Chem. Res. Toxicol. 1994, 7, 401-407
40 1
Effects of Glutathione on Alkylation and Cross-Linking of DNA by Mitomycin C. Isolation of a Ternary Glutathione-Mitomycin-DNA Adduct Mrinalni Sharma, Qiao-Yun He, 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 antitumor antibiotic, is known to alkylate DNA monofunctionally, and to generate DNA interstrand cross-links by bifunctional alkylation. Both processes are dependent on the reductive activation of MC. Glutathione (GSH) was shown here to cause three types of changes in the pattern of alkylation of DNA by MC: (i) GSH caused a decrease of both the overall covalent binding ratio of MC to Micrococcus luteus DNA and the extent of interstrand cross-linking of 32P-pBR322DNA, as the concentration of GSH was increased in the reaction media. Approximately 50 ?6 inhibition of cross-linking was observed at 20 mM GSH. It is likely that the inhibition is caused by the formation of MC-GSH conjugates competing with DNA alkylation, since both processes are triggered by reductive activation of MC [Sharma, M., and Tomasz, M. (1994) Chem. Res. Toxicol. (preceding paper in this issue)]. (ii) GSH causes a switch from monofuctional to bifunctional activation of MC by the prototype “monofunctional” MC-activating agents HdPt02 and NADPH:cytochrome c reductase/NADPH. This was seen by the predominance of bisadducts (i.e., cross-linked adducts) instead of the usual monoadducts in the enzymatic digests of MC-DNA complexes formed in the presence of GSH, as analyzed by HPLC. This finding suggests that GSH participates in the bifunctional activation of MC in vivo. (iii) A ternary MC-GSH-DNA adduct (6) was formed in the presence of GSH both withM. luteus DNA and with a synthetic duplex oligonucleotide; in this adduct the mitosene C1 is linked to N2 of guanine and the mitosene C10 is linked to GSH via sulfur. Adduct 6 was also formed from the DNA-bound MC monoadduct 2a. The overall inhibitory effect of GSH on alkylation and cross-linking of DNA by MC may be significant in drug resistance of tumor cells possessing elevated levels of GSH.
Introduction Mitomycin C (MC;l 1) is an antitumor antibiotic used clinicallyagainst cancer. It acta as a DNA-alkylatingagent, forming monoadducts and bisadducts with guanine residues at their N2-position,in the minor groove of DNA (Figure 1). Both DNA interstrand and intrastrand crosslinks are formed, as the consequence of bisadducta of MC with two guanines in opposite strands, and with two adjacent guanines in the same strand, respectively (1). The reactions with DNA are dependent on reductive activation of MC; hence, MC is regarded as the prototype bioreductive alkylating agent (2). The DNA-damaging activities of MC represent the primary basis of its cytotoxicity (1). Inactivation of antitumor drugs by GSH is believed to be one of the general mechanisms of resistance to drugs, which often develop in tumor cell populations. Specifically, resistance to MC has been correlated with elevated cellular GSH level in severalrecent studies (3-6). In search of a molecular basis for these observations, we demonstrated in cell-free model systems (7)that MC is susceptible to inactivation by GSH via conjugate formation. This conjugation process was dependent on metabolic reduction of MC, mediated by the same flavoreductases which activate MC to a DNA-reactive form. It appeared likely that the same reductively activated transient form of the
* Abstract published in Advance ACS Abstracts, April 1, 1994.
Abbreviations: MC, mitomycin C;M . luteus, Micrococcus luteus.
drug was the direct participant in the inactivation process via reaction with GSH and in the reactions which induce the cytotoxic DNA damage (7). In the present work we wanted to determine whether the alkylation of DNA by MC was indeed affected by GSH in some way: specifically,whether conjugation of MC with GSH and the alkylation of DNA can directly compete with one another. If so, such a competition could be a potential mechanism of GSH-mediated resistance to MC in tumor cells which have elevated levels of GSH. Reactions were conducted under physiological conditions in cell-free systems to characterize the interactions among MC, GSH, and DNA or oligonucleotides under reductive MC-activatingconditions. An inhibitory effect of GSH on DNA alkylation and cross-linking was demonstrated. A ternary GSH-MC-DNA adduct was isolated, and ita structure and mechanism of formation were determined. The unanticipated, powerful influence of GSH on monofunctional versus bifunctional activation of MC is also described.
