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Modulation of streptonigrin cytotoxicity by nitroxide sod mimics
Free Radical Biology & Medicine, Vol 17. No 5, pp 379-388, 1994 1994 Elsewer Science Ltd Pnnted in the USA All nghts reserved 0891-5849/94 $6.00 + 00
Pergamon
0891-5849(94)E0052-K
Original Contribution MODULATION OF STREPTONIGRIN CYTOTOXICITY BY NITROXIDE SOD MIMICS
MURALI C. KRISHNA,* RIVKA F. HALEVY, t RENLIANG ZHANG, t PETER L. GUTIERREZ, :~ and AMRAM SAMUNI t *Radiation Biology Branch, Clinical Oncology Program, Dwislon of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA; *University of Maryland Cancer Center, Baltimore, MD, USA, and *Molecular Btology, School of Medicine, Hebrew University, Jerusalem, Israel
(Recetved 13 May 1993; Revtsed 26 October 1993; Re-revtsed 22 February 1994; Accepted 23 February 1994)
Abstract Nltroxides are cell-permeable, stable radicals that react readily with paramagnetlc species such as transltaon metals or short-hved free radicals, though not generally with diamagnetic molecules Nltroxldes can undergo one-electron selective redox reactxons and thereby potenttally modify the actawty of cytotoxlc drugs. Streptomgnn (SN) toxicity requires bloreductlon to yield the semxquinone radical, and the toxicity is reportedly medmted by transition metals and oxygen-denved reactive species via redox-cyclmg of the semlquinone mtermedmte The present study shows that (1) nitroxldes protected isolated DNA and also aerated or hypoxic bacterial cells from SN toxicity; (2) H202 potentiated the hypoxlc cytotoxlcity of the drug but inhibited the damage to aerated cells; (3) pretreatment of cells with H202 conferred some protection, but not when the drug alone was preexposed to H202, and (4) desfernoxamme and 2,2-dlpyndyl, though neither dlethylenetnammo pentaacetate, exogenous catalase, or superoxlde dlsmutase, decreased SN-mduced cell kalling. The mechanisms by which mtroxldes protect from SN toxicity involve both a selective radical-radical reaction w~th SN semiqumone and the reoxldatlon of reduced cellular transition metal ions On the other hand, H~O2 appears to exert two opposing effects. (1) faclhtatlon of cell killing by the Fenton reaction and (2) lowenng the cellular level of reducing equwalents, thus mhthmng the bloreductlve activation of SN Keywords--EPR, Free radicals, H202, E. colt B, Semlqumone, Superoxlde, Spin-labels, Oxidative stress, Spin-trapping, DMPO
tives are widely used as biophysical probes in studying cell metabolism, 4 membrane fluidity, intracellular 02 level, 5'6 and as potential contrast agents in nuclear magnetic resonance imaging. 7'8 Being free radicals, nitroxides have been found also to react with and dismutate 02"- in a catalytic fashion similar to that of S O D . 9 It was anticipated, therefore, that SOD-mimics would protect cells against oxidative damage. Subsequent studies demonstrated that nitroxides and their respective hydroxylamines ~° inhibit hydrogen peroxide-induced damage under either air or hypoxia, to H202sensitive xth mutant of E. coli, 11 V79 Chinese hamster cells, 12 beating cultured cardiomyocytes, 13 and lipid membranes.I°'14 Moreover, hypoxic cytotoxicity of mitomycin c is and aerobic radiation damage in V79 cells were attenuated by nitroxides) 6 In addition, nitroxides were found to react with reduced transition metals such as Fe(II), though not with H202 or ten-butyl hydroperoxide directly.: Because nitroxides undergo one-electron reactions,
INTRODUCTION
The most common approaches used to assess and modulate free radical-mediated biologic damage induced by anticancer agents, focus on either detoxifying deleterious species or removal of redox-active transition metals that can catalyze and potentiate cell injury. Catalases and peroxidases are used to detoxify H202, change superoxide dismutase (SOD) to dismutate, remove O2"-, ~as chelating agents to bind the metals, 2 and as scayengers for trapping "OH. Success is, however, limited when intracellular compartments are not readily accessible to the protective agent. An alternative mechanism of protection has been demonstrated by a group of cell-permeable, relatively nontoxic 3 nitroxides having an unpaired electrorf. Nitroxide deriva-
Address correspondence to. Murall C Krlshna, Radlauon Biology Branch, Bldg 10, Rm. B3 B69, National Cancer Institute, NaUonal Institutes of Health, Bethesda, MD 20892, USA
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they react rapidly with other radical species, but not readily with diamagnetic molecules. This makes them a powerful research tool for the detection of free radical-mediated processes. More importantly, contrary to SOD or catalase, nitroxides penetrate intracellular spaces readily, x2 Streptonigrin is cellularly bioreduced to the respective semiquinone SN'-, x8 which in turn reduces molecular oxygen to yield superoxide and subsequently H202.19 E. coli B, unlike mammalian cells, is resistant to H202 up to several mM and was selected as a test-system to study the role of H202 in SN activity. The present results indicate that nitroxides protect ceils from SN under both aerated and hypoxic conditions. The protective effect appears to involve the termination of the radical chain reactions by a continuous recycling of the SN semiquinone to the parent drug and oxidation of reduced redoxactive transition metal ions. Although n202 potentiated SN-induced DNA cleavage in aerated cell-free systems, it remarkably protected aerobic bacterial cells from SN. The protection presumably reflects an inhibition of the bioreductive activation of SN to semiquinone indirectly through lowering cellular level of reducing equivalents. MATERIALS AND METHODS
Chemicals
Streptonigrin, 2,2-dipyridyl, and cytochrome c (cytc) reductase (EC 1.6.99.3) were obtained from Sigma; desferrioxamine (Desferal ®) was a gift from Ciba Geigy. Xanthine Oxidase (XO), reduced nicotinamide adenine dinucleotide (NADH), and catalase were purchased from Boehringer Biochemicals (Germany). Diethylenetriaminopentaacetic acid (DTPA), 5,5-dimethyl-l-pyrroline-Noxide (DMPO), 2,2,6,6-tetramethylpiperidine-l-oxyl (TEMPO), 4-hydroxyl-2,2,6,6-tetramethylpiperidine- 1oxyl (TEMPOL), and 4-amino-2,2,6,6-tetramethyl-piperidine-l-oxyl (TEMPAMINE) were purchased from Aldrich Chemical Co. (Milwaukee, WI). The DMPO solution was purified with activated charcoal, filtered, and its concentration was spectrophotometfically determined using 8.0 mM-~cm-t at 227 nm. All other chemicals were prepared and used without further purification. Deionized water was used throughout all experiments. Unless otherwise stated, the experiments were conducted at room temperature. Culture preparation and SN treatment of cells E. coli B cells were grown at 37°C in LB growth medium. Log phase cells in growth medium or washed cells in 1 mM phosphate buffer, pH7.4, containing 1 mM MgSO4 were treated for varying periods of time
with 10/zM SN in the absence or presence of nitroxide. The cells were sampled, diluted with phosphate buffer, plated on L-agar, incubated overnight at 37°C, and subsequently colonies were counted. Surviving fractions were calculated and presented graphically on semilogarithmic scale. Graphs represent typical results generally performed in triplicate. Error bars represent standard deviations of the mean and are shown when larger than the symbol. For studies of hypoxic toxicity, the cell suspension was bubbled 20 min with N2 prior to and during incubation with the drug. Electron paramagnetic resonance (EPR) measurements
Samples were drawn into a gas-permeable, 0.8 mm inner diameter, Teflon capillary. The capillary was inserted into a quartz tube and then placed within the EPR cavity. During the experiment, the sample within the spectrometer cavity was flushed with either air or N2, without disturbing the sample, and the EPR spectra were recorded on a Varian E9 X-band spectrometer, operating at 9.45 GHz, 100 kHz modulation frequency, 1 G modulation amplitude, and 10 mW microwave power. To study the decay kinetics of the nitroxide, the magnetic field was kept constant while the intensity of the EPR signal was followed. Spin-trapping of "OH and
02 °-
upon SN reduction
In experiments aimed to detect "OH and superoxide radicals, the reaction mixture was placed in the gaspermeable Teflon capillary containing 100 mM of the spin trap DMPO. For anoxic experiments, the air flow was substituted by N2. About 6 min was required to render the system anoxic. Bulk electrolysis
In bulk electrolysis experiments, an electrochemical reactor similar to that previously described2° was used. The cell consisted of a working electrode of graphite packed inside a porous Vycor glass tube (5 mM ID), through which the PBS solution containing SN was pumped (approximately 2 ml/min). An outer glass cylinder, with separate electrolyte, contained the platinum auxiliary electrode and a reference Ag/AgC1 electrode immersed in tetramethyl ammonium perchlorate. Controlled-potential electrolysis was performed with a CV27 Potentiostat (Bio Analytical Systems) using -0.7 V vs. Ag/AgC1 reference electrode at ambient temperature where a current of 1.6 mA was measured when 500 /~M SN was electrochemically reduced in 0.1 M
Nltroxldes modulate SN cytotoxlcRy tetramethylammonium perchlorate. For high resolution EPR spectra, 500 # M SN was electrolyzed in dimethylsulfoxide in the presence of 0.1 M tetraethylammonium perchlorate. Simulation of the EPR spectrum was performed assuming isotropic and coinciding A and g tensors.
