Chem. Res. Toxicol. 1994, 7, 390-400
390
Conjugation of Glutathione and Other Thiols with Bioreductively Activated Mitomycin C. Effect of Thiols on the Reductive Activation Rate Mrinalni Sharma and Maria Tomasz* Department of Chemistry, Hunter College, City University of New York, New York, New York 10021 Received November 18,199P
Mitomycin C (MC), a clinically used natural antitumor agent, was shown to form three monoconjugates (1la-13a) and two bisconjugates (14a,15a)with GSH upon reductive activation by rat liver microsomes, purified NADPH-cytochrome c reductase, or NADH-cytochrome c reductase or chemical reduction using HJPt02. Rat liver cytosol/NADH activated MC only at acidic p H (5.8), resulting in the formation of a single GSH-MC monoconjugate, 13a. The reductase responsible for cytosolic activation of MC to form this conjugate was DT-diaphorase. GSH itself did not reduce MC, and unreduced MC did not form conjugates with GSH. A moderate catalytic effect by glutathione S-transferase was demonstrated on the cytosol-activated reaction. Mercaptoethanol andN-acetylcysteine gave analogous sets of five MC-thiol conjugates under cytochrome c reductase or HJPt02 activation conditions. The structures of all 15 MCthiol conjugates (five each with GSH, mercaptoethanol, and N-acetylcysteine, respectively) were determined, using lH-NMR, UV, and mass spectroscopies, combined with analytical chemical and radiolabeling methods. The mechanism of formation of the conjugates features SNZ displacement of the carbamate of the reduced MC by GS-. The MC-GSH conjugates were noncytotoxic to the tumor cells tested. The conjugation of GSH with activated MC is likely to represent detoxication in mammalian cells. As another effect, GSH accelerates the rate of reduction of MC by “slow” reducing agents such as cytochrome c reductases and HJPt02. A mechanism is proposed to explain this effect, which involves further reduction of the initially formed MC semiquinone free radical by GSH.
Introduction Mitomycin C (MC;l 1) is an antitumor antibiotic used in clinical cancer chemotherapy against a variety of solid tumors, especially for the treatment of breast, prostate, and superficial bladder cancers (1). It is one of the few anticancer drugs active against colorectal and gastric carcinomas (2). However, development of resistance of tumor cells to MC has been recognized as a limiting factor to chemotherapeutic effectiveness, similarly to cases of other anticancer drugs ( 3 , 4 ) . Drug-resistant tumor cells usually display a variety of alterations in properties which can be potentially correlated with the observed resistance. A frequent type of alteration is increased intracellular level of glutathione (GSH) and/or glutathione S-transferase (GST) activity (5,6). In such cells, increased inactivation of the drug by conjugation with GSH is expected to occur, resulting in reduced cytotoxicity, provided the drug is subject to reaction with GSH. The main chemical basis of the detoxifiying activity of GSH lies in the high nucleophilic reactivity of its thiol group. A family of GST isoenzymes exist in mammalian cells which accelerate the reaction of GSH with a variety of electrophilic drugs, environmental carcinogens, and endogenous compounds (5). Accordingly, bifunctional alkylating agent type chlorambucil anticancer drugs, including melphalan (7), (a), andmechlorethamine (9,IO),as wellasother important drugs, such as adriamycin (11 ) and cis-diamminedichloAbstract publihed in Advance ACS Abstracts, April 1, 1994. MC, mitomycin C; MC., albomitomycin; N-acetylCys, N-acetylcysteine; GST, glutathione S-transferase;dT, thymidine; DIC,dicumarol. 1 Abbreviations:
roplatinum (11) (10-13), have been shown to display an inverse correlation between cytotoxic potency and cellular GSH level. DNA interstrand cross-linking in alkylating agent-resistant cells showed similar inverse correlation (9). Since MC, too, acts as a bifunctional DNA-alkylating agent (14, 151, it is likely to be subject to reaction with cellular GSH. Furthermore, such reactions may play a role in the resistance of tumor cell populations to MC. There is considerable indirect evidence for both of these assumptions. Thus GSH and N-acetyl cysteine (N-acetylCys) inhibited the alkylating activity of MC in cell-free rat hepatic or EMT6 mouse mammary tumor cell nuclei (16). In multi-drug-resistant mouse leukemia cells depletion of cellular GSH or inhibition of GST significantly increased the cells’ sensitivity to MC (17,181.Similarly, GSH depletion enhanced the cytotoxicity of MC in resistant mouse mammary tumor cells (19),as well as in a nonresistant human colon cancer line (20). In view of these findings, interactions of MC with GSH are likely to be important with respect to cellular resistance to the antitumor action of MC. Surprisingly, no reactions of MC with GSH or other thiols have been studied previously. A single MC-GSH conjugate was described in a different context (211. We undertook a study of the reactivity of MC with GSH in subcellular enzymatic systems. MC is a “bioreductive alkylating agent”, requiring reductive activation to a reactive electrophile (22). Reactions of GSH with such agents have not been described. The complex mechanism of MC activation involves enzymatic reduction of MC’s quinone function, followed by several consecutive trans-
0093-22~~/94/2707-0390$04.50/0 0 1994 American Chemical Society
Mitomycin C-Glutathione Conjugates
Scheme 1. Mechanism of the Reductive Activation of MC and Partitioning of the Active Form (4) to Inactive Metabolites and DNA Adducts8
6a:
1-a-OH
6b:
l-D.OH
OInset: Partitioning of 6 to monofunctional and bifunctionalactivation pathways.
