver–Burk plots. The microsomes were incubated with 0.156 mM CHB for 1 min. The microsomal GST activity was measured at 1.0 mM CDNB and 0.10–1.60 mM GSH.

2.6. LC/ESI/MS assay Assays were performed on a HP 1100 HPLC system with an Esquire-LC 00075 quadrupole mass spectrometer detection system. C18 Dikma Diamonsil ODS reverse-phase column (5 ␮m; 250 mm × 4.6 mm) was used. The gradient program was as follows at 0.5 ml/min: 0.1 mM ammonium acetate:methanol, 10 to 100% in 30 min. The sample injection volume was 10 ␮l. The mass spectrometer was equipped with an electrospray ionization source. Typical source conditions were as follows: ion polarity positive, capillary 4.5 kV, skimmer 38.3 V, dry inert gas N2 , gas temperature 300 ◦ C scan begin m/z 100.00 and scan end m/z 1200.00. This method offers a number of analytical advantages, including excellent selectivity, quantitation limits and improved sample throughput in the form of decreased runtime [35,36]. 2.7. Statistics

2.4. Reaction of CHB with GSH The incubation mixture contained CHB (1 mM), GSH (5 mM) and non-activated microsomal GST or microsomal GST that was stimulated approximately two-fold with 0.156 mM CHB in aqueous phosphate buffer (0.1 M, pH 7.4). The reaction was run at 37 ◦ C for 1 h, at which time the reaction was terminated and protein was removed by the addition of trichloroacetic acid (TCA) to a final concentration of 5%, followed by filtration. A non-enzymatic reaction was run in the absence of microsomal GST. Products of the incubation were assayed by reverse-phase high-performance liquid chromatography (HPLC) using the method described below.

Values were presented as x¯ ± S.D. of replicate measurements from three enzyme preparations. Data were statistically analyzed for significant differences using one-way analysis of variance (ANOVA) repeated measures and two-way Dunnett’s test. P < 0.05 was considered to be statistically significant.

3. Results 3.1. Activation of microsomal GST by NEM The microsomes were washed twice with 0.15 M Tris–Cl, pH 8.0, and then treated with 0.243 mM NEM

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Table 1 Effect of NEM treatment on GST activity in rat liver microsomes

Microsomal GST activity ( mol/min/mg protein)

0.12

Microsomal GST activity (␮mol/min mg−1 protein) Control

NEM treated

Activation-fold

0.026∗∗

Unwashed 0.127 ± 0.013 0.366 ± 2.88 Washed 0.053 ± 0.003 0.431 ± 0.018∗∗∗ 8.08 Data are x¯ ± S.D. (n = 3). ∗∗ P < 0. 01 vs. control. ∗∗∗ P < 0. 001 vs. control.

0.08 0.06 0.04 0.02 0.00

for 1 min. The microsomal GST was activated 8.08fold compared with the unwashed microsomal GST which was only activated 2.88-fold under the same condition (Table 1). Since NEM inhibits cytosolic GST activity but stimulates microsomal GST [37], this result ensured that the activity we followed was due to the microsomal enzyme and not to the low level of cytosolic transferases that contaminated the microsomes. 3.2. Effect of CHB on microsomal GST activity Incubation of microsomal GST with CHB increased GST activity in a concentration- and time-dependent manner, with the maximal increase in activity being about two-fold. As shown in Fig. 1, microsomal GST was stimulated maximally by 0.156 mM CHB. At higher concentration, the stimulation of the microsomal GST activity was decreased. The activation of microsomal GST occurred rapidly within 1 min (Fig. 2). To further examine the effect of CHB on the kinetic parameters of microsomal GST activity, we calculated 0.12 0.10 Microsomal GST activity ( mol/min/mg protein)

0.10

0.08

0

1

2

3

4

5

Time(min)

Fig. 2. Time-course of CHB on GST activity in rat liver microsomes. CHB treatment was performed with 0.156 mM CHB. Data are x¯ ± S.D. (n = 3).

Km and Vmax data for the enzyme. The apparent Km value for GSH was 0.391 mM in control, and CHB decreased the value for GSH to 0.167 mM. With GSH the Vmax values for the unstimulated and stimulated enzyme reactions were 0.110 ␮mol/min mg−1 protein and 0.146 ␮mol/min mg−1 protein, respectively (Table 2). Activation of microsomal GST could in principle be the result of a change in kinetic mechanism between the unactivated and activated forms of the enzymes. 3.3. Characterization of GSH conjugates of CHB by LC/ESI/MS Incubation of CHB with GSH in the presence of microsomal GST from rat liver resulted in the formation of a complex mixture that was separated by reversed-phase HPLC. LC/ESI/MS analysis of the mixture produced the protonated molecular ions at m/z 303.9, 267.8, 285.7, 845.8, 557 and 575.1, corresponding to CHB, CHBOH2 , CHBOH,

0.06

Table 2 Alteration of kinetic parameters of microsomal GST activities after CHB treatment

0.04 0.02

Microsomes

Km (mM)

Vmax (␮mol/min mg−1 protein)

Control CHB treated

0.391 ± 0.033 0.167 ± 0.001∗∗∗

0.110 ± 0.004 0.146 ± 0.005∗∗

0.00 0

1

2

3

4

5

Chlorambucil (mM)

Fig. 1. Concentration-dependence of CHB on GST activity in rat liver microsomes. CHB treatment was performed for 1 min. Data are x¯ ± S.D. (n = 3).