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Assay of in vitro DNA scission Covalently closed circular (ccc) pUC19 plasmid D N A was prepared and isolated as previously described. 2~ To assay for single-strand breaks, samples of 0.1 #g DNA/well were applied to horizontal, 0.9% agarose slab gels in pH 8 tris-acetate EDTA buffer containing 0.2 /zg/ml ethidium bromide and electrophoresed at a potential gradient of 2.5 V/cm. Following electrophoresis, the gels were illuminated with UV light and photographed using 665 Polaroid positivenegative film. To assess the degree of degradation, fragments of known sizes from digests of h-phage D N A by endonuclease Hind III have been included in the electrophoretic run. The untreated D N A consisted of both a major fraction of super-coiled and a minor relaxed ccc plasmid.
RESULTS In vitro effect of H202 and nitroxides on SN-induced scission of isolated DNA Streptonigrin has been previously found to cleave D N A in cell-free systems in a mechanism that requires one-electron reduction of the drug to SN'-, and involves O2"-, iron, H202, and "OH. 22 Positively charged nitroxides, such as TEMPAMINE, were previously found to confer greater protection to D N A than that demonstrated by neutral nitroxides. 23 The protective effect of T E M P A M I N E was examined in the present study by exposing isolated pUC19 D N A to SN upon its enzymic reduction by NADH. The D N A was incubated at 37°C in l0 m M HEPES buffer at pH 7 containing 0.2 m M N A D H and 100 # M SN, with either 0.05 U/ml XO or 40 lzg/ml cyt-c reductase for 10 min. The control experiments did not include SN. Subsequently, the treated D N A was electrophoresed on agarose slab gels and the extent of single-stranded breaks was determined by comparing the intensities of supercoiled and relaxed ccc D N A fractions. Under our experimental conditions, the relaxed circular and the linear forms of double-stranded pUC19 D N A have similar electrophoretic mobilities, hence their respective bands are hardly distinguishable. This was confirmed by running control samples of singly nicked plasmid (data not shown). As a result, the induction of either
Fig 1 TEMPAMINEprevents m vitro DNA cleavage induced upon enzymacreduction of streptomgnn. Isolated supercolled ccc pUC19 plasmld DNA m 10 mM HEPES buffer pH 7 was exposed at 37°C for l0 man to 100/zM SN and 0.2 mM NADH with either 0.05 U/ ml XO or 40 #g/ml cyt-c reductase. Control experiments did not include SN or enzyme. Lane 1. no addluves, Lane 2: + XO without SN; Lane 3. XO + SN; Lane 4- XO + SN + TEMPAMINE;Lane 5. + cyt-c reductase wtthout SN; Lane 6 cyt-c reductase + SN, Lane 7: cyt-c reductase + SN + TEMPAMINE,lane 8" + TEMPAMINE, without NADH; Lane 9' HmdII1digest of k-DNA. The DNA was electrophoresed for scisslon analysis as detailed in Methods
single- or double-stranded breaks increased the intensity of the trailing D N A band at the expense of that of the leading band of super-coiled DNA. A typical gel electrophoregram is illustrated in Fig. 1. The extent of D N A scission was evident by comparing untreated D N A (lane 1) with treated DNA. In the presence of XO and without SN, a partial breakage took place, whereas with SN all the D N A underwent scission (lane 3). In the presence of cyt-c reductase and without SN, no D N A scission took place (lane 5). However, with SN most of the D N A underwent scission (lane 6). The protective effect of the nitroxide is also illustrated in Fig. 1. T E M P A M I N E at 1 mM fully prevented SNfacilitated D N A breakage induced by either XO (lane 4) or cytochrome reductase (lane 7). The present results support the mechanism previously suggested for SNinduced D N A cleavage and demonstrate the protective effect of nitroxide. To test whether these conclusions
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Ttme of cell exposure to streptomgnn, roan
Fig. 2 Protecuon afforded by nitroxldes to E. coli B cells against streptomgrln-induced lolhng. Time-dependence of cell survival following aerobic incubation of 10 #M SN with 1 × 107 E coli B cells/ml m LB growth medium at 37°C, in the absence (11) and presence of 10 mM TEMPO (©), 1 mM TEMPAMINE (e), 1 mM TEMPOL (A), or 10 mM CHDO (n)
can be extrapolated to the cellular level, bacterial cells were studied.
Nitroxide effect on bactericidal activity of SN E. coli B at 1 × 107 cell/ml were exposed, in LB growth medium at 37°(2, to 10 #M SN, and the resuiting survival curves were determined. In Fig. 2, the effect of nitroxides on the rate of SN-induced cell killing in the presence of air is displayed. Nitroxides at 10 mM showed no toxicity in the absence of SN (data not shown). All nitroxides tested protected E. coli cells. Figure 2 shows that hydrophilic nitroxides, TEMPOL, and TEMPAMINE provide greater protection than the more lipophilic derivatives such as CHDO or TEMPO. Addition of 200 U/ml SOD or 130U/ml catalase did not affect the aerobic toxicity of SN (data not shown). Similarly, the cell-permeable Mn(III)-desferrioxamine chelate (Mn-DF), which possesses SOD-mimic activity, 24'25 did not protect the cells (data not shown). In fact, 180 #M Mn-DF slightly potentiated SN toxicity, possibly because of dissociation of some of the chelate.
Effect of chelating agents Transition metals, particularly iron, are known to potentiate SN cytotoxicity in various test-systems, including bacterial cells. 26"27In the present work, the effect of 200 #M DTPA, Desferal (DF), and 2,2-dipyridyl on the rate of SN-induced cell killing was studied
and the results are displayed in Fig. 3. DF provided partial protection upon 30 mln preincubation. Dipyridyl provided better protection, whereas DTPA, which does not enter ceils, was without effect. These results are consistent with previous conclusions implicating intracellular transition metals in SN cytotoxicity.2s'29 H202 effect on the aerobic and hypoxic cytotoxictty
of SN Wild-type E. coli B, unlike mammalian cells, is relatively resistant to H202, and was selected to study SN toxicity. The cytotoxicity of SN requires molecular oxygen, 3° as demonstrated in Fig. 4 (open symbols). H202 at subtoxic concentrations (___ 2 mM) protected aerobic E. coli B cells from SN toxicity (Fig. 4, solid lines), whereas under hypoxia, H 2 0 2 did not protect and even slightly increased the rate of cell killing (Fig. 4, dashed lines). To determine whether the protective effect of H202 against aerobic toxicity of SN results f r o m H202 reaction with SN, the reagents were incubated together in the absence of cells. After 20 min, the solution was checked for any change in SN absorption spectrum. Because no spectral changes were observed, direct reaction between SN and H202 during this period seemed unlikely. To confirm this conclusion, SN was preincubated aerobically for 20 min with H202 in LB medium in the absence of cells. Then, log-phase E. coli B cells were added to a final concentration of 1 )< 107 cells/ml together with 130 U/ml catalase to remove the H202, and the time-dependence of cell survival was determined. The rate of cell killing did not
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Nitroxides modulate SN cytotoxlclty
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Time of cell exposure to streptomgnn, nun F:g 3. Effect of cell-permeable and nonpermeable chelating agents on SN toxicity" Time-dependence of E. colt B survivmg fraction following exposure at 37°C to 10 #M SN m the absence (solid symbols) and the presence (open symbols) of 200/zM chelatmg agents: DTPA (circles); DF (squares); dipyrldyl (mangles).
differ from that observed using fresh SN untreated with H202.