formation steps (Scheme 1; steps leading from 1to 4, the reactive "quinone methide" form; for review, see ref 23). A priori, a variety of potential reactions with GSH may be envisaged GSH could interact with various intermediates of the reductive activation pathway or it may form conjugates directly with nonreduced MC, preventing its activation;alternatively, it may act as a reductive activator of MC, in analogy to the activation of various enediyne antitumor antibiotics by GSH (24). Furthermore, MC could form a monoconjugate with GSH which then acts as a toxic monofunctional DNA alkylator. Precedents for this last scheme are provided by 1,2-dibromoethane (25), 1,3-bis(chloroethyl)-N-nitrosourea(26), and Cr(V1) derivatives (27). To elucidate the actual mechanism, we isolated and characterized MC-GSH conjugates formed in cell-free systems and found that their formation was mediated by quinone oxidoreductases known to be involved in the reductive metabolism of MC in intact cells. GST had a moderate catalytic effect on the conjugation. A novel, accelerating effect of GSH on the rate of enzymatic reduction of MC is abo described. These findings,together with an analysis of the chemical mechanism of formation of the conjugates, provide a plausible molecular basis for the detoxication of MC by GSH. Experimental Section Materials. MC was supplied by Bristol-Myers Squibb Co. (Wallingford, CT). Mercaptoethanol,N-acetyl-Cys,GSH, NADH, dicumarol, triphenyltin chloride, and NADH-cytochrome c reductase were obtained from Sigma, St. Louis, MO. NADPHcytochrome c reductase was received from Dr. Wayne Backes, Louisiana State Medical School, New Orleans, LA. NADPH was purchased from Boehringer Mannheim, Indianapolis, IN. Radiolabeled GSH (glycine-2-SH; specific activity 1.0 Ci/mmol) was obtained from NEN DuPont, Boston, MA.
Chem. Res. Toxicol., Vol. 7, No. 3, 1994 391 Rat liver microsomes and cytosol from male Sprague-Dawley rata (200-250 g weight; not fasted) were prepared by the method of Cederbaum et al. (28) and were a gift from Dr. Arthur Cederbaum, Mount Sinai School of Medicine, New York, NY. The pelleted microsomes were resuspended in 0.125 M KC1 and stored at -150 "C. Protein content of one preparation was 13 mg/mL, and the specific activity of NADPH-cytochrome c reductase was 0.245 unit/mL, as determined by published assay procedures (refs 29 and 30, respectively). The cytosol fraction was dialyzed at 4 OC in 0.1 M potassium phosphate (pH 7.0) and stored at -150 OC. Its protein content was 20 mg/mL (29). An assay (31) indicated 1.8 units of DT-diaphorase activity/mL. Complete inhibition of DT-diaphorase activity was observed in the presence of 1pg of dicumarol/lO pL of cytosol. Mitosenes26a, 6b, and 7 were prepared from MC by catalytic hydrogenation as previously described (32). Neutral stock solutions of GSH and N-acetyl-Cys were prepared freshly before use by dissolution of these substances in 0.015 M Tris-HC1 (pH 7.4), followed by neutralization with 0.1 N NaOH to pH 7.4, to give 0.13 M GSH or N-acetyl-Cys concentration. Spectroscopic Methods. 'H-NMR spectra were measured on a General Electric QE-300 NMR spectrometer (300 MHz). UV spectra were obtained on a Varian Cary 3 spectrophotometer. Chemical ionizationand FAB mass spectra were determined using a VG7070 double-focusing mass spectrophotometer at the NIH Regional Mass Spectroscopy Facility at The Rockefeller University, New York, NY. HPLC. A Beckman Model 338 gradient system equippedwith Model 265A absorbance detector and Model 427M peak area integrator was used. Reverse-phase columns (Beckman Ultrasphere ODs; 0.4 X 25 cm or 1.0 X 25 cm) were employed; flow rates were 1.0 and 2.0 mL/min, respectively. Elution gradients. system A, linear gradient of 0-15% acetonitrile in 0.03 M ammonium acetate in 35 min, continuing with 15% acetonitrile isocratically; System B, linear gradient of 0-15% acetonitrile in HzO in 35 min, continuing with 15% acetonitrile isocratically. For milligram-scale isolation of conjugates, a preparative Rainin HPLC assembly, including an HPLX solvent delivery system, was used, employing a Dynamax C-18 column (2.1 X 25 cm) (Rainin, Woburn, MA) and a 12 mL/min flow rate. Conditions of Reactions of GSH with MC. (i) Rat liver microsomes/NADPH as activating agent: The microsomal suspension (5 pL) was diluted by addition of 0.5 mL 0.2 M sodium phosphate (pH 7.1) buffer, followed by deaeration by purging with argon for 10 min at 0 OC. This solution was transferred to 1.5 mL of similarly deaerated solution containing MC, NADPH and GSH in the above buffer, to give 0.5 mM MC and 2.5 mM NADPH as final concentrations. The concentration of GSH was variable. Incubation at room temperature continued under anaerobic conditions. (ii) Rat liver cytosolic fraction/NADH as activating agent: The reaction mixture (2.0 mL) contained 500 pL of dialyzed cytosol, MC (1 mM), NADH (5 mM), and GSH (varying concentration), in 0.5 M potassium phosphate buffer (pH 5.8), and was incubated at room temperature aerobically. The inhibitors DIC (dicumarol) (70 pM) or triphenyltin chloride (1 mM) were included in some experiments. (iii) NADPHcytochrome c reductase as activating agent: To a deaerated solution containing 0.5 mM MC, 0.52 mM NADPH, and varying concentrations of GSH in 0.015 M Tris-HC1 (pH 7.4) buffer was added 0.27 unit/mL of a deaerated solution of NADPHcytochrome c reductase. Incubation continued at 37 OC under positive argon or helium pressure. (iv) NADH-cytochrome c reductase/NADH as activator: The procedure was analogous to that in (iii). (v) Catalytic hydrogenation (HdPtOZ) as activating agent: A solution containing 0.5 mM MC, varying concentrations of GSH, and solid PtOz (90 pglpmol of MC) in 0.015 M Tris-HC1 (pH 7.4) was deaerated by purging with helium gas, followed by 2 The term 'mitosene" refers to a structure as in 6a, 6b,or 7, without substituents at the 1-,2-, and 7-positions. 'Mitosane" is as in 1, without substituents at the 1-,2-, 7-, and Sa-positions (33).