Experimental Section Materials. Micrococcus luteus (M. luteus) DNA wasobtained from ICN Biomedicals (CostaMesa, CA) and was sonicatedbefore use. The oligonucleotidesd(TIACGT1T) and d(ACACGTCAT) were synthesized using a DNA synthesizer (Applied Biosystems Model 380B). All reagents for the synthesis were purchased from Applied Biosystems (Foster City, CAI. The crude products (110-Fmol scale) were purified by HPLC, both at the “trityl-on”
0893-228~/94/2707-0401$04.50/0 0 1994 American Chemical Society
402 Chem. Res. Toxicol., Vol. 7, No. 3, 1994
OH
Sharma et al.
2 a: 1"-a b: 1"-p
activation
OCONH?
1; MC
\\
bilunctional aclivation
+ DNA
5
OH
Figure 1. Structure of mitomycin C and its DNA adducts. stage and after removal of the trityl group, according to the manufacturer's protocol (Users Bulletin, No. 13, Revised, April 1,1987). Enzymes used for DNA and oligonucleotide digestions and their sources were as follows: DNase I (code D), snake venom diesterase (Crotalus adamanteus phosphodiesterase I), and Escherichia coli alkaline phosphatase (type 111-R),Worthington, Freehold, NJ. Reagents for the assay of cross-linked DNA and their sources were the following: pBR322 DNA, Klenow fragment of DNA polymerase I, and restriction endonucleaseEcoRI were purchased from GIBCO BRL (Frederick, MD), and [ C ~ - ~ ~ P I ~was ATP obtained from DuPont BiotechnologyDivision,Wilmington, DE. Reagents for agarose gel electrophoresiswere obtained from Sigma (St.Louis, MO). All other materials were obtained as described previously (7). Preparation of Linearized a'-End-Labeled pBR322 DNA (8). pBR322 DNA was treated with EcoRI restriction endonuclease as follows: In 625 pL of 50 mM Tris-HC1 (pH 8.0)-10 mM MgClz-lOO mM NaCl buffer 125 pg of DNA was incubated with 250 units of EcoRI restriction endonuclease at 37 "C for 2 h. After addition of 310 pL of 7.5 M NH40Ac the DNA was precipitated by adding 2 volumes of ethanol. After standing at -20 "C for a few hours, the sample was centrifuged for 10 min a t 12 OOO rpm, the pellet was washed with 70% ethanol a t -20 "C, and after centrifuging it was lyophilized. 3'-End-labeling: (30 TP Linearized DNA (5 pg) was incubated with [ ( U - ~ ~ P I ~ A pCi) and 5 units of Klenow fragment of DNA polymerase I for 20 min at room temperature in 57 pL of 50 mM Tris-HC1 (pH 8.0)-10 mM MgC12-100 mM NaCl buffer, followed by precipitation by ethanol as above. Cross-Linking of aZP-pBR322 DNA with MC in the Presence of GSH under Reductive Activating Conditions. (i) Reactions employing varying concentrations of GSH: A mixture containing approximately 10 ng of 32P-pBR322DNA, lo00 ng of calf thymus DNA, 2 nmol of MC, and varying amounts of GSH in 21 pL of 5 mM Tris-HC1 (pH 7.4)-16 mM EDTA was deaerated by gentle bubbling with argon for 2 min. Under continued flushing with argon 4 pL of a freshly made, deaerated NazSlO, solution (7.5 mM) in 0.1 M Tris-HC1 (pH 7.41-2 mM EDTA buffer was added to give final concentrations of 120 pM DNA, 80 pM MC, 0-40 mM GSH, and 1.2 mM NazSz04in a total
volume of 25 p L of 20 mM Tris-HC1 (pH 7.4)-14 mM EDTA buffer. The sample was capped tightly and left at room temperature for 1h. The reaction was terminated by the addition of an equal volume of "stop solution" (0.6 M sodium acetate-1 mM EDTA, 300 pg/mL tRNA) and 3 volumes of cold ethanol. Precipitation of the DNA followed the procedure given above. (ii) Reactions in the presence or absence of 20 mM GSH, using varying concentrations of MC: A mixture containing 10 ng of 32P-pBR322DNA, 1000ng of calf thymus DNA, 0.5 pmol of GSH, and varying amounts (0-2 nmol) of MC in 21 p L