Effect of preincubation of cells with induced killing of E. coli
H202 o n
of cellular reducing equivalents, H202 is repressing SN bioreductive activation, then preincubation of cells with H2Oz before exposing them to SN should provide protection. To investigate this possibility, E. coli cells were preincubated in LB growth medium at 37°C with 2 mM H202 in the absence of SN for 15 min. After this incubation, 130 U/ml catalase was added to the cells followed by 10 #M SN. Catalase and SN were added also to the cells in the control system (Fig. 5,
SN-
An alternative explanation for the protective effect of H202 against SN toxicity could be a depletion of cellular reducing equivalents. If, by lowering the level
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Time o f cell exposure to streptonigrln, m m Fig. 4. The effect of H202 on the anoxic and aerobic cytotoxlcity of strcptonigrin. E. colt B cells were incubated with l0 #M SN in the absence (open symbols) and in the presence (solid symbols) of 2 mM H202 m Davis medium at 37°C, under anoxlc (©) and aerobic conditions (A).
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Time of exposure to streptonlgnn, wan Fig. 5 Effect of prelncubatlon with H202 on SN-mduced cytotoxlclty E. coli B cells were prelncubated at 37°C in growth medium m the absence (open) and the presence (closed) of 2 m M H202. After 15 nun, 130 U/ml catalase has been added, to remove H202, followed by the addltaon of 10/.tM SN The cells were sampled after various u m e intervals, diluted and plated for the determination of colomes forming abihty
circles), which had not been preexposed to H202. The cells of both systems were sampled at various time intervals and survival was determined. Figure 5 shows that preexposure of the cells to H202 rendered them more resistant to SN. The reaction between SN semiquinone radical (S'-) and nitroxide
Because SN toxicity depends on its bioreductive activation to SN'-, the protection by nitroxides could be attributed to direct inactivation of the semiquinone intermediate. Therefore SN was enzymatically reduced in the absence and presence of nitroxide. When 100 #M TEMPOL was incubated under air at room temperature in 10 mM phosphate buffer, pH7, with 40 /zM SN, 1 mM NADH, and 0.1 U/ml XO, the EPR signal rapidly decayed. However, this decay was prevented by 100 U/ml SOD (data not shown), which suggests that 02"- is responsible for this spin-loss. The rate of EPR signal decay under hypoxia resembled that observed under air; however, it was unaffected by SOD (Fig. 6), which indicates a direct reduction of the TEMPOL by SN'-. In the absence of SN, no loss in the EPR signal intensity of TEMPOL was observed in both aerobic and hypoxic conditions. The EPR signal of TEMPOL, lost upon SN reduction both under air and hypoxia, could be restored by adding one-electron oxidant such as ferricyanide. The reaction between
H202
and SN'-
The protective effect of H 2 0 2 against SN-induced damage could result from direct reaction with SN semi-
quinone. To test this, SN'- was generated by a controlled-potential bulk electrolysis using an electrochemical reactor (see Methods section). The EPR signal observed following electrochemical reduction of SN in dimethylsulfoxide containing 0.1 M tetraethylammonium perchlorate under strict anoxia is demonstrated in Fig. 7. The SN'- spectrum consisted of 18 well defined lines, among which a triplet due to a relatively large a N could be observed (Fig. 7, top). The fact that the group of lines that makes up the high field triplet is smaller, indicates a slow tumbling of the SN'radical. The hyperfine coupling constants validated by spectral simulation (Fig. 7, bottom) are: a"cocH3 = 0.266 G for three equivalent protons, aNNH2 = 1.53 G, aHNH2 = 0.63 G, and aHNH2 = 0.576 G. The difference in the hyperfine coupling constants observed for SN'radical in the present study to that reported earlier34 is due to the difference in solvent. In aqueous solution using 500 #M SN in PBS as electrolyte, the EPR lines became somewhat broader (data not shown). At higher values of modulation amplitude, a broad singlet was observed as shown in Fig. 8 (top). Subsequently, the SN'- solution was mixed 1:1, either with water (Fig. 8, top) or with freshly prepared 10 mM H 2 0 2 in water (Fig. 8, bottom) within the sealed quartz EPR cell, and rescanned for EPR signal using overmodulating setting. To avoid traces of molecular oxygen, which can rapidly eliminate SN'-, the H202 solution was freshly prepared prior to the experiment and all the solutions were thoroughly bubbled with ultra pure nitrogen prior to and during mixing. As seen in Fig. 8 (bottom), H202 totally eliminated the SN'- signal. To identify the products of SN'- reaction with H202,
..._.~_.___~.
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_.=
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No H202
I
10
I
20
I
30
Time, wan Fig. 6 Spin-loss of TEMPOL induced upon the enzymatic reduction of SN. Decay kinetics of the EPR signal of 100 # M TEMPOL upon hypoxlc incubation at RT in 10 m M phosphate buffer pH 7 containmg 40 # M SN, 1 m M NADH, and 0.1 U/ml XO, m the absence and the presence of H202 In the absence of SN, no change of the nitroxide EPR signal was observed
Nltroxldes modulate SN cytotoxioty
385
H202 concentration was required to enable it to better 2 GAUSS
1
compete against 0 2 for the SN semiquinone. The failure of SOD to eliminate the DMPO/'OH signal indicated that this spin-adduct did not result from a decomposition of DMPO/'OOH, but rather trapping of authentic "OH radicals. This observation suggests that SN'- reduces H202 to produce "OH rather than oxidizing it to 02"-. With SN'-, unlike the reaction of H202 with adriamycin semiquinone, 31 DMPO/'OH was observed both in the absence and in the presence of either 50/~M DF or 70/~M DTPA, thus excluding the role of redoxactive transition metals in the reductive cleavage of H202 by SN'-. DISCUSSION
Role of nitroxides
Fig 7. The EPR spectra of SN'- semiqulnone. (Top) SN'- was generated by a controlled-potential bulk electrochemical reduction of SN in dimethylsulfoxide containing 0 1M tetraethyl ammonium perchlorate under strict anoxm. (Bottom) The simulated spectrum plotted using a"coc,3 = 0.266 G for 3 equivalent protons, attN.2 = 1.53 G, an~m = 0 63 G, and anr~,2 = 0.576 G
streptonigrin was enzymatically reduced in the presence of the spin-trap DMPO. A phosphate buffer 0.1 M, pH 7, containing 100 #M SN, 63 mM DMPO, 2.2 mM NADH, and 0.2 U/ml XO in a gas-permeable Teflon capillary was placed in the spectrometer cavity, and scanned for EPR signal. In the presence of air, the 12-line signal characteristic of DMPO/'OOH, the O2"spin-adduct of DMPO, was observed. Upon changing the gas from air to N2, the DMPO/'OOH signal disappeared. However, when 415 U/ml SOD and 0.4 M H202 were included in the reaction system, only the 1:2:2:1 four-lines spectrum of DMPO/'OH ('OH adduct of DMPO) was observed (data not shown). Generally, much lower SOD concentration is sufficient to effectively compete for O2"- against DMPO. However, because 02"- rapidly reacts with and destroys DMPO spin adducts, 9 higher [SOD] was needed to protect the DMPO/'OH from destruction by superoxide. High
Contrary to SOD and catalase, nitroxides conferred full protection to bacterial cells exposed to SN (Fig. 2). The protective effect of TEMPOL and TEMPAMINE exceeded those of the more lipophilic nitroxides TEMPO and CHDO. Moreover, the positively charged TEMPAMINE exhibited the largest protective effect. These results support the position of DNA being a critical target. Because SN, like paraquat and menadione, is an effective generator of intracellular superoxide radicals, 26"32 a straightforward explanation for the nitroxides' protective effect is their SOD-mimicking activity. Accordingly, nitroxides protect by removing intracellular superoxide. The failure of Mn-DF to pro-
_2 J---SN"
+
H20
SN" ÷ H202
10 Gauss
Fig. 8. H202-1nducedspin-loss of SN semiquinoneradical. The EPR signal of streptonignn senuquinone rathcal, recorded under overmodulating setting, followingelectrochemicalreductionof 500/zM SN m PBS under anoxaa. Subsequently,the SN'- solution within the sealed quartzEPR cell has been nuxed 1:1 etther wtth water (Top spectrum)or with 10 mM H202(Bottomspectrum)and rescannedfor EPR signal.