392 Chem. Res. Toxicol., Vol. 7, No. 3, 1994 bubbling Hz gas for 5.0 min through the solution then again helium for 5.0 min. The mixture was exposed to air and then filtered for removal of PtOz. Synthesis and Determination of the Specific Radioactivity of MC-['H]GSH Conjugates. [3H]GSH was diluted from 1.0 mCi/pmol to 0.17 pCi/pmol specific radioactivity by the addition of [3H]GSH to a neutral stock solution of nonradiolabeled GSH. A mixture of MC (0.45 mM), 13H]GSH(12 mM), and PtOz (90 pglpmol of MC) in 0.015 M Tris-HC1 (pH 7.4) was hydrogenated. The resulting conjugates were separated by HPLC of the reaction mixture (see Figure 3b for conditions), and the five collected fractions were lyophilized. Each fraction was analyzed for absorbance at 310 nm and for radioactivity; the latter was measured in Scintiverse liquid scintillation fluid (Beckman), using a Beckman LS 6800 counter. Values of pmol of GSH/pmol of mitosene residue were calculated from the data by using E310 = 11500 for the 7-aminomitosene chromophore (33). Reactions of MC with mercaptoethanol and N-acetylCys were conductedanalogouslyto those of MC with GSH. Details are given in the legend of Figure 4. Reactions of GSH with Mitosenes.2 (i) In the absence of a reductive activating agent: Mitosenes (6a, 6b, or 7; 0.5 mM) and GSH (12 mM) were incubated at room temperature in 0.015 M Tris-HC1(pH7.4) buffer under aerobic or anaerobicconditions. (ii) HZ/PtOZ as activating agent: A mixture of mitosenes (0.5 mM), GSH (0 or 12 mM), and PtOz (90 pglpmol of MC) in 0.015 M Tris-HC1(pH 7.4) was hydrogenated for 5.0 min followingthe hydrogenation protocol described for the MC reactions above. (iii)NADH-cytochrome c reductase as activator: To a deaerated solution containing 0.5 mM mitosene, 0.62 mM NADH, and 12 mM GSH in 0.015 M Tris-HC1 (pH 7.4) buffer was added 0.27 unit/mL deaerated NADH-cytochrome c reductase. Incubation was continued for 1 h at 37 "C under anaerobic conditions. Preparative-scale synthesis of MC-thiol conjugates were achieved using 75 mg of MC as starting material and HdPt02 as reducing agent. The products were purified by preparative HPLC. Acetylation of MC-thiol conjugates for use in NMR was carried out as follows: Several milligrams of conjugate, dried by lyophilization, was mixed with 0.6 mL of dry pyridine and 50 pL of acetic anhydride. The mixture was purged with helium through a rubber septum equipped with gas inlet and outlet. A crystal of (dimethy1amino)pyridine was added as catalyst, and the mixture was stirred for 2 h at room temperature under positive helium pressure and then evaporated to dryness on the rotary evaporator, followed by addition of water and lyophilization.The acetylated conjugates were purified by preparative-scale HPLC, using 20-30% acetonitrile in 0.03 M NHdOAc as isocratic eluant. Determination of the Extent of Reactions of MC by HPLC. Thymidine (0.5mM) which is inert under the reaction conditions described herein was included in reaction mixtures to serve as internal standard for peak area determination in the HPLC. The extent of reaction of MC was calculated from the formula:
Sharma and Tomasz a MC
I
dT
I
I:'
b 60, 13a
J
1 J 0
C
13a
/I
d
I
I
I
I
10
20
30
40
mln
(area of MC/area of dT)(100) 76 MC reacted = 100 - (area of MC/area of dT),,,,
Results Formation of MC-GSHConjugates in the Rat Liver Microsome/NADPH System. I n t h e absence of GSH MC was reductively metabolized t o t h e mitosenes 6a, 6b, and 7 (Figure la), in accordance with our earlier report (32). The amount of MC converted t o t h e metabolites was 72%. I n t h e presence of 25 m M GSH t h e same metabolites a n d a new substance, identified as t h e G S H conjugate 13a, were formed; the latter was t h e most prominent product. Overall, 89 % of MC was metabolized under these conditions. (Figure lb). Further addition of
Figure 1. HPLC analysis of MC-GSH conjugates formed in the rat liver microsomes/NADPHactivation system. Thymidine (dT; 0.5 mM) was included in all reaction mixtures depicted in this figure, as quantitative internal standard. HPLC eluant: system A. (a) No GSH was added; incubation time was 2 h. The peak marked MC. is albomitomycin C (M. Kasai, personal communication), present to a small extent in all solutions in equilibrium with MC. (b) 25 mM GSH was added; incubation time was 2 h. (c) 50 pL of fresh microsomes waa added to the reaction mixture in (b) after 2 h and incubation continued for 1h longer. (d) The initial reaction mixture contained 100 pL/mL of microsomes and 25 mM GSH. Incubation time was 2 h. fresh microsomes (50 pL) to this mixture resulted in complete conversion of all metabolites and unreacted MC t o t h e three GSH conjugates lla-13a (Figure IC).At an initially higher concentration of microsomes (100pL/mL) a n d 25 m M GSH, MC was quantitatively converted to
Chem. Res. Toxicol., Vol. 7, No. 3, 1994 393
Mitomycin C-Glutathione Conjugates I
a
I
a
I
I
h III(
II
yc
I1 I
mln
Figure 2. HPLC analysis of formation of MC-GSH conjugates in the rat liver cytosol/NADH activation system. HPLC eluant: system A. (a) 50 mM GSH was included in the reaction mixture; incubation time was 30 min. (b) Same as (a), except incubation time was 2 h. (c) No GSH was included; incubation time was 2 h. (d) 50 mM GSH was included; incubation time was 30 min. (e) Same as (d), except 1mM triphenyltin chloride was included.
