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(The FASEB Journal. 2000;14:1215-1223.)
© 2000 FASEB

The pro-oxidative activity of SOD and nitroxide SOD mimics

TAL OFFER1, ANGELO RUSSO* and AMRAM SAMUNI

Molecular Biology, Hebrew University - Hadassah Medical School, Jerusalem, 91120, Israel; and
* Radiation Biology Branch, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA

1Correspondence: Molecular Biology, Faculty of Medicine, Hebrew University-Hadassah Medical School, Jerusalem, 91120, Israel. E-mail: talof{at}md2.huji.ac.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 NITROXIDE SOD MIMICS
 ADVERSE EFFECTS
 POTENTIAL MECHANISMS
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Native Cu,Zn-SOD and synthetic SOD mimics sometimes demonstrate an apparently anomalous bell-shaped dose-response relationship when protecting various biological systems from oxidative stress. Several mechanisms have been proposed to account for such an effect, including: overproduction of H2O2, peroxidative activity of SOD, and opposing roles played by O2·- in both initiation and termination of radical chain reactions. In the present study, ferrocyanide and thiols, which are susceptible to one-electron and two-electron oxidation, respectively, were subjected to a flux of superoxide in the presence and absence of SOD or SOD mimics. The results show that 1) either O2·-/HO2· or H2O2 alone partially inactivates papain, whereas when combined they act synergistically; 2) nitroxide SOD mimics, but not SOD, exhibit a bell-shaped dose-response relationship in protecting papain from inactivation; 3) SOD, which at low dose inhibits superoxide-induced oxidation of ferrocyanide, loses its antioxidative effect as its concentration increases. These findings offer an additional explanation for the pro-oxidative activity of SOD and SOD mimics without invoking any dual activity of O2·- or a combined effect of SOD and H2O2. The most significant outcome of an increase in SOD level is a decrease of [O2·-]steady state, rather than any notable elevation of [H2O2]steady state. As a result, the reaction kinetics of the high oxidation state of each catalyst is altered. In the presence of ultra-low [O2·-]steady state, the oxidized form of SOD [Cu(II),Zn-SOD] or SOD mimic (oxo-ammonium cation) does not react with O2·- but rather oxidizes the target molecule that it was supposed to have protected. Consequently, these catalysts exert an anti- or pro-oxidative effect depending on their concentration.—Offer, T., Russo, A., Samuni, A. The pro-oxidative activity of SOD and nitroxide SOD mimics.


Key Words: papain • hydrogen peroxide • superoxide • oxidative damage • thiol


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
NITROXIDE SOD MIMICS
 ADVERSE EFFECTS
 POTENTIAL MECHANISMS
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
STABLE NITROXIDE RADICALS are membrane-permeable, low molecular weight antioxidants (LMWA) that protect many biological systems from diverse oxidative insults. Protective effects have been reported for laboratory animals (1 2 3 4 5) , bacterial and mammalian cells, and isolated biomolecules such as enzymes, proteins, lipids, or DNA, (6 , 7) .


   NITROXIDE SOD MIMICS
TOP
 ABSTRACT
 INTRODUCTION
 NITROXIDE SOD MIMICS
ADVERSE EFFECTS
 POTENTIAL MECHANISMS
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Unlike monofunctional antioxidants such as catalase, which protects by removing H2O2, or superoxide dismutase (SOD), which dismutates superoxide, nitroxides operate through diverse pathways (some are illustrated in Scheme 1 ).



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  		Scheme 1. NO CAPTION

Besides the SOD mimetic activity of nitroxides, which catalytically remove superoxide radicals (8) , nitroxides can oxidize reduced transition metals that potentiate damage by producing reactive oxygen-derived species (ROS) (6 , 9) , detoxify hypervalent metals such as the ferryl-heme species (10) , facilitate heme-mediated catalytic removal of H2O2 (10) , trap carbon-centered radicals (11) , and terminate radical chain reactions (12) . Through competing with NO for O2·- the nitroxides might also indirectly lower the production of peroxynitrite and elevate [NO]steady state.