386
M. C KRISHNAet al
tect could result from a dissociation of the chelate, which gives rise to redox active Mn ions. In addition, the increase of SN hypoxic toxicity by H202 (Fig. 4) and the protective effect of chelators (Fig. 3) confirm the mediatory role of transition metals, and substantiate the suggestion that nitroxides also protect by oxidizing reduced metals such as Fe(II), as previously found. 11'~2 According to this mechanism, nitroxides compete with H202 for the reduced metal ion, Fe(II) + R'RN - O" + H + Fe(III) + R'RN - OH,
(1)
to inhibit the Fenton reaction that yields "OH radicals and/or hypervalent metal ions (Eq. 2a and 2b). Fe(II) + H202 --+ Fe(II) - H202 --' Fe(III) + OH- + "OH Fe(II) - HzOz ~ Fe(IV) + 2OH-.
(2a) (2b)
Becasure SN activity requires bioreductive activation of the drug, 27'33'34 an alternative explanatmn can be envisaged in which the reaction of the nitroxlde with SN'- functions to terminate the radical chainreaction, and hence prevents the ultimate damage. The formation of DMPO/'OH in the presence of SOD, DF, and H202, combined with the results displayed in Fig. 8, show that the semiquinone is readily oxidizable not only by 02
(3)
SN'- + 02 ~ SN + 02"but also by H202: SN'- + H202 --+ SN + O H - + "OH.
(4)
Reactions 3 and 4 both yield secondary radicals. Conversely, the oxidation of the semiqumone by nitroxide is a radical-radical reaction which terminates the radical chain reaction. SN'- + R'RN - O" + H + SN + R'RN
-
OH.
(5)
The present results indicate that nitroxides are capable of protecting the cells not only by catalytically removing O2"-, 9'35 and by maintaining (reaction 1) redoxactive metal ions in their higher oxidation state, ~z but also by competing with oxygen and H202 for the SN'radical (reaction 5).
Role o f H202
The H2Oz-induced enhancement of SN hypoxic cytotoxicity and increased DNA degradation in vitro (Figs. 1 and 4) is consistent with the commonly adopted site-specific mechanism underlying the deleterious effect of the drug (SN toxicity under hypoxia could also increase by 02 generated through H202 decomposition). According to the site-specific mechanism of damage, redoxactive metal ions are coordinated to, amongst other things, critical cellular sites. Hence, the reactions take place locally and the active species are formed site-specifically in close proximity to the critical sites, rather than randomly in the bulk. The production of "OH radicals in reactions 2 and 4 implies that Increasing H202 concentration should facilitate the damage. The protective effect of H202 against the aerobic cytotoxicity of SN seen in Fig. 4 is not consistent with this prediction. In fact, 2 mM H202 protected the cells from SN. A similar protection of mammalian cells by H202 against the cytotoxicity of tumor necrosis factor has been previously found. 36 This protective effect was observed following a pretreatment of the cells with HzO2, but not when tumor necrosis factor alone was preincubated with H202. The inhibition of SN-induced killing is attributable to the effect of H202 on the level of cellular reducing equivalents. Because the affinity of catalase for H202 is low, a major pathway of H202 metabolism in cells involves the reduction of H202 by glutathione (GSH) and GSH peroxidase. Accordingly, the reduction of GSSG to GSH is mediated by GSSG reductase, which uses NADPH. Thus, the GSH cycle is coupled with the oxidation of glucose-6-phosphate and 6-phosphogluconate, which provides NAD(P)H for reduction of GSSG. Previous studies demonstrated that exogenously added GSH does not protect E. coli cells from H2Oz-induced damage. 37 On the other hand, treatment of E. coli with H202 caused an 80% decrease in the cellular GSH, and increased the export of GSSG and the inactivation of GSH reductase. 38'39 Another key study of HzOz effect on NAD(P)H in E. coli cells showed that the concentration of these reducing equivalents in H202-treated E. coli cells also diminished via a direct reaction with the peroxide. 4° Obviously, an H202-induced depletion of cellular GSH and NAD(P)H can lower the rate of SN reduction and inhibit the cellular damage.
CONCLUSION
The results of the present study extend our understanding of the protective effects of nitroxides and help
Nitroxides modulate SN cytotoxlclty
elucidate the roles of redoxactive metals in streptonigrin toxicity. They also indicate that H202 may, under certain circumstances, act as a protective agent, and call for greater caution in extrapolating conclusions drawn from cell-free to cellular systems. Acknowledgements - - The helpful dxscusslons with Dr A. Russo and Dr. J.B Mitchell are gratefully acknowledged This research was supported by Grant 89-00124 from the Umted States-Israel Binatlonal Science Foundation (BSF), Jerusalem, Israel
REFERENCES
1 McCord, J. M ; Fridovlch, I. Superoxlde dismutase' The first twenty years (1968-1988). Free Radtc. Btol Med. 5:363-369, 1988 2 Halhwell, B. Protection against tissue damage m vivo by desfernoxamine. What is its mechanism of actaon0 Free Radtc. Btol Med. 7:645-651; 1989. 3 Ankel, E. G.; Lal, C. S ; Hopwood, L. E , Zlvkovlc, Z. Cytotoxicity of commonly used nitroxlde radical spin probes. Ltfe Sct 40:495-498; 1987 4. Bacic, G ; Nilges, M. J.; Magm, R. L ; Walczak, T ; Swartz, H M. In VlVOlocahzed ESR spectroscopy reflecting metabolism. Magn. Reson. Med. 10:266-272; 1989 5. Chan, H. C.; Glockner, J. F., Swartz, H. M Oximetry in cells and tissues using a mtroxlde-hposome system. Btochim. Btophys Acta 1014:141-144, 1989 6 Froncisz, W ; LaL C S., Hyde, J S. Spin-label oxlmetry Kineuc study of cell resplrauon using a rapid-passage T 1-sensitive electron spin resonance display Proc. Natl Acad Sct USA. 82:411 415, 1985. 7 Bennett, H. F ; Brown, R D., III; Keana, J. F.; Koenog, S H., Swartz, H M. InteracUons of mtroxides with plasma and blood Effect on l/T1 of water protons Magn. Reson. Med 14:40-55; 1990. 8 Chan, H. C.; Sun, K. Q ; Magin, R L.; Swartz, H. M Potentml of albumin labeled with mtroxlde as a contrast agent in magnetic resonance imaging and spectroscopy Btoconjug, Chem. 1:3236; 1990 9 Samum, A , Krlshna, C. M.; Rlesz, P , Fmkelstem, E ; Russo, A. A novel metal-free low molecular weight superoxlde dlsmutase rmmlc J. Btol Chem 263:17921-17924; 1988 10. Zhdanov, R. I , Komarov, P. G Stencally hindered hydroxyla mines as bioacuve spin labels Free Radtc Res. Commun 9:367-377, 1990. 11 Samum, A ; Godmger, D , Aronovltch, J , Russo, A.; Mitchell, J. B. Naroxldes block DNA SClSStOn and protect cells from oxldaUve damage. Btochemtstry 30:555-561; 1991. 12. M~tchell, J B , Samuni, A ; Krlshna, M. C.; DeGraff, W G., Ahn, M. S., Samuni, U , Russo, A. Biologically acUve metalindependent superoxide dismutase rmmlcs Btochemtstry 29:2802-2807, 1990 13 Samum, A., Wmkelsberg, D.; Pmson, A., Hahn, S. M ; Mitchell, J B.; Russo, A. NltrOxlde stable radicals protect beating cardlomyocytes against oxidative damage. J. Chn Invest. 87:15261530; 1991 14. Nllsson, U A , Olsson, L. I , Carhn, G., Bylund, F A. Inhthltlon of lipid peroxidatlon by spin labels. Relationships between structure and function J Btol Chem. 264:11131-11135, 1989. 15. Krishna, M C , DeGraff, W ; Tamura, S., Gonzalez, F. J., Samum, A , Russo, A., Mitchell, J B. Mechanisms of hypoxlc and aerobic cytotoxlclty of roatomycm C m Chinese hamster V79 cells Cancer Res 51:6622-6628; 1991 16 Mitchell, J B , DeGraff, W.; Kaufman, D ; Krlshna, M C , Samuni, A , Fmkelsteln, E.; Ahn, M S.; Hahn, S M.; Gamson, J , Russo, A. InhibiUon of oxygen-dependent radiauon-mduced
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37. Greenberg, J. T.; Demple, B. Glutathione in Eschertchta colt ~s dispensable for resistance to H202 and gamma radiation. J. Bacteriol. 168:1026-1029; 1986. 38. Link, E. M. The mechanism of pH-dependent H202 cytotox~c~ty in vitro. Arch. Biochem. Btophys. 265:362-372; 1988. 39. Romero, M. J.; Canada, A. T. The evaluation of Escherichta coli as a model for oxidant stress in mammalian hepatocytes' Role of glutathlone. Toxtcol. Appl Pharmacol. 111:485-95; 1991. 40. Imlay, J. A., Lmn, S. DNA damage and oxygen radical toxicity. Science 240:1302-1309; 1988.