five GSH conjugates 1 la-15a (Figure Id). No conversion of MC occurred in the absence of NADPH (data not shown). Formation of a Single MC-GSH Conjugate in the Rat Liver Cytosol/NADH System. In the absence of GSH MC was reductively metabolized to the single mitosene 7 at pH 5.8 (Figure 2c). No metabolism occurred at pH 7.0 (data not shown). This unusual low-pH dependence is characteristic of the reductive metabolism of MC by DT-diaphorase of rat liver or human tumor cells (34,211.Therefore, the results suggestthat DT-diaphorase is the sole MC-metabolizing enzyme present in the rat cytosol. The observed inhibition of the metabolism by DIC (data not shown) supports this hypothesis. At pH 5.8, in the presence of 50 mM GSH, mostly 7 was accumulated at 30-min incubation time (Figure 2a). After 2-h incubation, both 7 and its conjugate 13a were present (Figure2b). The increase of the 13a:7 ratio with incubation time indicates that some or possibly all of the conjugate 13a is formed from 7. Addition of the GST inhibitor triphenyltin chloride (35)resulted in 3-fold suppression of the MC-GSH conjugate 13a, as calculated from the ratios of 13a to 7 in the absence and presence of the inhibitor (0.508 and 0.175; Figure 2, panels d and e, respectively). Repeating the cytosolic incubations under anaerobic conditions gave qualitatively similar results. No conversion of MC was observed in the absence of NADH. Formation of MC-GSH Conjugates under NADPHCytochrome c Reductase/NADPH Activating Conditioar. In the presence of 12 mM GSH, MC was metabolized quantitatively to five GSH conjugates marked lla-
20
30
40
min
Figure 3. HPLC analysis of MC-GSH conjugates formed under reductive activation of MC by NADPH-cytochrome c reductase/ NADPH or HdPt02. HPLC eluant: system B in (a) and (b), system A in (c). (a) NADPH-cytochrome c reductase activation: 12 mM GSH was included; incubation time was 1h. (b) HdPt02 activation system: 12 mM GSH was included. (c) Same as (b) except no GSH was included.
15a in the HPLC pattern of the reaction mixture (Figure 3a). Activation by catalytic hydrogenation instead of reductase/NADPH resulted in the formation of the same products (Figure 3b). In the absence of added GSH both the reductase and H2/Pt02 treatments converted MC to the three known metabolites 6a, 6b, and 7 (Figure 3c). No conversion of MC was observed in control experiments in which reductase or catalytic hydrogenation was omitted from otherwise complete systems. NADH-cytochrome c reductase, a commercially available enzyme, gave essentially identical product patterns to those obtained with NADPH-cytochrome c reductase under the same reaction conditions (data not shown.) Formation of Conjugates of MC with N-Acetyl-Cys and Mercaptoethanol. Using either Hz/Pt02 or NADPH-cytochrome c reductase as MC activating agents, both thiols formed five conjugates with MC, in complete analogy to GSH (Chart 1, Figure 4). The relative proportion of bisconjugates 14 and 15 to monoconjugates 11-13 was highest in the mercaptoethanol and lowest in the GSH series, indicating steric hindrance to thiol attack at C1 of MC.
394 Chem. Res. Toxicol., Vol. 7, No. 3, 1994
Sharma and Tomasz a
Chart 1. Structures of MC-GSH, MC-N-Acetyl-Cys, and MC-Mercaptoethanol Conjugates 1Ib
l~ 12b
R
0
1
0
15b
2
0
3
-
0
0
,CONHCH&OOH a: GSH conjugates;
R=
-cHz-CH .NHCOCHzCHzCH(NH,)COOH ,COOH
b: N-acetyl Cys conjugates; R = -CH~-CH .NHCOCH, c. Mercaptoethanol conjugates; R = - C H & H ~ O H
P a
Determination of the Structures of MC-Thiol Conjugates (Chart 1). UV spectraof the GSH,N-acetylCys, and mercaptoethanol conjugates (Figure 5 ) showed the presence of the characteristic 7-aminomitosene2chromophore (Amu 310-315 and 245-255 nm) which is readily distinguishable from the 7-aminomitosane chromophore (Am= 367 and 220 nm) such as that in MC (33). An assay for the -SH group (36) was applied to all 15 conjugates listed in Chart 1,together with GSH itself as positive control. All conjugates tested negative for free -SH group. Mass spectroscopy (mlz): Mercaptoethanol conjugates llc-16c each gave satisfactory mass spectra (CI negative mode), with predominant ions as follows: l l c , 337 (M-); 1 2 ~337 , (M-), 1 3 ~321 , (M-); 1 4 ~397 , (M-), 319 [(M SCH&H20H)-]; 16c, same as 14c. N-acetyl Cys conjugates gave the following m/z values (FAB negative mode): l l b , 421 [(M - HI-]; 12b, none; 13b, 405 [(M - HI)-]; 13b (W-acetyl derivative), 446 [(M - 2H)-I, 447 [(M - H))-l; 14b or 16b (C1 stereochemistry not identified), 566 (M H))-, 588 (M - 2H Na)-. GSH conjugates failed to give mass spectra. Use of PHI-labeled GSH for characterization of MCGSH conjugates: The molar ratio of GSH to the 7-aminomitosene chromophoreof each of the five conjugates (1la15a)was calculated to give the following results: 1la, 0.84: 1; 12a, 1.03:l; 13a, 1.05:l; 14a, 2.3:l; lSa, 1.85:l. These ratios indicated that 1la-13a are monoconjugatesand 14a and 16a are bisconjugates of GSH. 1H-NMRof MC-Thiol Conjugates. 'H-NMRspectra were determined for all conjugates depicted in Chart 1,as well as their peracetates. All were assigned by direct comparison with spectra of the known standard compounds 6a, 6b, and 7, and their peracetates (32,37).lHNMR spectra of standard GSH, N-acetyl-Cys, and mer-
+
1 0 2 0 3 0
mln
mln
Figure 4. HPLC analysis of conjugates of MC formed with N-acetyl-Cysand mercaptoethanol. (a)N-acetyl-Cysconjugates, NADPH-cytochrome c reductase as activation system. A solution of 0.45 mM MC, 0.67 mM NADPH, 0.6 unit/mL reductase, and 22 mM N-acetyl-Cys was incubated in 0.015 M Tris-HC1 (pH 7.4) for 1 h at 37 OC. HPLC eluant: linear gradient of 8-18% acetonitrile in 0.03 M ammonium acetate in 45 min, then continuing with 18 % acetonitrile isocratically. (b) Same conjugates, formed under HdPt02 activating conditions. A solution of 0.45 mM MC and 22 mM N-acetyl-Cys in the above buffer was hydrogenated for 5 min at room temperature. (c)Mercaptoethanol conjugates; NADPH-cytochrome c reductase as activation system. 0.75 mM MC, 0.75 mM NADPH, 0.45 unit of reductase, and 75 mM mercaptoethanol were incubated in 0.015 M TrisHC1 (pH 7.4) for 1h at 37 O C . HPLC eluant: linear gradient of 12-20% acetonitrile in 0.03 M ammonium acetate in 20 min, then continuing with 20% acetonitrile isocratically. (d) Same conjugates as in (c) except HdPt02 was employed, as the activation system.