of buffer was deaerated and treated with 4 p L of 7.5 mM NazSzO4 as in (i), resulting in final concentrations of 120 pM DNA, 0-80 r M MC, 20 mM GSH, and 1.2 mM NazS204 in 25-pLtotal reaction volume. Further procedures were the same as in (i). Each reaction was repeated without any GSH added to the reaction mixture. Assay of Cross-Linked *ZP-pBR322 DNA by Neutral Agarose Gel Electrophoresis. This was based on the method developed by Hartley and co-workers(9). The lyophilized sample from the above experiments was dissolved in 2 pL of H20, and then 20 p L of strand-separation buffer (30% Me2SO-1 mM EDTA-0.02 % bromophenol blue-0.02 % xylene cyanol) was added. The DNA was denatured by heating the solution a t 90 "C for 3 min and then chilling in ice water. Samples were loaded onto a 1.0% agarose gel (20 cm long) and electrophoresed for 16 h at 40 V. The running buffer was 40 mM Tris-HC1-20 mM acetic acid-2 mM EDTA (pH 8.1). Gels were dried a t 80 "C onto 3-mm filter paper using a Bio-Rad Model 583 gel drier under vacuum. Autoradiography was accomplished using X-OMAT AR film (Kodak) with intensifying screen overnight at -70 "C. The autoradiogram was scanned by a soft laser scanning densitometer (Biomed Instruments, Inc., Model SL-DNA). The % cross-linked DNA was calculated from the densities of the bands corresponding to denatured (fast-moving) and renatured (slow-moving)32P-pBR322DNA in the sample. Formation of MC-DNA Complexes under Various Conditions. (i) HdPt02 as activator: A mixture containing MC (0.7 mM), M.luteus DNA (0.7 mM), GSH (50 mM), and solid PtO2 (90 pglpmol of MC) in 0.015 M Tris-HC1 (pH 7.4) buffer a t room temperature was deaerated by purging it with argon for 10 min. H2 gas was bubbled through the solution for 5 min, followed by purging again with argon (5 min). The mixture was exposed to air and filtered, and the filtrate was chromatographed over a Sephadex G-100 column, using 0.02 M NHhHCO, as eluant. For 10 pmol of DNA (mononucleotide) a 2.5 X 56 cm column was employed. The complex was isolated from the void volume fraction by lyophilization. (ii) NazSzO4 as activator: A mixture of MC (0.7 mM), M . luteus DNA (0.7 mM), and GSH (50 mM) in 0.015 M Tris-HC1 (pH 7.4) buffer was purged with argon or helium for 10 min at room temperature. Aqueous NazSzOl solution in 1.5 molar excess of MC, made freshly in water and kept under continuous purging with argon or helium, was added in 5 equal portions at 10-min intervals from a syringe to the reaction mixture. The decolorized mixture was then exposed to air, turning purple. The complex was isolated as in (i). (iii) Activation by NADPH:cytochrome c reductase/NADPH: A mixture of MC (0.7 mM), DNA (0.7 mM), GSH (5 or 50 mM), and NADPH (0.84 mM) in 0.015 M Tris-HC1 (pH 7.4) buffer a t 37 "C was deaerated as above. In a separate flask NADPH: cytochrome c reductase in a small volume of the above buffer was similarly deaerated at 0 "C and then added to the reaction mixture (1unit/pmol of MC). The solution was incubated a t 37 "C for 60 min and then exposed to air, and the complex was isolated as above. MC-duplex oligonucleotide complexes were prepared analogouslyto MC-DNA complexes. Synthesis and purification of the monoadduct-substituted oligonucleotide 7(2a) (Chart 1) were described previously (10). Preparation of Ternary Adduct 6 (Scheme 1). The starting material 2a was obtained as previously described (IO). A solution of 2a (0.5 mM) and GSH (13 mM) in 0.015 M Tris-HC1 (pH 7.4) buffer was supplemented with Pt02 (90 pglpmol of 2a) and hydrogenated for 5 min using the same procedure as given above