ABBREVIATIONS
C C C - - c o v a l e n t l y c l o s e d circular CHDO--2-spirocyclohexane-5,5-dimethyl-3oxazolidinoxyl cyt-c--cytochrome c
DF--desferrioxamine DMPO--5,5-dimethyl-l-pyrrohne-N-oxide E P R - - e l e c t r o n p a r a m a g n e t i c resonance GSH--glutathione G S S G - - o x i d i z e d glutathione HX--hypoxanthine L B - - L u r i e Broth Mn-DF--Mn(III)-desferrioxamine P B S - - p h o s p h a t e buffered saline S O D - - superoxide dismutase S N - - streptonigrin T E M P O - - 2,2,6,6-tetramethyl-piperidinoxyl TEMPOL--4-hydroxyl-2,2,6,6-tetramethylpiperidinoxyl TEMPAMINE--4-amino-2,2,6,6-tetramethylpiperidinoxyl X O - - x a n t h i n e oxidase
Pergamon
0891-5849(94)E0052-K
Original Contribution MODULATION OF STREPTONIGRIN CYTOTOXICITY BY NITROXIDE SOD MIMICS
MURALI C. KRISHNA,* RIVKA F. HALEVY, t RENLIANG ZHANG, t PETER L. GUTIERREZ, :~ and AMRAM SAMUNI t *Radiation Biology Branch, Clinical Oncology Program, Dwislon of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA; *University of Maryland Cancer Center, Baltimore, MD, USA, and *Molecular Btology, School of Medicine, Hebrew University, Jerusalem, Israel
(Recetved 13 May 1993; Revtsed 26 October 1993; Re-revtsed 22 February 1994; Accepted 23 February 1994)
Abstract Nltroxides are cell-permeable, stable radicals that react readily with paramagnetlc species such as transltaon metals or short-hved free radicals, though not generally with diamagnetic molecules Nltroxldes can undergo one-electron selective redox reactxons and thereby potenttally modify the actawty of cytotoxlc drugs. Streptomgnn (SN) toxicity requires bloreductlon to yield the semxquinone radical, and the toxicity is reportedly medmted by transition metals and oxygen-denved reactive species via redox-cyclmg of the semlquinone mtermedmte The present study shows that (1) nitroxldes protected isolated DNA and also aerated or hypoxic bacterial cells from SN toxicity; (2) H202 potentiated the hypoxlc cytotoxlcity of the drug but inhibited the damage to aerated cells; (3) pretreatment of cells with H202 conferred some protection, but not when the drug alone was preexposed to H202, and (4) desfernoxamme and 2,2-dlpyndyl, though neither dlethylenetnammo pentaacetate, exogenous catalase, or superoxlde dlsmutase, decreased SN-mduced cell kalling. The mechanisms by which mtroxldes protect from SN toxicity involve both a selective radical-radical reaction w~th SN semiqumone and the reoxldatlon of reduced cellular transition metal ions On the other hand, H~O2 appears to exert two opposing effects. (1) faclhtatlon of cell killing by the Fenton reaction and (2) lowenng the cellular level of reducing equwalents, thus mhthmng the bloreductlve activation of SN Keywords--EPR, Free radicals, H202, E. colt B, Semlqumone, Superoxlde, Spin-labels, Oxidative stress, Spin-trapping, DMPO
tives are widely used as biophysical probes in studying cell metabolism, 4 membrane fluidity, intracellular 02 level, 5'6 and as potential contrast agents in nuclear magnetic resonance imaging. 7'8 Being free radicals, nitroxides have been found also to react with and dismutate 02"- in a catalytic fashion similar to that of S O D . 9 It was anticipated, therefore, that SOD-mimics would protect cells against oxidative damage. Subsequent studies demonstrated that nitroxides and their respective hydroxylamines ~° inhibit hydrogen peroxide-induced damage under either air or hypoxia, to H202sensitive xth mutant of E. coli, 11 V79 Chinese hamster cells, 12 beating cultured cardiomyocytes, 13 and lipid membranes.I°'14 Moreover, hypoxic cytotoxicity of mitomycin c is and aerobic radiation damage in V79 cells were attenuated by nitroxides) 6 In addition, nitroxides were found to react with reduced transition metals such as Fe(II), though not with H202 or ten-butyl hydroperoxide directly.: Because nitroxides undergo one-electron reactions,
INTRODUCTION
The most common approaches used to assess and modulate free radical-mediated biologic damage induced by anticancer agents, focus on either detoxifying deleterious species or removal of redox-active transition metals that can catalyze and potentiate cell injury. Catalases and peroxidases are used to detoxify H202, change superoxide dismutase (SOD) to dismutate, remove O2"-, ~as chelating agents to bind the metals, 2 and as scayengers for trapping "OH. Success is, however, limited when intracellular compartments are not readily accessible to the protective agent. An alternative mechanism of protection has been demonstrated by a group of cell-permeable, relatively nontoxic 3 nitroxides having an unpaired electrorf. Nitroxide deriva-
Address correspondence to. Murall C Krlshna, Radlauon Biology Branch, Bldg 10, Rm. B3 B69, National Cancer Institute, NaUonal Institutes of Health, Bethesda, MD 20892, USA
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they react rapidly with other radical species, but not readily with diamagnetic molecules. This makes them a powerful research tool for the detection of free radical-mediated processes. More importantly, contrary to SOD or catalase, nitroxides penetrate intracellular spaces readily, x2 Streptonigrin is cellularly bioreduced to the respective semiquinone SN'-, x8 which in turn reduces molecular oxygen to yield superoxide and subsequently H202.19 E. coli B, unlike mammalian cells, is resistant to H202 up to several mM and was selected as a test-system to study the role of H202 in SN activity. The present results indicate that nitroxides protect ceils from SN under both aerated and hypoxic conditions. The protective effect appears to involve the termination of the radical chain reactions by a continuous recycling of the SN semiquinone to the parent drug and oxidation of reduced redoxactive transition metal ions. Although n202 potentiated SN-induced DNA cleavage in aerated cell-free systems, it remarkably protected aerobic bacterial cells from SN. The protection presumably reflects an inhibition of the bioreductive activation of SN to semiquinone indirectly through lowering cellular level of reducing equivalents. MATERIALS AND METHODS
Chemicals
Streptonigrin, 2,2-dipyridyl, and cytochrome c (cytc) reductase (EC 1.6.99.3) were obtained from Sigma; desferrioxamine (Desferal ®) was a gift from Ciba Geigy. Xanthine Oxidase (XO), reduced nicotinamide adenine dinucleotide (NADH), and catalase were purchased from Boehringer Biochemicals (Germany). Diethylenetriaminopentaacetic acid (DTPA), 5,5-dimethyl-l-pyrroline-Noxide (DMPO), 2,2,6,6-tetramethylpiperidine-l-oxyl (TEMPO), 4-hydroxyl-2,2,6,6-tetramethylpiperidine- 1oxyl (TEMPOL), and 4-amino-2,2,6,6-tetramethyl-piperidine-l-oxyl (TEMPAMINE) were purchased from Aldrich Chemical Co. (Milwaukee, WI). The DMPO solution was purified with activated charcoal, filtered, and its concentration was spectrophotometfically determined using 8.0 mM-~cm-t at 227 nm. All other chemicals were prepared and used without further purification. Deionized water was used throughout all experiments. Unless otherwise stated, the experiments were conducted at room temperature. Culture preparation and SN treatment of cells E. coli B cells were grown at 37°C in LB growth medium. Log phase cells in growth medium or washed cells in 1 mM phosphate buffer, pH7.4, containing 1 mM MgSO4 were treated for varying periods of time