---
I '.. \
I
I
3w
4QQ
3w
4w
I
2w
11I
I
nm
Figure 5. UV spectra of (top) the five MC-GSH conjugates and (bottom) the five MC-N-acetyl-Cys conjugates. Solvent: HzO.
captoethanol were also determined, under the same conditions as those of the conjugates, in order to facilitate the assignments. A list of selected chemical shifts which are characteristic of the new structures is as follows:3 (i) MC-GSH conjugates in D20: lla: 1.73 (8, C6-CH31, 2.08 (m, Glu-j3-CH2), 2.45 (t, Glu-y-CHz), 2.80 and 3.08 (m, Cys-p-CHz), 3.70 (8, Gly-a-CHz),3.75 (t, Glu-a-CH), 3 Chemicalshifts are given in 6 (ppmdownfield fromtetramethylsilnne as external standard). In the case of multiplets the chemical shift refers to the center of a multiplet.
Chem. Res. Toxicol., Vol. 7, No. 3, 1994 396
Mitomycin C-Glutathione Conjugates
3.88 (AB qt, ClO-Hz),4.15-4.30 (m, three protons: C3-Ha, C2-H, C3-H@), 4.7 (m, Cys-a-CH),4.97 ( 8 , C1-H); 12a: 3.78 (AB qt, ClO-H2), 5.0 (d, C1-H); 13a: 2.9 (m, Cl-Hb), 3.3 (m, C1-Ha), 3.8 ( 8 , CleH2); 13a peracetate: 2.0 (8, acetylCH3), 2.07 (8, acetyl-CHa); 14a: 3.90 (AB qt, ClO-H3,4.7 (br s, C1-H). (ii) MC-N-acetyl-Cys conjugates in perdeuterated methanol: 12b: 1.78 ( 8 , CG-CHa), 1.85 (8, N-acetyl of Cys), 2.50 and 2.85 (m, Cys-@-CHz),3.7 (C3-Ha, partially hidden under HDO peak), 3.80 (AB qt, C10-H2), 4.0 (br, C2-H), 4.25 (br, C3-Ha), 4.68 (d, C1-H), 6.42 (s, C7-NHz), 7.3 (d, Cys-NH); 13b: 2.5 (m, C1-HB),2.9 (m, C1-Ha),3.8 (s,C10H2); l l b peracetate: 5.84 ( 8 , C1-H); 12b peracetate: 6.00 (d, C1-H); 14b (or 15b; assignment uncertain): 4.55 (br s, C1-H); 14b (or 15b) peracetate: 4.6 (s, C1-H). (iii) MC-mercaptoethanol conjugates in perdeuterated Me2SO: 1 IC: 2.66 (t, mercaptoethanol-/3-CHz),3.45 (t, mercaptoethanol-a-CHz), 3.8 (9, C10-H2), 4.6 (s, C1-H); 1 ICperacetate: 2.05 (8, mercaptoethanol-a-O-acetyl-CH3), 3.8 (9, ClO-HZ), 5.8 ( 8 , C1-H); 12c peracetate: 6.02 (d, C1-H); 1 3 ~ :2.9 (dd, C1-Ha), 3.82 (9, c10-H~);1 3 ~ peracetate: 3.05 (dd, C1-Ha), 3.80 (s, C10-H2), 3.95 (dd, C3-H@),4.16 (t, mercaptoethanol-a-CHz), 4.35 (dd, C3Ha), 4.78 (br m, C2-H); 14c: 3.80 (AB qt, ClO-HZ),4.7 ( 8 , C1-H); 14c peracetate: 3.80 (AB qt, C10-H3, 5.0 ( 8 , C1HI. The main supporting features of the NMR for the structures of the conjugates are the following: (a) Loss of the C10-carbamate is indicated by lack of the ClOa-NH2 signal observable a t approximately 6.5 ppm in deuterated methanol and MezSO. (b) Upfield shift of the typical AB quartet of the ClO-HZ protons from approximately 5.0 ppm (38) to approximately 3.7-3.9 ppm, observed in all conjugates, is consistent with the change of -CH20C(O)NH2 to -CH& Aesignment of this shift to the presence of a C10-sulfur linkage is in agreement with the NMR data published for other C10-sulfur-substituted mitosenes (39-41). C10-decarbamoyl mitosenes2 exhibit similar upfield shifts (42). (c) C10-H2 of conjugates 13a-c forms a singlet rather than an AB quartet. This is consistent with C1 being a -CHz- group in these conjugates rather than -CH(OH)-(32). Futhermore, appearance of two highfield protons assigned to Cl-H2 of these conjugates (2.9 and 3.3 ppm) confirms the Cl-H(OH) c 1 - H ~change (32). (d) The a or @ stereochemistry of the C1-OH was deduced from comparing the appropriate pairs 11 and 12: Uponacetylation, the C1 proton of C1-a-acetates is a broad singlet further upfield from the C1 proton of C1-@-acetates (a doublet), in analogy to the C1-a- and C-l-@-acetatesof the known standard pair, 6a and 6b (32,37). Formation of GSH Conjugates from the Metabolite Mitosenes2 6a, 6b, and 7. In the absence of a reducing agent these mitosenes were unreactive toward GSH. Under HdPt02 reducing conditions rapid and quantitative formation of single products lla, 12a, and 13a were observed, respectively (Figure 6b-d). Their identity with GSH conjugates obtained directly from MC under the same conditions (Figure 6a) was established by coelutions in HPLC. The decarbamoyl derivative of 7 (32) did not react with GSH (data not shown). The metabolite mitosenes 6a, 6b, and 7 were also converted to their respective GSH conjugates lla, 12a, and 13a under microsomal reducing conditions as seen indirectly by the following: Figure l b shows accumulation of 6a, 6b, and 7 (as well as a small amount of conjugate 13a) in the
-
b
1
b
r
'
0
;
o
;
o
l
o
mln
Figure 6. HPLC analysis of formation of MC-GSH conjugates from metabolite mitosenes. (a) Formation of conjugates from MC under HdPt02 activation in the presence of 12 mM GSH. (b) Formation of conjugate from metabolite 6a under HdPt02 treatment. (c) Formation of conjugate from mitosene 6b under HdPt02 treatment. (d) Formation of conjugate from m i h e n e 7 under HdPt02 treatment. HPLC eluant: system A.