Data are x¯ ± S.D. (n = 3). ∗∗ P < 0. 01 vs. control. ∗∗∗ P < 0. 001 vs. control.

J. Zhang et al. / Chemico-Biological Interactions 149 (2004) 61–67

Fig. 3. Catalysis effect of microsomal GST on CHB-GSH conjugation reaction. Microsomal GST was prepared and incubated with 1 mM CHB in physiological saline pH 7.4 containing 3 mM GSH for 1 h at 37 ◦ C and the reaction products were analyzed by HPLC all as described in the methods. Unshaded bars, no microsomal GST added; shaded bars, microsomal GST added; horizontal bars, microsomal GST and 0.156 mM CHB coincubation for 1 min added. Data are x¯ ± S.D. (n = 3). ∗∗ P< 0.01, ∗∗∗ P <0. 001.

CHBSG2 , CHBSGOH and CHBSG. These data are in good agreement with those of other investigators [11]. 3.4. Effect of microsomal GST on CHB–GSH conjugation reaction Integration of the HPLC/UV peak areas demonstrated that the enzyme-catalyzed reaction of CHB with GSH produced a significant increase in the relative of GSH conjugates formed as compared with the nonenzymatic reaction. Interestingly, we found the activation effect of CHB on rat liver microsomal GST activity, and microsomal GST which was stimulated approximately two-fold with CHB had a stronger catalytic effect (Fig. 3). A marked decrease in the amounts of peaks CHBOH2 , CHBOH and CHB were observed in the enzymatic reaction.

4. Discussion The effectiveness of alkylating agents in the cancer treatment is limited by the frequent development of drug resistance. Several different mechanisms have been identified that mediate tumor cell resistance to alkylating agents [3–6,23–30]. One such mechanism

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involves increased inactivation of the alkylating agents in drug resistant tumor cells due to elevation of GSH levels and/or overexpression of cytosolic GSTs. GSTs can catalyze the addition of GSH to a wide variety of electrophilic compounds generally leading to their detoxification [9–12]. It is believed that the GSTs catalyzed GSH conjugation of alkylating agents reduced their interaction with DNA, which is a critical event in anti-neoplastic effects of alkylating agents. The results presented here demonstrate that CHB is a substrate for the microsomal GST, which is in good agreement with the previous study [11]. CHB reacts with GSH to produce several conjugates that are formed by nucleophilic displacement of one or both chlorines from the nitrogen mustard side-chain. The presence of microsomal GST may serve to increase the relative nucleophilicity of the cysteine sulfhydryl moiety of GSH and therefore provide increased competition for non-enzymatic hydrolysis of the active alkylating agent. While the property of these adducts from CHB and GSH have not been investigated, it would be anticipated that these conjugates are less reactive than CHB itself. If so, conjugation with GSH could provide a mechanistic basis for the development of ADR for CHB in tumor cells. There are many examples in which the development of resistance to alkylating agents is associated with increased expression of cytosolic GST. Altered expression of cytosolic GST-␣, -␮ and -␲ has been associated with the drug resistant phenotype in laboratory models [23–30]. Recent work has investigated this relationship through transfection, permitting the modification of only one cellular trait. Manohoran et al. [25] demonstrated that transfection of the cytosolic GST-␣ cDNA into Cos cells conferred resistance to an alkylating molecule. The same laboratory later transfected cytosolic GST-␲, -␣ or -␮ into mouse C3H/10 T1/2 -CL8 fibroblasts and subsequently analyzed these cells for resistance to alkylating agents. They found that transfection of cytosolic GST-␣ provided the greatest level of resistance to chlorambucil and melphalan (1.3–2.9fold) [26]. These studies demonstrated that overexpression of cytosolic GSTs contributed to resistance. With respect to microsomal GST, Clapper et al. investigated the subcellular compartmentalization of GST in Walker 256 rat mammary carcinoma cells which are either sensitive or resistance to CHB, and found an approximate five-fold increase in GST activity localized in microsomes of resistance cell line as

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compared to the sensitive parent cell line [34]. This result suggested a potential involvement of the induction of a specific mGST in the establishment of cellular resistance to CHB. However, the relationship between activation of microsomal GST and the development of ADR is still uncertain. There are now numerous examples of activation of the microsomal GST in vitro and in vivo [38]. It can be activated by SH-alkylating reagents such as NEM. This activation occurs by covalent binding of NEM to a single cysteine residue (Cys-49) in each polypeptide chain of the protein. In our experiments, we demonstrated an activation effect of CHB on rat liver microsomal GST and an alteration of kinetic parameters of microsomal GST activities after CHB treatment. Interestingly, microsomal GST, which was stimulated approximately two-fold with CHB, had a stronger catalytic effect on the conjugation of CHB and GSH. Thus, microsomal GST may play a potential role in the metabolism of CHB in biological membranes, and in the development of ADR. Further studies will address the presence of CHB–GSH conjugates in sensitive and resistant cells and the possible biochemical mechanisms responsible for the development of ADR.

Acknowledgments This work was supported by the National Natural Science Foundation of China No. 30070904.

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