with 10/zM SN in the absence or presence of nitroxide. The cells were sampled, diluted with phosphate buffer, plated on L-agar, incubated overnight at 37°C, and subsequently colonies were counted. Surviving fractions were calculated and presented graphically on semilogarithmic scale. Graphs represent typical results generally performed in triplicate. Error bars represent standard deviations of the mean and are shown when larger than the symbol. For studies of hypoxic toxicity, the cell suspension was bubbled 20 min with N2 prior to and during incubation with the drug. Electron paramagnetic resonance (EPR) measurements
Samples were drawn into a gas-permeable, 0.8 mm inner diameter, Teflon capillary. The capillary was inserted into a quartz tube and then placed within the EPR cavity. During the experiment, the sample within the spectrometer cavity was flushed with either air or N2, without disturbing the sample, and the EPR spectra were recorded on a Varian E9 X-band spectrometer, operating at 9.45 GHz, 100 kHz modulation frequency, 1 G modulation amplitude, and 10 mW microwave power. To study the decay kinetics of the nitroxide, the magnetic field was kept constant while the intensity of the EPR signal was followed. Spin-trapping of "OH and
02 °-
upon SN reduction
In experiments aimed to detect "OH and superoxide radicals, the reaction mixture was placed in the gaspermeable Teflon capillary containing 100 mM of the spin trap DMPO. For anoxic experiments, the air flow was substituted by N2. About 6 min was required to render the system anoxic. Bulk electrolysis
In bulk electrolysis experiments, an electrochemical reactor similar to that previously described2° was used. The cell consisted of a working electrode of graphite packed inside a porous Vycor glass tube (5 mM ID), through which the PBS solution containing SN was pumped (approximately 2 ml/min). An outer glass cylinder, with separate electrolyte, contained the platinum auxiliary electrode and a reference Ag/AgC1 electrode immersed in tetramethyl ammonium perchlorate. Controlled-potential electrolysis was performed with a CV27 Potentiostat (Bio Analytical Systems) using -0.7 V vs. Ag/AgC1 reference electrode at ambient temperature where a current of 1.6 mA was measured when 500 /~M SN was electrochemically reduced in 0.1 M
Nltroxldes modulate SN cytotoxlcRy tetramethylammonium perchlorate. For high resolution EPR spectra, 500 # M SN was electrolyzed in dimethylsulfoxide in the presence of 0.1 M tetraethylammonium perchlorate. Simulation of the EPR spectrum was performed assuming isotropic and coinciding A and g tensors.
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Assay of in vitro DNA scission Covalently closed circular (ccc) pUC19 plasmid D N A was prepared and isolated as previously described. 2~ To assay for single-strand breaks, samples of 0.1 #g DNA/well were applied to horizontal, 0.9% agarose slab gels in pH 8 tris-acetate EDTA buffer containing 0.2 /zg/ml ethidium bromide and electrophoresed at a potential gradient of 2.5 V/cm. Following electrophoresis, the gels were illuminated with UV light and photographed using 665 Polaroid positivenegative film. To assess the degree of degradation, fragments of known sizes from digests of h-phage D N A by endonuclease Hind III have been included in the electrophoretic run. The untreated D N A consisted of both a major fraction of super-coiled and a minor relaxed ccc plasmid.
RESULTS In vitro effect of H202 and nitroxides on SN-induced scission of isolated DNA Streptonigrin has been previously found to cleave D N A in cell-free systems in a mechanism that requires one-electron reduction of the drug to SN'-, and involves O2"-, iron, H202, and "OH. 22 Positively charged nitroxides, such as TEMPAMINE, were previously found to confer greater protection to D N A than that demonstrated by neutral nitroxides. 23 The protective effect of T E M P A M I N E was examined in the present study by exposing isolated pUC19 D N A to SN upon its enzymic reduction by NADH. The D N A was incubated at 37°C in l0 m M HEPES buffer at pH 7 containing 0.2 m M N A D H and 100 # M SN, with either 0.05 U/ml XO or 40 lzg/ml cyt-c reductase for 10 min. The control experiments did not include SN. Subsequently, the treated D N A was electrophoresed on agarose slab gels and the extent of single-stranded breaks was determined by comparing the intensities of supercoiled and relaxed ccc D N A fractions. Under our experimental conditions, the relaxed circular and the linear forms of double-stranded pUC19 D N A have similar electrophoretic mobilities, hence their respective bands are hardly distinguishable. This was confirmed by running control samples of singly nicked plasmid (data not shown). As a result, the induction of either
Fig 1 TEMPAMINEprevents m vitro DNA cleavage induced upon enzymacreduction of streptomgnn. Isolated supercolled ccc pUC19 plasmld DNA m 10 mM HEPES buffer pH 7 was exposed at 37°C for l0 man to 100/zM SN and 0.2 mM NADH with either 0.05 U/ ml XO or 40 #g/ml cyt-c reductase. Control experiments did not include SN or enzyme. Lane 1. no addluves, Lane 2: + XO without SN; Lane 3. XO + SN; Lane 4- XO + SN + TEMPAMINE;Lane 5. + cyt-c reductase wtthout SN; Lane 6 cyt-c reductase + SN, Lane 7: cyt-c reductase + SN + TEMPAMINE,lane 8" + TEMPAMINE, without NADH; Lane 9' HmdII1digest of k-DNA. The DNA was electrophoresed for scisslon analysis as detailed in Methods
single- or double-stranded breaks increased the intensity of the trailing D N A band at the expense of that of the leading band of super-coiled DNA. A typical gel electrophoregram is illustrated in Fig. 1. The extent of D N A scission was evident by comparing untreated D N A (lane 1) with treated DNA. In the presence of XO and without SN, a partial breakage took place, whereas with SN all the D N A underwent scission (lane 3). In the presence of cyt-c reductase and without SN, no D N A scission took place (lane 5). However, with SN most of the D N A underwent scission (lane 6). The protective effect of the nitroxide is also illustrated in Fig. 1. T E M P A M I N E at 1 mM fully prevented SNfacilitated D N A breakage induced by either XO (lane 4) or cytochrome reductase (lane 7). The present results support the mechanism previously suggested for SNinduced D N A cleavage and demonstrate the protective effect of nitroxide. To test whether these conclusions
382
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Fig. 2 Protecuon afforded by nitroxldes to E. coli B cells against streptomgrln-induced lolhng. Time-dependence of cell survival following aerobic incubation of 10 #M SN with 1 × 107 E coli B cells/ml m LB growth medium at 37°C, in the absence (11) and presence of 10 mM TEMPO (©), 1 mM TEMPAMINE (e), 1 mM TEMPOL (A), or 10 mM CHDO (n)
can be extrapolated to the cellular level, bacterial cells were studied.