   ADVERSE EFFECTS
TOP
 ABSTRACT
 INTRODUCTION
 NITROXIDE SOD MIMICS
 ADVERSE EFFECTS
POTENTIAL MECHANISMS
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
There is increasing evidence for the protective activity of nitroxides, yet information concerning their potential adverse effects is scarce. Nitroxides have previously been found to demonstrate toxicity that depends on dose and exposure time. They exert mutagenicity in Salmonella typhimurium (13) , potentiate the mutagenic effect of H2O2 in Salmonella typhimurium strain TA 104 (14) , cause stress and eventually some necrosis in thymocytes (5) , increase the activity of cytochrome P450 oxidase, elevate the production of O2·- and H2O2 in human tumor cell lines (15) , and inhibit cell growth possibly by triggering an apoptotic mechanism (16) .

SOD, whose activity in diverse organs and organisms is relatively constant, demonstrates a bell-shaped dose-response in several biological systems (17 18 19 20 21) . McCord reported that cultured cells expressing increased amounts of SOD fare much better when challenged with oxidative stress, but are worse off under normal conditions, stating that "It would be very easy for an organism to produce more SOD if more were better, but for some reason that did not happen" (21) . Cytotoxic effects due to increased peroxidase activity (22) and lipid peroxidation (21 , 23) were found in such cells. SOD was also reported to amplify sensitivity to ionizing radiation (24) . Apparently higher than normal levels of SOD, as in transgenic mice or Down syndrome patients, can exert adverse effects or even be lethal (22) .


   POTENTIAL MECHANISMS
TOP
 ABSTRACT
 INTRODUCTION
 NITROXIDE SOD MIMICS
 ADVERSE EFFECTS
 POTENTIAL MECHANISMS
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The bell-shaped curve of the SOD dose-response has intrigued several researchers, who have offered various potential mechanisms. The proposed explanations included a cytotoxic effect due to ·OH formation through the peroxidase activity of SOD (25) , excessive formation of H2O2 (20 , 22 , 26 27 28) , and a modulation of lipid peroxidation due to opposing effects of superoxide that can presumably induce initiation as well as termination of radical chain reactions (21 , 23) .

It has previously been shown that piperidinyl, such as 4-OH-2,2,6,6-tetramethylpiperidinoxyl (TPL), and pyrrolidinyl derivatives are capable of catalyzing removal of O2·-/HO2· while switching between the radical and oxo-ammonium cation (denoted TPL+) forms of the nitroxide (29) .




Thus, the net reaction yields H2O2 and O2 as in the enzymatic or nonenzymatic dismutation of superoxide:

We hypothesize that in systems where nitroxides protect by acting as SOD mimics, they might also exhibit a pro-oxidative effect resembling that of SOD. Since the steady-state concentration of superoxide is inversely proportional to the concentration of SOD or of the SOD mimic, any elevation in the level of the catalyst is accompanied by a respective decrease in O2·- level. In the presence of a sufficiently high level of SOD (or SOD mimic), most of the superoxide radicals are removed through dismutation yielding O2 and H2O2, while maintaining the catalyst in its oxidized form. Hence, a further increase in [SOD] (or [SOD mimic]) does not practically alter the yield of H2O2. On the other hand, this increase can significantly lower the steady-state level of O2·-. A decrease in [O2·- ]steady state might promote a pro-oxidative effect of the catalyst itself. Because nitroxides react with O2·-/HO2· but not with H2O2 or with O2, no other processes compete with reaction 1. As [O2·- ]steady state decreases, the TPL+ generated through reaction 1 might predominantly react with one-electron or two-electron donating groups of essential biological targets inducing a pro-oxidative effect, rather than with O2·- through reaction 2.