Glutathione Effects on DNA Alkylation by Mitomycin
Chart 1. Structures of Oligonucleotides and Their Adducts. M I
5’ - ACACGTCAT
5’ - ACACGTCAT
7
7(24
5’- T I ACGT I T
M.GSH I 5’ - ACACGTCAT 9
S
0 M mitomycin residue attached to G asjn 2a. M-GSH: mitomycin-GSH conjugate residue attached to G as in 6.
Table 1. Effect of GSH on the Binding Ratio of MC to M.luteus DNA binding ratio (mol of MC/mol of DNA mononucleotide) 60 mM GSH 0 mM (% of the 0 mM activating agent GSH GSH value) 0.190 0.047 (25) HdPt02 0.10 0.060 (60) NazSzO4 NADPHcytochrome c 0.12 0.036 (30) reductase/NADPH
Scheme 1. Scheme of Synthesis of Adduct 6
in detail. The mixture was filtered, and the filtrate was concentrated and applied to a reverse-phase HPLC column (1.0 X 25 cm; Beckman ODs-C-18 Ultrasphere). Adduct 6 was eluted at 26 min by gradient elution using 0-15% acetonitrile in 0.03 M ammonium acetate, pH 7.0, in 20 min at a 2 mL/min flow rate. No other UV-absorbingpeak waa seen in the HPLC tracing. The collected sample from HPLC was desalted by lyophilization. Conversion of DNA-or Oligonucleotide-BoundMonoadduct 2a to Ternary Adduct 6. A solution of the monoadducted DNA or oligonucleotide(1A m unit) in 0.5 mL of 0.1 M Tris-HC1 (pH 7.4) buffer in the presence or absence of 50 mM GSH was deaerated as above and then treated with a deaerated solution of Na&O, (2 mM) at room temperature in the case of DNA and at 0 OC in the case of the oligonucleotides. Incubation under anaerobic conditionscontinued for 40 min. The reaction mixture was chromatographed over a Sephadex G-100 (DNA) or G-25 (oligonucleotide) column, and the DNA- or oligonucleotidecontaining fractions were lyophilized. Enzymatic digestion of MC-DNA and MC-oligonucleotide complexes and HPLC analysis of digests were performed as described previously (12, 13). Quantitative analysis of oligonucleotidesand MC-nucleoside adducts by UV spectrophotometry was described in detail elsewhere (13). For the analysis of M.luteus DNA tm = 7000 was used. Electroepray mass spectroscopy was performed as previously described (14), at The Rockefeller University NIH Regional Mass Spectrometry Facility, New York, NY.
Results Inhibition of the Extent of Overall Binding of MC to M.luteus DNA in the Presence of GSH. This was measured by determining the binding ratio of the MCDNA complex (mol of C boundlmol of DNA mononucleotide) using a UV spectrophotometric method (15). The data in Table 1indicate that the binding ratio is lowered
Chem. Res. Toxicol., Vol. 7, No. 3, 1994 403
by 40-7595 in the presence of 50 mM GSH under three different conditions used for activating MC. Inhibition of Interstrand Cross-Linking of DNA by MC. This was demonstrated with linear pBR322 DNA as substrate for the cross-linkingreaction using a sensitive assay for DNA cross-linking developed by Hartley and co-workers (9). This is based on the spontaneously reversible denaturation of cross-linked DNA. Such renatured DNA can be separated from the denatured, noncross-linkedDNA molecules by agarose gel electrophoresis. The extent of cross-linkingof DNA by MC under anaerobic Na2S204 activation decreased markedly with increasing concentration of GSH (Figure 2a,b). GSH (20 mM) induced a 5 0 4 0 % decrease of cross-linking over a wide range of MC concentration (Figure 2c). Alteration of DNA Adduct Distribution Patterns of MC-DNA Complexes by GSH. M.luteus DNA was treated with MC under HdPt02 activating conditions, which induce the monofunctional activation mode of the drug (16). Accordingly, monoadduct 2a was formed as the major product, as seen by HPLC analysis of the enzymatic digest of the complex (Figure 3a). When the same reaction included 50 mM GSH, the binding ratio of totaladducts decreased from 0.028 to 0.016 and the adduct pattern was also different: Cross-link adduct 4 and an unknown adduct marked 6 were formed as major products, while 2a was completely absent (Figure 3b). A strikingly similar pair of results was obtained when NADPH: cytochrome c reductase/NADPH was used as reducing agent instead of HdPt02 (Figure 3c,d). At a lower concentration of GSH (5mM) the cross-link adduct 4 was the sole product formed (Figure 3e). Similarly, when