microsomal incubation mixture. Upon addition of fresh microsomes, however, all three mitosenes were further convertedto GSH conjugates (Figure IC).Similarly,initial accumulation of 7 in the cytosolic reduction mixture was followed by further conversion to 13a (Figure 2b,c). IncreasedRate of Enzymatic or Hz/PtOz-Mediated Reduction of MC in the Presence of GSH. Qualitatively, this phenomenon is seen by comparison of the HPLC patterns in Figure 3: In the absence of GSH under either reducing condition much of the MC was recovered unreacted (Figure 3c) while the presence of 12 mM GSH induced essentially complete conversion of MC (Figure 3a,b). In a kinetic experiment, under reduction by HdPt02 the fraction of MC converted in a given time increased as a function of concentration of added GSH (Figure 7a). For example, in the absence of GSH only 35% MC was reduced in 5.0 min while in the presence of 12 mM GSH 92% (almost a 3-fold amount) of the drug was reduced. The same phenomenon was observed reproducibly under enzymatic reducing conditions. Thus, a kinetic experiment, employing NADH-cytochrome c reductase/NADH, indicated that in the absence of GSH only 27% MC was reduced after 20 min, in contrast to 87 5% (3.2-fold amount) of reduction in the presence of 12 mM GSH (Figure 7b). In the cytosolic reaction, in the absence of GSH after 2-h incubation MC was partially converted to 7; the ratio of the HPLC peak area of 7 to that of MC was 1.9 (Figure 2c). In the presence of 5 mM GSH this ratio increased to
396 Chem. Res. Tonicol., Vol. 7, No. 3, 1994
Sharma and Tomasz
Discussion
and biochemical aspects of the interactions between MC and GSH, using subcellular enzymatic systems. The following is concluded from the results. Neither GSH nor two simpler thiols, N-acetyl-Cys and mercaptoethanol, reduce MC under physiological conditions. Thus it is demonstrated explicitly for the first time, to our knowledge, that thiols alone cannot activate MC to a reactive electrophile. Unreduced MC does not form conjugates with GSH or other thiols even in the presence of GST. When MC is reduced independently by other reducing agents, however, three monoconjugates and two bisconjugates of GSH to MC are formed. This was demonstrated using a variety of reducing conditions relevant to activation of MC in cells. Thus, NADPH-cytochrome c reductase (43), rat liver microsomes (32), and cytosol, each supplemented with reduced pyridine nucleotide, promoted the formation of GSH conjugates. Catalytic hydrogenation in neutral aqueous buffer, previously shown to generate the same reductive metabolites of MC as microsomal reduction (32), gave the same array of GSH conjugates as the enzymes or the microsome^.^ GST may catalyze the conjugation, as suggested by the 3-fold inhibitory action of PhsSnCl, a known inhibitor of GST (351, on the formation of the conjugate 13a in the cytosol which contains GST. More direct evidence is needed to confirm this point. The structures of the five GSH conjugates and the analogousconjugatesof N-acetyl-Cys and mercaptoethanol (Chart 1)were determined by a combination of 'H-NMR, mass and UV spectroscopies, and radiolabeling experiments. A set of chemical transformations in which each of the three known reductive metabolites of MC (6a, 6b, and 7) was converted upon reduction to a single distinct GSH monoconjugate, lla, 12a, and 13a, respectively (Figure 6), independently confirmed the assigned structures of 1la-13a. This was especially helpful with respect to the C1 stereochemistry of l l a and 12a, which was otherwise based only on similarities to 6a and 6b (acetates) in the 'H-NMR. The C1 stereochemistry of the bisconjugate pairs could not be distinguished by NMR. Our tentative assignment is based on their relative elution times from HPLC: C1-a-substituted CP-6-aminomitosenes are generally eluted before C1-&substituted ones (e.g., 41,44), including the monoconjugates described here. One of the MC-GSH conjugates (13a)was also described by Ross and his co-workers while our work was in progress (21). It was formed from MC upon reduction by purified rat liver DT-diaphorase, and it is the same sole product isolated and identified from the rat liver cytosolic reduction system in the present work. Mechanism of Formation of the Conjugates. An unexpected feature of the conjugate structures is that the Cl0-carbamate group of MC is replaced by an alkylthio group under the same reducing conditions which, in the absence of thiols, activate only the C1 position of MC (32, 34, 45; see also the present results). The lack of C10 activation under such (Hz or flavoreductase) conditions has been explained by a mechanism (Scheme 1)in which the C1-position is activated via formation of the quinone methide 4, followed by nucleophilic substitution at C1 to give 5a and 5b, or by C1 protonation to give 7. Subsequent activation of C10, via El-elimination of the carbamate
In view of recent reports implicating GSH as a modulator of MC cytotoxicity and resistance of tumor cells, the present study was designed to determine the chemical
It may be unexpected that catalytic hydrogenation was not inhibited but rather accelerated by the thiols.This effect is contrary to the wellknown 'poisoning" of P t and Pd catalysts by thiols in organic solvents.