The present study was aimed at elucidating the mechanism underlying the potential adverse effects of increasing doses of nitroxide SOD mimics and of Cu,Zn-SOD. Since nitroxides can modify oxidative damage through several modes of action other than SOD mimetic activity, diethylenetriamine-pentaacetic acid (DTPA) was always included in the reaction systems to bind redox-active metals and prevent the Fenton reaction. In most cases, superoxide toxicity is mediated indirectly through powerful oxidants derived from HO2·/O2·-. In several cases, however, superoxide radicals are directly responsible for biological injury (30) . To examine the antioxidant/pro-oxidant properties of SOD mimics and of Cu,Zn-SOD, one-electron (ferrocyanide) and two-electron (thiol) oxidizable substrates, which are susceptible to damage inflicted by superoxide radicals per se, have been selected as targets and subjected to a flux of superoxide.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 NITROXIDE SOD MIMICS
 ADVERSE EFFECTS
 POTENTIAL MECHANISMS
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Materials
Catalase (H2O2 oxidoreductase; EC 1.11.1.6), xanthine oxidase (EC 1.2.3.2 xanthine: oxygen oxidoreductase; XO), and Cu, Zn superoxide dismutase (SOD) were purchased from Boehringer Mannheim (Mannheim, Germany). The nitroxides TPL and 4-amino-2,2,6,6-tetramethylpiperidinoxyl (TPA) were purchased from Aldrich Chemical (Milwaukee, Wis.). Hypoxanthine (HX), N-carbobenzoxy-glycine p-nitrophenyl ester (CBZ), ferricytochrome c, and papain (EC 3.4.22.2) were obtained from Sigma (St. Louis, Mo.). Glutathione (GSH) was purchased from Fluka AG Buchs SG (Buchs, Switzerland). D-Mannitol was purchased from Difco Laboratories (Molesley, Surrey, U.K.). Potassium ferrocyanide was obtained from BDH (Poole, Dorset, U.K.). Deionized water was used for preparation of solutions for all the experiments. Fresh solutions of 0.5 mg/ml CBZ in 20% acetonitrile were prepared daily. Production of superoxide and H2O2 by the enzymatic generator HX/XO was determined spectrophotometrically using the SOD-inhibitable reduction of cytochrome c and the iodometric assay, respectively.

Papain reactivation and inactivation
Papain reactivation was carried out by incubating the enzyme with 0.1 mM DTPA and 5 mM thiol such as GSH or dithiothreitol for 30 min. The esteratic activity of papain was assayed using CBZ as a substrate and following spectrophotometrically at 400 nm the rate of its hydrolysis. Papain inactivation was induced by subjecting it to a flux of O2·- and H2O2 generated by HX/XO, which results in a progressive loss of activity due to the oxidation of the thiol at the enzyme active site. The reactivated papain, generally at 0.4 mg/ml, was exposed to 1 mM HX and 0.2 mU/ml XO. Whereas at 5 mM GSH inhibits the oxidative inactivation of papain, 0.5 mM GSH facilitates it as previously reported for other SH enzymes (31) . Hence, GSH was not removed from but rather was retained in the exposure system at ~0.1–0.5 mM. Another reason for the inclusion of GSH in the exposure system was to inhibit O2-induced oxidation of papain, which was quite rapid in the absence of the thiol. The exposure systems always contained 100 µM DTPA to prevent the participation of transition metal ions and avoid generation of ·OH radicals or hypervalent metals through the Fenton reaction. Under such conditions, O2·-/HO2· and H2O2 formed by HX/XO are the only species mediating papain oxidative inactivation.

During the XO-catalyzed oxidation of HX, H2O2 is formed directly through a two-electron reduction of O2 as well as indirectly through dismutation of O2·-/HO2· radicals, which are generated by one-electron reduction of O2. Since the total catalytic activity of XO, the relative rates of these two pathways, and the [HO2· ]/[O2·- ] ratio depend on the pH, those changes had to be taken into account when analyzing the pH effect on papain inactivation. The exposure system of papain was vortexed periodically to introduce oxygen and sampled to assay for its residual esteratic activity. The rate of papain inactivation was determined from the initial linear phase of the plot displaying the progressive loss of its activity after the addition of XO.

Ferrocyanide oxidation
A typical experiment consisted of exposing K4Fe(CN)6 to 1 mM HX and ~7 mU XO/ml in the absence and presence of nitroxide. To minimize nonspecific oxidation of ferrocyanide by air, a fresh anoxic solution was prepared before each experiment. The reaction mixture always contained 100 µM DTPA to avoid Fenton chemistry and 100 U catalase/ml to prevent any possible effect of H2O2 and to regenerate the oxygen needed as an electron acceptor for HX oxidation. The oxidation of ferrocyanide was followed spectrophotometrically at 430 nm using a dual-beam Uvikon 860 (Kontron, Switzerland). The initial rate of ferrocyanide oxidation was measured because after ferricyanide accumulates, it can be reduced by superoxide and by urate, formed through xanthine oxidation, as well as through direct electron transfer from XO when the O2 level decreases.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 NITROXIDE SOD MIMICS
 ADVERSE EFFECTS
 POTENTIAL MECHANISMS
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Papain inactivation and the protective effect of antioxidants
The enzymatic activity of papain after incubation with HX/XO was assayed using CBZ as a substrate; typical progressive loss of activity is displayed in Fig. 1 . Catalase and SOD together provided full protection from inactivation, whereas each enzyme alone only partially decreased the rate of papain inactivation, indicating that either H2O2 or HO2·/O2·- can oxidize the papain SH and inactivate the enzyme (32 , 33) . The individual contributions of H2O2 and HO2·/O2·- toward the oxidative inactivation depended on the pH. Figure 2 shows the relative rate of papain inactivation measured in the presence of TPL, SOD, or catalase as a fraction of the rate of inactivation observed in their absence, demonstrating the pH dependence of the protective effect of the three antioxidants. To check the generality of the nitroxide effect, TPL was replaced by TPA, and the results were similar (data not shown). Figure 2 shows that the protective effect of SOD and of TPL increases whereas that of catalase decreases as the pH decreases. These results suggest that the relative contribution of HO2·/O2·- to papain inactivation increases as the pH is lowered.