duplex oligonucleotide 718 was treated with MC under HdPt02 or NADPH:cytochrome c reductase reducing conditions in the absence of GSH, monoadduct2a was the major adduct formed in both cases. In the presence of 50 mM GSH, however, the amount of adducts was lower and the adduct distribution pattern was also altered, indicating 4 and 6 as the major adducts under both HdPt02 and the cytochrome c reductase activating conditions (data not shown). Structure of 6, a Ternary Adduct of GSH, MC, and Deoxyguanosine. This substance, obtained originally from DNA or oligonucleotidestreated with MC and GSH under reductive conditions (Figure 3), was also obtained quantitatively when the known MC-dG adduct 2a (15) was treated with GSH under HdPt02 reduction (Scheme 1). Its UV and CD spectra were very similar to those of 2a, indicating that this substance is a composite of the 7-aminomitoseneand deoxyguanosine chromophores and, furthermore, that it is a closely related derivative of 2a (10; Figure 4). The electrospray mass spectrum showed two prominent peaks at mlz = 816.75 and 408.25, corresponding to (M + HI+ and (M + 2HI2+of 6, respectively. A thiol-group assay (17)was negative, corroborating the structure as 6. These data, taken together with the mode of formation of 6 from 2a which was completely analogous to that of simpler GSH-mitosene conjugates (7),led to the assignment of structure 6 to the tertiary adduct with confidence. Conversion of Monoadduct 2a to Ternary Adduct 6 by GSH in a n Oligonucleotide and in DNA. The monoadduct-substituted oligonucleotide 7(2a) (Chart 1) was reduced with HdPt02 in the presence of 50 mM GSH and then analyzed for adducts by HPLC after diges-
404 Chem. Res Tolcicol., Vol. 7, No.3,1994
Shurmcr et al.
a 1 2 3 4 5 6 7 8 9
ds DNA
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Figare 2. Inhibition of MC-induced cross-linking of linear V-pBR322 DNA by GSH. (a) 1%agarose gel electrophoresis of linear V-pBR322 DNA cross-linked by MC in the presence of GSH, followed by heat denaturation. Complete system: DNA was treated with MC, GSH, and Na&Od, followed by heat denaturation as described in the Experimental Section. Lanes 1-9 contained the following: 1,control linear V-pBR322 DNA; 2, control linear V-pBR322 DNA, heatdenatured; 3, complete system minus Na&O4; 4-9, complete system; GSH concentrations are 0,5,10,20,30, and 40 mM, respectively. (b) Plot of % cross-linking as a function of GSH concentration, measured by densitometry of the autoradiogram of the gel in (a). (c) % crosslinked DNA as function of MC concentration, in the absence ( 8 ) or presence (+) of 20 mM GSH, measured by densitometryof the autoradiogram of a gel run of reactions similar to those in (a), except that the MC concentrations varied (see Experimental Section).
tion. The same was repeated in the presence of an equimolar amount of the complementary oligonucleotide
digests of MC-DNA complexes: Effect of GSH on adduct patterns. The digests originated from complexes formed under the following conditions: (a) MC-M. luteus DNA complex, Hz/ Pt02 activation,in the absence of GSH. (b) MC-M. luteus DNA complex, HdPt02 activation in the presence of 50 mM GSH. (c) MC-M. luteus DNA complexes, NADPHcytochrome c reductase/NADPH activation in the absence of GSH. (d) Same as (c), in the presence of 50 mM GSH. (e) Same as (c), in the presence of 5 mM GSH. HPLC conditions: A reverse-phase column (0.46 X 25 cm; Beckman ODS-C-18 Ultrasphere) was used,in conjunction with a Beckman Model 332 HPLC system. Gradient elution system: 618%acetonitrilein 0.03 M potassium phosphate, pH 5.4, in 60 min, flow rate 1.0 mL/min.
8. Conversion of adduct 2a to 6 in the singlestranded oligonucleotidewas quantitative (Figure54b). In contrast, no conversion of 2a to 6 took place in the duplex oligonucleotide, although 2a was partially converted to the cross-link 4 (Figure 5c). Monoadduct-substituted M. luteus DNA was treated with NADPH:cytochrome c reductase (0.2 unit/Am unit of DNA) and NADPH (0.2 pmol/Am unit of DNA) at 50 mM GSH concentration. HPLC of the digest indicated conversion of the monoadducttoboththecross-link(4)andtemaryadduct6 (Figure 5d).