1Wl
:L 10
15
mM GSH
1w-l
b
Y 8
" 7
o
i
o
m
m
u
,
s
o
min
Figure 7. Effect of GSH on the rate of reductive activation of MC. (a) MC was activated by HdPt02 at GSH concentrations which varied from 0 to 12 mM under the general conditions described in the ExperimentalSection.The extent of the reaction of MC ("% MC reacted") was determined by HPLC utilizing added dT in each reaction mixture as internal standard as described in the Experimental Section. (b)MC was activated by NADH-cytochrome c reductaee/NADH in the presence or absence of GSH. Conditions were as described in the Experimental Section; the GSH concentration was 12 mM. Aliquota were removed for HPLC at various times during 40-min incubation and frozen in liquid nitrogen before HPLC analysis. '% MC reacted" was determined as in (a).0 ,no GSH present; +,12 mM GSH. Table 1. Cytotoxicity of MC-GSH Conjugates in Vitro ICw (cMP compd
HCT1lGb
MC (1)
0.194
1la
>443
12a 13a
519
>910
HCT/VM4Bb 0.121 2443 496
>910
Determined using the XTT dye reduction method after 72-h drug exposure. Human colon carcinoma cell lines; HCT/VM46 is resistant to etoposide. 0
*
5.0, indicating 2.6-fold increase of the rate of conversion of MC to 7 (data not shown). In both reactions 7 was the only observed product. A t 50 mM GSH concentration a 3.8-fold increase of the rate of conversion of MC was calculated from the data (Figure 2b, compared to Figure 2c). Cytotoxicity of MC-GSH Conjugates. ICs0 values in human colon tumor cells are shown in Table 1,together with those of MC for comparison. The high values indicate a lack of cytotoxicity of the conjugates.
Chem. Res. Toxicol., Vol. 7, No. 3, 1994 397
Mitomycin C-Glutathione Conjugates
Scheme 2. Proposed Mechanism of Formation of MC-GSH Conjugates 2-3-
HzN&
1H3C OH
at C1
at C1
1"
w
H3C
I
I H
NH2
11,lZ (monoconjugates)
at C10
h
IOz
I
I
at C10
I"
I
1" I t
2 NHz
1: [red] 2: R S a t C 1 0
at C10
HzN + ;)f
4
NHZ
13 (monoconjugate)
from 5a or 5b, is slower and therefore in HdPtO2-or flavoreductase-catalyzedreductions this second activation step is prevented by the fast reoxidation of 5a or 5b by excess unreduced MC (inset in Scheme l), driven by the lower redox potential of mitosenes2 as compared to mitosanes such as MC (46-48). The C10 activation step has been observed only under fast reduction, e.g., by NazSzOr, which removes excess unreduced MC quickly (46,48). In the presence of thiols, however, C10 substitution is apparently faster than the reoxidation step, even when the "slow" reducing agents HdPt02 or flavoreductases are employed. Most likely the mechanism is s N 2 displacement of the carbamate by the excellentnucleophile RS-. The s N 2 process is not manifested with relatively poor nucleophiles such as water or phosphate or when MC is reoxidized. Schiltz and Kohn (49) reported another instance of sN2 displacement of the C10-carbamate of reduced MC by a strong nucleophile, i.e., the bisulfite ion. Our proposed mechanism accounting for the formation of all observed thiol-MC conjugatesis summarized in Scheme 2. Accordingly, reduction activates the C1-position in the usual manner via quinone methide 4. Addition to C1 by water, proton, or RS- will compete with one another (paths A-C, respectively). As yet another competing path (D), the C10-carbamate is displaced by GSH (but not by the other nucleophiles) by the s N 2 mechanism. The C1substituted products of paths A-C, still in their reduced states, can also undergo s N 2 substitution a t C10 by RS-, to give the final mono- or bisconjugates as indicated. The intermediate product of path D, also still reduced, may undergo any of the usual C1 additions (by H20, H+, or RS-), to yield mono- and bisconjugates. Strong evidence for the s N 2 step a t C10 was provided by demonstrating the displacement of the carbamate by GSH directly from the mitosenes 6a, 6b, and 7 upon their reduction to give conjugates lla-13s (Figure 6b-d); in contrast, no displacement of the carbamate by water took place under the same reducing conditions either in the presence or in the absence of GSH (data not shown). This indicates that the El-elimination mechanism of C10
''
H3C H2N@NH2
CHzSR N '
SA
11, 12, 13, 14 or 15, respectively bisconj (mono-ugates) and
14,15 (bisconjugates)
activation (inset, Scheme 1)was not operative. It should be noted that path D represents a mode of inactivation which is unique to GSH. On this path, sN2 attack at C10 precedes a C1 attack and is not subject to competition by the solvent. Experimental evidence suggesting the existence of path D is provided by the observed product distributions as follows: In the presence of GSH the C1 protonation product increases relative to C1 nucleophilic products. This can be seen upon comparison of Figure 3c with Figure 3b or Figure l a with Figure lb. This change of distribution requires that in the presence of GSH the C1 reactions proceed, at least partly, via a new precursor, different from 4, which favors C1 protonation over C1 nucleophilic attack. Path D, i.e., the C10-thio-substituted quinone methide, fulfills this requirement.6 The attack of thiols at C1 appears to be sterically hindered, since at equal thiol concentration the ratio of bisconjugates to monoconjugates was 4 times higher in the case of mercaptoethanol than in that of GSH (data not shown). Apparently, the smaller thiol competes more effectively with water for addition at C1 of 4 than a larger one. It is concluded from this mechanistic analysis that thiols react differently with activated MC from other biological nucleophilessuch as H20, phosphate ions, deoxyguanosine, or DNA, namely, by displacement of the C10-carbamate by s N 2 rather than by the less efficient, E l mechanism. The s N 2 displacement of the carbamate does not represent a general activation of the C10-position of MC, however. Rather, this is inactivation of the potential DNA crosslinking function by the thiol. Another distinct effect of GSH on the activation of MC observed here is an increase of the rate of the reduction itself (the first step in Scheme l), under both enzymatic and H2/Pt02 ~atalysis.~ The following explanation is 6 Path D is analogous to the demonstrated facile S Ndisplacement ~ of the C10-carbamate by GSH in mitosenes 6a, 6b,and 7 upon reduction. The analogyis even more compellingif one assumesthat the displacement takes place in the reduced aziridinomitosene tautomer of 4, Le., in 3 (Scheme 1).