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  		Figure 1. Sample plots for the oxidative inactivation of papain. Papain 0.4 mg/ml was incubated with 1 mM HX and 0.2 mU/ml XO in 100 mM phosphate buffer, at pH 7.5, containing 0.5 mM GSH and 100 µM DTPA in the presence of catalase or SOD at 30°C. The residual enzymatic activity was determined by recording spectrophotometrically at 400 nm the rate of hydrolysis of carbobenzoxy-glycine p-nitrophenyl ester (CBZ). Open symbols: HX/XO alone; closed symbols: in the presence of 260 U/ml catalase (triangles), 25 U/ml SOD (squares), and without HX/XO (circles).



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  		Figure 2. Catalase, SOD, and nitroxide inhibit papain inactivation. Papain 0.4 mg/ml was incubated with 1 mM HX and 0.2 mU XO/ml in 100 mM phosphate buffer (at pH 5.5, 6.5, and 7.5) containing 0.5 mM GSH and 100 µM DTPA for 30 min at 30°C in the presence of catalase (triangles), SOD (circles), or TPL (squares). The residual enzymatic activity was determined at several times by recording spectrophotometrically at 400 nm the hydrolysis of CBZ as detailed in Materials and Methods. The rates of papain inactivation were determined from the initial linear phase of the plots as seen in Fig. 1 . The relative rates (+ error bars) of papain inactivation (means of 4 independent experiments) are presented as a fraction of the control rate of inactivation measured without antioxidant.

Dose-response relationship of SOD mimics and of SOD
The effect of nitroxide concentration on the rate of papain inactivation in the presence of HX/XO has been studied. Control experiments showed that TPL, by itself does not affect the activity of papain. Figure 3 shows an inverted bell-shaped dose-response curve. At low [TPL] (< 40 µM), the protective effect increased as the nitroxide concentration increased, achieving a constant value (<40% protection) over a concentration range of three orders of magnitude (40 µM <= [TPL] <= 15 mM). However, for high [TPL] (>= 20 mM), the protective effect was significantly lower (Fig. 3) . A different dose-response relationship was demonstrated by SOD. It lowered the rate of papain inactivation by ~30% at all doses of SOD tested without exhibiting bell-shaped behavior (Fig. 4 ).



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  		Figure 3. Opposing effects of nitroxide on the rate of papain inactivation. Papain 50 µg/ml was incubated with 1 mM HX and 0.2 mU/ml XO in 20 mM phosphate buffer, pH 6.5, containing 125 µM GSH and 100 µM DTPA at 30°C. The relative rate of papain inactivation induced by HX/XO in the presence of various concentrations of TPL is presented as a fraction of the rate measured in control experiment in the absence of antioxidant.



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  		Figure 4. SOD inhibits the inactivation of papain. The relative residual rate of papain inactivation induced by HX/XO at pH 6.5 in the presence of SOD is presented as a fraction of the rate measured in control experiment without antioxidant. The conditions are the same as detailed for Fig. 3 .

Catalase acts synergistically with SOD or SOD mimics
Although catalase alone only partially inhibited the rate of papain inactivation at pH 5.5, it provided full protection when added together with nitroxides or SOD. Even at a ‘nonprotective concentration’ of 100 mM, TPL fully blocked papain inactivation induced by HX/XO when given together with catalase. The failure of catalase, when given alone, to better protect the papain could be ascribed to its own inactivation by superoxide, which can be reversed by SOD (30) . Therefore, the accumulation of H2O2 under the present experimental conditions was studied. The HX/XO reaction mixture in phosphate buffer incubated at room temperature with and without catalase was sampled at various points of time and assayed for H2O2. No hydrogen peroxide was detected when 1000 U/ml catalase was included in the reaction mixture, whereas 3 and 2 µmol H2O2/min were detected after 30 min in control (without catalase) at pH 7.5 and pH 5.5, respectively. In other words, catalase effectively removed all H2O2 formed in the absence of SOD.