Discussion MC in its native form (1) is inert toward DNA. Reduction by flavoreductaaesor certain chemical reducing agents activates MC to a mono- and bifunctional alkylator of DNA, resulting in monoadducts and bisadducts of deoxyguanosine (I; Figure 1). In cell-free systems, the reduction conditions determine whether MC is activated monofunctionally at C1 or bifunctionally at Cl and ClO. In the former circumstance the DNA-bound monoadduct 2a is the final product, while in bifunctional activation, the initially formed 2a is further converted to the 10decarbamoylated adduct 3, or cross-link adducts 4 or 5. Bifunctional activation in cell-freesystemsis inhibited by the presence of both air and excess unreduced MC (I63
Glutathione Effects on DNA Alkylation by Mitomycin
2.0
n
nm
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I \I
-'"I
-16.0
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v ,
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520
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wavelength (nm)
Figure 4. Upper: Ultraviolet spectra of adducts 2a and 6 in 0.01 M Tris-HC1, pH 7.4. Lower: Circular dichroism spectra of 2a and 6 in water. (- - -) 2a; (-) 6. a
,
0
,
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,
C
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Figure 5. HPLC analysis of MC adducts in nuclease digests of MC-DNA and MC-oligonucleotide complexes: Conversion of DNA-boundadduct 2a to ternary adduct 6. The digestsoriginated from the following complexes and conditions: (a) MC-oligonucleotide complex 7(2a) as control; (b) MC-oligonucleotide complex 7(2a) treated with HdPt02 and 50 mM GSH; (c) MCduplex oligonucleotidecomplex 7(2a)/8, treated with HdPt02 and 50 mM GSH; (d)MC-M. luteus DNA complex,treated with NADPH:cytochrome c reductese/NADPHand 50 mM GSH. The HPLC of the adduct digest of the untreated MC-M. luteus DNA complex is shown in Figure 3c. HPLC conditions: Same as in Figure 3. 18). In cultured tumor cells, all four adducts are formed side by side, indicating that in the intact cell both types of activation occur (19,20). The fundamental similarity between MC-DNA adduct patterns and their dynamics observed in tumor cells and in the cell-free model systems
Chem. Res. Toxicol., Vol. 7, No. 3, 1994 406
developed in our laboratory (20) validates the use of the latter for testing the effects of agents on adduct formation. In the present work we examined the effects of GSH on overall DNA alkylation and on cross-linking alone. Since GSH itself does not reduce MC (3, several different reducing agents were used to activate MC: monofunctional MC activators HdPt02 and NADPH:cytochrome c reductase, as well as the bifunctional activator, anaerobic NazSz04 (16). Three methods were used to assess the extent of reaction of MC with DNA (i) measurement of the MC/DNA covalent binding ratio, indicating the amount of DNA-bound drug in any adduct form; (ii) assay of cross-linked DNA which indicated specifically the amount of the DNA interstrand cross-link adduct 4; and (iii) separation and quantitation of individual adducts by HPLC, formed by digestion of MC-DNA complexes. The latter method reveals both the distribution and quantity of individual adducts. Inhibition of MC-DNA adduct formation by GSH was observed by using each of these methods. Since we showed previously that in the absence of DNA activated MC reacts with GSH, forming inactive the observed inhibition mono- and bis-GSH conjugates (7), is likely to be due to competition of GSH with DNA for reaction with the activated MC. A similar inhibition of the reaction between cis-dichloro(ethy1enediamine)plati"(11) and cell-free DNA by GSH was described by Eastman (21). It should be noted that the inhibition in our case is relatively modest: for example, 50 mM GSH inhibited the overall reaction between 0.7 mM MC and 0.7 mM DNA only to the extent of 40-75%; similarly, 20 mM GSH inhibited the cross-linking of 0.08 mM DNA by 0.08 mM MC only by 50-60 % . It is clear from these results that activated MC reacts faster with DNA than with GSH, most likely due to its relatively weak but well-defined noncovalent affinity to DNA ( 1 ) . In the cell, however, high levels of GSH could also inhibit DNA-MC adduct formation indirectly, simply by deactivating MC by conjugation before MC could reach its DNA target. Whether this occurs in cells is not known (7). The inhibitory effect of GSH on MC-DNA adduct formation could in principle protect DNA from MC-induced damage in the cell and thus contribute to the modulation of MC cytotoxicity by GSH levels, observed in certain tumor cells (3-6). This hypothesis could be criticallytested by analysis of MC-DNA adduct formation and DNA cross-linking in intact cells (20) having elevated levels of GSH. In addition to decreased formation of all adducts a striking effect of GSH was observed on adduct distribution. Using either M.luteus DNA or the duplex oligonucleotide 7/8 as substrates, the major adduct formed under the activating conditions HdPt02 or cytochrome c reductase was 2a, indicative of monofunctional activation of MC (Figure 3a,c). When GSH was also present, however, the major product was cross-link adduct 42in each case (Figure 3b,d,e). This shows that GSH modulates the reductive activation of MC by changing it from the monofunctional to the bifunctional mode (Figure 1). As mentioned above, GSH alone does not reduce MC, but it accelerates the rate of reduction of the drug initiated by other reducing agents (7). It is likely that this effect of GSH is responsible for the observed change in adduct distribution since fast MC reduction rate in general favors bifunctional activation
* Under the enzymatic DNA-digestionconditionsused in the present work the intrastrand cross-link adduct 5 is hydrolyzed to adduct 4 ( I ) . The latter is therefore a composite measure of the intrastrand and interstrand cross-links.