Nitroxide effect on bactericidal activity of SN E. coli B at 1 × 107 cell/ml were exposed, in LB growth medium at 37°(2, to 10 #M SN, and the resuiting survival curves were determined. In Fig. 2, the effect of nitroxides on the rate of SN-induced cell killing in the presence of air is displayed. Nitroxides at 10 mM showed no toxicity in the absence of SN (data not shown). All nitroxides tested protected E. coli cells. Figure 2 shows that hydrophilic nitroxides, TEMPOL, and TEMPAMINE provide greater protection than the more lipophilic derivatives such as CHDO or TEMPO. Addition of 200 U/ml SOD or 130U/ml catalase did not affect the aerobic toxicity of SN (data not shown). Similarly, the cell-permeable Mn(III)-desferrioxamine chelate (Mn-DF), which possesses SOD-mimic activity, 24'25 did not protect the cells (data not shown). In fact, 180 #M Mn-DF slightly potentiated SN toxicity, possibly because of dissociation of some of the chelate.
Effect of chelating agents Transition metals, particularly iron, are known to potentiate SN cytotoxicity in various test-systems, including bacterial cells. 26"27In the present work, the effect of 200 #M DTPA, Desferal (DF), and 2,2-dipyridyl on the rate of SN-induced cell killing was studied
and the results are displayed in Fig. 3. DF provided partial protection upon 30 mln preincubation. Dipyridyl provided better protection, whereas DTPA, which does not enter ceils, was without effect. These results are consistent with previous conclusions implicating intracellular transition metals in SN cytotoxicity.2s'29 H202 effect on the aerobic and hypoxic cytotoxictty
of SN Wild-type E. coli B, unlike mammalian cells, is relatively resistant to H202, and was selected to study SN toxicity. The cytotoxicity of SN requires molecular oxygen, 3° as demonstrated in Fig. 4 (open symbols). H202 at subtoxic concentrations (___ 2 mM) protected aerobic E. coli B cells from SN toxicity (Fig. 4, solid lines), whereas under hypoxia, H 2 0 2 did not protect and even slightly increased the rate of cell killing (Fig. 4, dashed lines). To determine whether the protective effect of H202 against aerobic toxicity of SN results f r o m H202 reaction with SN, the reagents were incubated together in the absence of cells. After 20 min, the solution was checked for any change in SN absorption spectrum. Because no spectral changes were observed, direct reaction between SN and H202 during this period seemed unlikely. To confirm this conclusion, SN was preincubated aerobically for 20 min with H202 in LB medium in the absence of cells. Then, log-phase E. coli B cells were added to a final concentration of 1 )< 107 cells/ml together with 130 U/ml catalase to remove the H202, and the time-dependence of cell survival was determined. The rate of cell killing did not
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Time of cell exposure to streptomgnn, nun F:g 3. Effect of cell-permeable and nonpermeable chelating agents on SN toxicity" Time-dependence of E. colt B survivmg fraction following exposure at 37°C to 10 #M SN m the absence (solid symbols) and the presence (open symbols) of 200/zM chelatmg agents: DTPA (circles); DF (squares); dipyrldyl (mangles).
differ from that observed using fresh SN untreated with H202.
Effect of preincubation of cells with induced killing of E. coli
H202 o n
of cellular reducing equivalents, H202 is repressing SN bioreductive activation, then preincubation of cells with H2Oz before exposing them to SN should provide protection. To investigate this possibility, E. coli cells were preincubated in LB growth medium at 37°C with 2 mM H202 in the absence of SN for 15 min. After this incubation, 130 U/ml catalase was added to the cells followed by 10 #M SN. Catalase and SN were added also to the cells in the control system (Fig. 5,
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An alternative explanation for the protective effect of H202 against SN toxicity could be a depletion of cellular reducing equivalents. If, by lowering the level
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384
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Time of exposure to streptonlgnn, wan Fig. 5 Effect of prelncubatlon with H202 on SN-mduced cytotoxlclty E. coli B cells were prelncubated at 37°C in growth medium m the absence (open) and the presence (closed) of 2 m M H202. After 15 nun, 130 U/ml catalase has been added, to remove H202, followed by the addltaon of 10/.tM SN The cells were sampled after various u m e intervals, diluted and plated for the determination of colomes forming abihty
circles), which had not been preexposed to H202. The cells of both systems were sampled at various time intervals and survival was determined. Figure 5 shows that preexposure of the cells to H202 rendered them more resistant to SN. The reaction between SN semiquinone radical (S'-) and nitroxide
Because SN toxicity depends on its bioreductive activation to SN'-, the protection by nitroxides could be attributed to direct inactivation of the semiquinone intermediate. Therefore SN was enzymatically reduced in the absence and presence of nitroxide. When 100 #M TEMPOL was incubated under air at room temperature in 10 mM phosphate buffer, pH7, with 40 /zM SN, 1 mM NADH, and 0.1 U/ml XO, the EPR signal rapidly decayed. However, this decay was prevented by 100 U/ml SOD (data not shown), which suggests that 02"- is responsible for this spin-loss. The rate of EPR signal decay under hypoxia resembled that observed under air; however, it was unaffected by SOD (Fig. 6), which indicates a direct reduction of the TEMPOL by SN'-. In the absence of SN, no loss in the EPR signal intensity of TEMPOL was observed in both aerobic and hypoxic conditions. The EPR signal of TEMPOL, lost upon SN reduction both under air and hypoxia, could be restored by adding one-electron oxidant such as ferricyanide. The reaction between
H202
and SN'-
The protective effect of H 2 0 2 against SN-induced damage could result from direct reaction with SN semi-
quinone. To test this, SN'- was generated by a controlled-potential bulk electrolysis using an electrochemical reactor (see Methods section). The EPR signal observed following electrochemical reduction of SN in dimethylsulfoxide containing 0.1 M tetraethylammonium perchlorate under strict anoxia is demonstrated in Fig. 7. The SN'- spectrum consisted of 18 well defined lines, among which a triplet due to a relatively large a N could be observed (Fig. 7, top). The fact that the group of lines that makes up the high field triplet is smaller, indicates a slow tumbling of the SN'radical. The hyperfine coupling constants validated by spectral simulation (Fig. 7, bottom) are: a"cocH3 = 0.266 G for three equivalent protons, aNNH2 = 1.53 G, aHNH2 = 0.63 G, and aHNH2 = 0.576 G. The difference in the hyperfine coupling constants observed for SN'radical in the present study to that reported earlier34 is due to the difference in solvent. In aqueous solution using 500 #M SN in PBS as electrolyte, the EPR lines became somewhat broader (data not shown). At higher values of modulation amplitude, a broad singlet was observed as shown in Fig. 8 (top). Subsequently, the SN'- solution was mixed 1:1, either with water (Fig. 8, top) or with freshly prepared 10 mM H 2 0 2 in water (Fig. 8, bottom) within the sealed quartz EPR cell, and rescanned for EPR signal using overmodulating setting. To avoid traces of molecular oxygen, which can rapidly eliminate SN'-, the H202 solution was freshly prepared prior to the experiment and all the solutions were thoroughly bubbled with ultra pure nitrogen prior to and during mixing. As seen in Fig. 8 (bottom), H202 totally eliminated the SN'- signal. To identify the products of SN'- reaction with H202,
..._.~_.___~.