398 Chem. Res. Toxicol., Vol. 7, No. 3, 1994
Sharma and Tomasz
proposed. As shown above, GSH alone does not reduce MC. This is in accordance with the known unfavorable thermodynamics of the equilibrium Q + RS-
~t Q'-
+ RS'
(1)
where Q is quinone (50). On the other hand, semiquinone radicals QZT are known to react with thiols, such as GSH, to form thiyl radicals (51, 52): QH'
+ GSH
-
QH,
+ GS'
(11)
In the reduction of MC by cytochrome c reductases and by H2/Pt02, one-electron (H) transfer generates MC semiquinone radical (QH), according to the known mechanism of electron transfer from these agents. On the other hand, the MC semiquinone radical is known to disproportionate rapidly to MC and MC hydroquinone under physiological conditions (53). The subsequent nonenzymatic steps of the MC activation cascade (Scheme 1) such as elimination of the Sa-methoxyl group, etc., take place at the reduction level of the hydroquinone as was shown by pulse radiolytic reduction studies (53, 54).6 Based on this, as well as on analogy to reactions of other quinones with GSH (52),it is proposed that GSH intercepts the disproportonation of the MC semiquinone radical by further reducing the latter as in eq 11. Thus, the efficiency of the net reduction of MC to the hydroquinone is increased. The process is summarized as Q+H'-QH'
+ GSH QH, + GS' Q + H' + GSH QH, + GS'
QH' net:
-
(111)
-
(11) (IV)
This mechanism applies to one-electron reduction by cytochrome c reductase or HdPtOz, as in eq 111. However, increased reduction rate is also seen in the cytosolic metabolism of MC, in which DT-diaphorase, an obligatory two-electron transferring agent, is the reductive catalyst (see Results). The GSH effect (eq 11) can still apply, considering that the hydroquinone of MC is known to be autoxidized under aerobic conditions to quinone via Q H as intermediate (e.g. ref 47). GSH prevents this autoxidation by rereducing Q H (eq 11), with the result of increased efficiency of aerobic MC activation by DTdiaphorase. Significance. Paradoxically, reductive metabolism of MC (Scheme 1) leads both to cytotoxic DNA lesions and to detoxication by formation of inactive metabolites 6a, 6b,and 7 and their corresponding C1-phosphates (32,45, 55). This duality is analogous to that of the oxidative metabolism of xenobiotics via cytochrome P-450 enzymes, which generates both carcinogenic DNA damage and detoxified metabolites. In further analogy, both the reduction of MC and oxidation of the xenobiotic, e.g., aflatoxin (56),lead to highly reactive electrophiles which are then partitioned between DNA alkylation and detoxication by hydrolysis or other reactions with low-molecularweight cellular nucleophiles. In the case of oxidative metabolism, one of these nucleophiles is GSH, forming nontoxic drug conjugates, thereby contributing to the 6 In view of these findings, in our more recent publications (23,41)the mechanism of reductive activation of MC is depicted at the hydroquinone rather than the semiquinone reduction level.
detoxication pathway, at the expense of DNA alkylation (57). We show here, for the first time, that the reductively formed reactive metabolites of MC are subject to similar conjugation with GSH. Inactivation of the DNA-reactive electrophile 4 by water, phosphate, and protonation may be considered a "base-level" process while ita inactivation by GSH may be modulated by the concentration of GSH and/or GST. Does GSH conjugation represent a significant detoxication pathway of MC in addition to the "baselevel" detoxication path? On the basis of mechanistic reasoning, the S~2-typeattack of GSH on C10 in path D (Scheme 2) is the most obvious mechanism for additional inactivation of 4, since this reaction is not subject to competition by the solvent and is thus specific to GSH. The quantitative significance of path D relative to A-C is not established by the present work. However, positive qualitative evidence for inactivation by GSH in our systems is provided by the finding that the cytotoxic DNA lesions, i.e., alkylation and cross-linking, mediated by 4, are suppressed by GSH (58). Thus, the conjugation certainly represents detoxication rather than activation or "no effect" of MC. It is significant in this respect that the GSH conjugates lla, 12a, and 13s were found to be noncytotoxic. Since GSH is abundant in mammalian cells 11-10 mM; generally higher in tumor cells; up to a 50-fold increase was observed in some drug-resistant lines (12)3, the reactions observed here are expected to occur in viuo. They provide a likely molecular basis for the previously observed inverse correlation between GSH level and MC toxicity in various MC-sensitive and -resistant tumor cells, as well as for inhibition of alkylating activity of MC in the nucleus (16). Characterization of the array of GSH-MC conjugates and their potential urinary mercapturate metabolites (i.e., the N-acetyl-Cys conjugates) provides biomarkers for their detection in biological samples, enabling one to test detoxication of MC by GSH in viuo directly. Another distinct effect of GSH on the reductive metabolism of MC is acceleration of the reduction of MC. This effect is likely to result in increased rates of metabolic removal of MC before it reaches ita DNA target in the nucleus and, as such, it may be another mechanism of MC detoxication by GSH. The effect is also significant in light of the finding (58)that GSH helps cytochrome c reductases to activate MC to a DNA-cross-linking rather than DNAmonoalkylating species. It is likely that the faster MC reduction kinetics is responsible for this switch to bifunctional activation (58, 15). The reactions of activated MC with GSH, N-acetylCys, and mercaptoethanol under physiologicalconditions suggest an analogous mechanism for covalent binding of MC to cellular proteins. There is evidence that such binding occurs in cells (59). The reported inhibition of DT-diaphorase by MC in a manner resembling suicide inactivation, via covalent binding of MC to the enzyme (60),may similarly involve reaction of a thiol group with activated MC in situ.
Acknowledgment. We thank Roselyn Lipman for excellent technical assistance with the cytosol and microsome experiments. Dr. D. M. Vyas, Bristol-Myers Squibb Co., Wallingford, CT, Dr. W. Backes, Louisiana State University Medical School, New Orleans, LA, and Dr. A. I. Cederbaum, Mt. Sinai School of Medicine, New York, are gratefully acknowledged for donation of mitomycin C, NADPH-cytochrome c reductase, and rat liver
Mitomycin C-Glutathione Conjugates
subcellular fractions, respectively. Our thanks are due also to Dr. Craig Fairchild, Bristol-Myers Squibb Co., Wallingford, CT, for cytotoxicity determinations of the GSHMC conjugates, and to Dr. Brian T. Chait, The Rockefeller University, New York, for mass spectroscopic determinations. This work was supported by a grant from the National Cancer Institute (CA28681) and by a “Research Centers in Minority Institutions” award (RR03037) from the Division of Research Resources, NIH.
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