406 Chem. Res. Toxicol., Vol. 7, No. 3, 1994 Scheme 2. Alternative Mechanisms of Formation of the Ternary GSH-MC-DNA Adduct 6 MC(activated) + GSH
+
DNA MC-GSH
conjugate
(16, 18). It is interesting that both hydrogenation- and reductase enzyme-mediated reductions of MC were accelerated by GSH (7). Since now it is shown that both of these systems switch from monofunctional to bifunctional activation of MC in the presence of GSH, this further supports a common, nonenzymatic mechanism for the acceleration, as proposed in the preceding report (7).It is possible that the activation of MC in vivo involves the concerted action of both cytochrome c reductase and GSH. A third effect of GSH on MC-DNA adduct formation is the appearance of a new, ternary GSH-MC-DNA adduct, 6, among theproducts,formed with bothM. luteus DNA and the duplex oligonucleotide 718. There were two alternative mechanisms to be considered for its formation (Scheme 2): (a) adduct 2a was formed with DNA and subsequently, while still in the reduced state, it reacted with GSH a t ClO"; (b) the reduced form of MC first reacted with GSH at its C10 position by an s N 2 mechanism (3, and the resulting GSH-MC conjugate, still activated at the C1 position, then attacked DNA at guanine N2. Both mechanisms have known precedents involving other bifunctional electrophilic agents: the first, in the trapping by GSH of DNA monoadducts of cis-dichloro(ethy1enediamine)platinum(II) (21),and the second, in the formation of the ternary adduct S-[2-(W-guanyl)ethyllGSHby GSH reacting first with the mutagen l,&dibromoethane, followed by attack by the resulting conjugate on DNA (22). Since conversion of adduct 2a to 6 upon reduction was readily demonstrated using either free 2a or oligonucleotide- and DNA-bound 2a (Figure 51, mechanism a accounts for the formation of the ternaryadduct, in analogy to the case of the cis-platinum drugs. It is noteworthy that, in duplex DNA, the reductive conversion of 2a to the cross-link adduct 4 is much more favorable than to ternary adduct 6, due, undoubtedly, to steric hindrance to an external s N 2 attack by GSH (7) on adduct 2a hidden in the minor groove. Consistent with this, conversion to 6 in the single-stranded oligonucleotide adduct 7(2a) (no steric hindrance) was quantitative (Figure 5 ) . It remains to be seen whether the ternary adduct 6 also occurs in intact cells. The present results indicate that this is chemically feasible and provide the authentic standard to be used in search of adduct 6. Analogous ternary GSH adducts were detected in intact cells upon treatment with cisplatin (23)and 1,Pdibromoethane (24). Formation of ternary adducts of thiol-containing proteins may also be envisaged;this would account for the observed MC-mediated DNA-protein cross-links in intact cells (25). In summary, GSH was shown to modestly inhibit DNA alkylation and cross-linking by MC, most likely by direct competition with DNA for the activated form of MC. Two additional effects of GSH on these processes were discovered. One is a change in adduct distribution, from predominantly monoadducts to predominantly bisadducts (cross-links) formed in the cell-free activation systems. Another effect is formation of a new, ternary MC-GSHDNA adduct. The inhibitory effect of GSH on the overall
Sharma et al.
MC-induced DNA damage demonstrated here suggests a molecular rationale for the inverse relationship between MC cytotoxicity and GSH level observed in tumor cells (3-6). Acknowledgment. We thank Dr. D. M. Vyas, BristolMyers Squibb Co., Wallingford, CT, for donation of mitomycin C and Drs. Brian T. Chait and Urooj A. Mirza, The Rockefeller University, New York, for the mass spectroscopic determination. 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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