10 mMH202
_.=
,.d O
~
No H202
I
10
I
20
I
30
Time, wan Fig. 6 Spin-loss of TEMPOL induced upon the enzymatic reduction of SN. Decay kinetics of the EPR signal of 100 # M TEMPOL upon hypoxlc incubation at RT in 10 m M phosphate buffer pH 7 containmg 40 # M SN, 1 m M NADH, and 0.1 U/ml XO, m the absence and the presence of H202 In the absence of SN, no change of the nitroxide EPR signal was observed
Nltroxldes modulate SN cytotoxioty
385
H202 concentration was required to enable it to better 2 GAUSS
1
compete against 0 2 for the SN semiquinone. The failure of SOD to eliminate the DMPO/'OH signal indicated that this spin-adduct did not result from a decomposition of DMPO/'OOH, but rather trapping of authentic "OH radicals. This observation suggests that SN'- reduces H202 to produce "OH rather than oxidizing it to 02"-. With SN'-, unlike the reaction of H202 with adriamycin semiquinone, 31 DMPO/'OH was observed both in the absence and in the presence of either 50/~M DF or 70/~M DTPA, thus excluding the role of redoxactive transition metals in the reductive cleavage of H202 by SN'-. DISCUSSION
Role of nitroxides
Fig 7. The EPR spectra of SN'- semiqulnone. (Top) SN'- was generated by a controlled-potential bulk electrochemical reduction of SN in dimethylsulfoxide containing 0 1M tetraethyl ammonium perchlorate under strict anoxm. (Bottom) The simulated spectrum plotted using a"coc,3 = 0.266 G for 3 equivalent protons, attN.2 = 1.53 G, an~m = 0 63 G, and anr~,2 = 0.576 G
streptonigrin was enzymatically reduced in the presence of the spin-trap DMPO. A phosphate buffer 0.1 M, pH 7, containing 100 #M SN, 63 mM DMPO, 2.2 mM NADH, and 0.2 U/ml XO in a gas-permeable Teflon capillary was placed in the spectrometer cavity, and scanned for EPR signal. In the presence of air, the 12-line signal characteristic of DMPO/'OOH, the O2"spin-adduct of DMPO, was observed. Upon changing the gas from air to N2, the DMPO/'OOH signal disappeared. However, when 415 U/ml SOD and 0.4 M H202 were included in the reaction system, only the 1:2:2:1 four-lines spectrum of DMPO/'OH ('OH adduct of DMPO) was observed (data not shown). Generally, much lower SOD concentration is sufficient to effectively compete for O2"- against DMPO. However, because 02"- rapidly reacts with and destroys DMPO spin adducts, 9 higher [SOD] was needed to protect the DMPO/'OH from destruction by superoxide. High
Contrary to SOD and catalase, nitroxides conferred full protection to bacterial cells exposed to SN (Fig. 2). The protective effect of TEMPOL and TEMPAMINE exceeded those of the more lipophilic nitroxides TEMPO and CHDO. Moreover, the positively charged TEMPAMINE exhibited the largest protective effect. These results support the position of DNA being a critical target. Because SN, like paraquat and menadione, is an effective generator of intracellular superoxide radicals, 26"32 a straightforward explanation for the nitroxides' protective effect is their SOD-mimicking activity. Accordingly, nitroxides protect by removing intracellular superoxide. The failure of Mn-DF to pro-
_2 J---SN"
+
H20
SN" ÷ H202
10 Gauss
Fig. 8. H202-1nducedspin-loss of SN semiquinoneradical. The EPR signal of streptonignn senuquinone rathcal, recorded under overmodulating setting, followingelectrochemicalreductionof 500/zM SN m PBS under anoxaa. Subsequently,the SN'- solution within the sealed quartzEPR cell has been nuxed 1:1 etther wtth water (Top spectrum)or with 10 mM H202(Bottomspectrum)and rescannedfor EPR signal.
386
M. C KRISHNAet al
tect could result from a dissociation of the chelate, which gives rise to redox active Mn ions. In addition, the increase of SN hypoxic toxicity by H202 (Fig. 4) and the protective effect of chelators (Fig. 3) confirm the mediatory role of transition metals, and substantiate the suggestion that nitroxides also protect by oxidizing reduced metals such as Fe(II), as previously found. 11'~2 According to this mechanism, nitroxides compete with H202 for the reduced metal ion, Fe(II) + R'RN - O" + H + Fe(III) + R'RN - OH,
(1)
to inhibit the Fenton reaction that yields "OH radicals and/or hypervalent metal ions (Eq. 2a and 2b). Fe(II) + H202 --+ Fe(II) - H202 --' Fe(III) + OH- + "OH Fe(II) - HzOz ~ Fe(IV) + 2OH-.
(2a) (2b)
Becasure SN activity requires bioreductive activation of the drug, 27'33'34 an alternative explanatmn can be envisaged in which the reaction of the nitroxlde with SN'- functions to terminate the radical chainreaction, and hence prevents the ultimate damage. The formation of DMPO/'OH in the presence of SOD, DF, and H202, combined with the results displayed in Fig. 8, show that the semiquinone is readily oxidizable not only by 02
(3)
SN'- + 02 ~ SN + 02"but also by H202: SN'- + H202 --+ SN + O H - + "OH.
(4)
Reactions 3 and 4 both yield secondary radicals. Conversely, the oxidation of the semiqumone by nitroxide is a radical-radical reaction which terminates the radical chain reaction. SN'- + R'RN - O" + H + SN + R'RN
-
OH.
(5)
The present results indicate that nitroxides are capable of protecting the cells not only by catalytically removing O2"-, 9'35 and by maintaining (reaction 1) redoxactive metal ions in their higher oxidation state, ~z but also by competing with oxygen and H202 for the SN'radical (reaction 5).
Role o f H202
The H2Oz-induced enhancement of SN hypoxic cytotoxicity and increased DNA degradation in vitro (Figs. 1 and 4) is consistent with the commonly adopted site-specific mechanism underlying the deleterious effect of the drug (SN toxicity under hypoxia could also increase by 02 generated through H202 decomposition). According to the site-specific mechanism of damage, redoxactive metal ions are coordinated to, amongst other things, critical cellular sites. Hence, the reactions take place locally and the active species are formed site-specifically in close proximity to the critical sites, rather than randomly in the bulk. The production of "OH radicals in reactions 2 and 4 implies that Increasing H202 concentration should facilitate the damage. The protective effect of H202 against the aerobic cytotoxicity of SN seen in Fig. 4 is not consistent with this prediction. In fact, 2 mM H202 protected the cells from SN. A similar protection of mammalian cells by H202 against the cytotoxicity of tumor necrosis factor has been previously found. 36 This protective effect was observed following a pretreatment of the cells with HzO2, but not when tumor necrosis factor alone was preincubated with H202. The inhibition of SN-induced killing is attributable to the effect of H202 on the level of cellular reducing equivalents. Because the affinity of catalase for H202 is low, a major pathway of H202 metabolism in cells involves the reduction of H202 by glutathione (GSH) and GSH peroxidase. Accordingly, the reduction of GSSG to GSH is mediated by GSSG reductase, which uses NADPH. Thus, the GSH cycle is coupled with the oxidation of glucose-6-phosphate and 6-phosphogluconate, which provides NAD(P)H for reduction of GSSG. Previous studies demonstrated that exogenously added GSH does not protect E. coli cells from H2Oz-induced damage. 37 On the other hand, treatment of E. coli with H202 caused an 80% decrease in the cellular GSH, and increased the export of GSSG and the inactivation of GSH reductase. 38'39 Another key study of HzOz effect on NAD(P)H in E. coli cells showed that the concentration of these reducing equivalents in H202-treated E. coli cells also diminished via a direct reaction with the peroxide. 4° Obviously, an H202-induced depletion of cellular GSH and NAD(P)H can lower the rate of SN reduction and inhibit the cellular damage.
CONCLUSION
The results of the present study extend our understanding of the protective effects of nitroxides and help
Nitroxides modulate SN cytotoxlclty
elucidate the roles of redoxactive metals in streptonigrin toxicity. They also indicate that H202 may, under certain circumstances, act as a protective agent, and call for greater caution in extrapolating conclusions drawn from cell-free to cellular systems. Acknowledgements - - The helpful dxscusslons with Dr A. Russo and Dr. J.B Mitchell are gratefully acknowledged This research was supported by Grant 89-00124 from the Umted States-Israel Binatlonal Science Foundation (BSF), Jerusalem, Israel
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ABBREVIATIONS
C C C - - c o v a l e n t l y c l o s e d circular CHDO--2-spirocyclohexane-5,5-dimethyl-3oxazolidinoxyl cyt-c--cytochrome c
DF--desferrioxamine DMPO--5,5-dimethyl-l-pyrrohne-N-oxide E P R - - e l e c t r o n p a r a m a g n e t i c resonance GSH--glutathione G S S G - - o x i d i z e d glutathione HX--hypoxanthine L B - - L u r i e Broth Mn-DF--Mn(III)-desferrioxamine P B S - - p h o s p h a t e buffered saline S O D - - superoxide dismutase S N - - streptonigrin T E M P O - - 2,2,6,6-tetramethyl-piperidinoxyl TEMPOL--4-hydroxyl-2,2,6,6-tetramethylpiperidinoxyl TEMPAMINE--4-amino-2,2,6,6-tetramethylpiperidinoxyl X O - - x a n t h i n e oxidase