To ensure that the protective effect of catalase was due to its enzymatic activity rather than to some contaminants, the enzyme preparation was further purified by eluting it through a Sephadex G-25 column. The protection provided by purified catalase did not differ from that provided by an untreated enzyme preparation. In another control experiment where an equivalent concentration of heat-inactivated catalase was used, no protection was observed. This excluded the possibility that the observed synergism resulted from some nonspecific interaction with the protein.

The effect of carbonate, nitrite, and mannitol
It was previously suggested that H2O2 could mediate SOD toxicity by producing ·OH at the catalytic site of the enzyme (25) . Therefore, mannitol, a LMWA commonly used for scavenging ·OH, was added to check for a potential involvement of authentic hydroxyl radicals. When mannitol was included together with a high concentration of TPL, no further protection of papain against inactivation was observed.

Other studies showed that peroxidative activity of SOD treated with H2O2 is mediated through a strongly bound oxidant, such as enzyme-CuOH2+ (34) , rather than a free ·OH released from the enzyme (35) . It was also demonstrated that carbonate significantly potentiates the peroxidative effect of SOD in the presence of H2O2 due to the formation of a diffusible carbonate radical (36) . Therefore, the protection of papain by high and low [SOD] from the HX/XO-induced inactivation was studied with and without an addition of 20 mM carbonate and/or 1 mM nitrite to the exposure system. No significant effect of carbonate and nitrite, alone or in combination, on the protective effect of SOD was observed.

The oxidation of ferrocyanide by TPL+
Ferrocyanide, a one-electron reductant that competes for TPL+, was selected as a detector of the pro-oxidative effect of the catalyst. The kinetic experiments were conducted by following spectrophotometrically the rate of ferricyanide accumulation in the presence of a constant flux of superoxide. Ferrocyanide was exposed to HX/XO in the presence of DTPA and catalase in 20 mM phosphate buffer pH 6.5. The rate of oxidation of ferrocyanide was determined by following the initial increase in absorption at 430 nm. With 100 µM ferrocyanide, the rate of oxidation was negligible as most of O2·-/HO2· disappeared through disproportionation. Since nitroxides react rapidly with HO2· (reaction 1) and the oxo-ammonium cations can oxidize ferrocyanide, the maximal rate of oxidation was achievable by including nitroxide in the reaction mixture (the maximal rate of oxidation was also observed when using several mM ferrocyanide). Figure 5 demonstrates the dependence of the rate of oxidation of either 0.1 or 1 mM ferrocyanide on [TPL]. Evidently, TPL as low as 10 µM was sufficient to achieve maximal rate of ferrocyanide oxidation.



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  		Figure 5. Effect of nitroxide on the rate of ferrocyanide oxidation induced by O2·-/HO2·. The rate of ferrocyanide oxidation in the presence of 1 mM HX and 7 mU/ml XO, 5 U/ml catalase and 100 µM DTPA in 20 mM phosphate buffer pH 6.5 at 25°C has been measured as detailed in Materials and Methods in the presence of various TPL concentrations. Closed symbols: 950 µM K4Fe(CN)6; open symbols: 95 µM K4Fe(CN)6.

The effect of SOD
When SOD was included in the reaction system, the rate of ferrocyanide oxidation was lower. In the presence of SOD, which removes superoxide, fewer radicals react with nitroxide to yield TPL+, which in turn can oxidize ferrocyanide.

Competition kinetics
To study the competition kinetics between TPL (reaction 1) and SOD (reaction 3) for superoxide, the experiment was repeated using increasing doses of SOD. To analyze the data, the maximal rate (denoted V) of ferrocyanide oxidation, which equals the flux of O2·- and obtained in the absence of SOD, was divided by the lower rate (denoted v) of reaction observed in the presence of SOD. The values of V/v were plotted as a function of [SOD] according to expression I:

The decrease in the rate of ferrocyanide oxidation as [SOD] increases is reflected in the INSET for Fig. 6 , which shows a linear dependence of V/v on [SOD], in compliance with expression I, indicating a simple competition between reactions 1 and 3.



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  		Figure 6. Competition kinetics. Opposing effects of SOD on the rate of ferrocyanide oxidation. The rates of oxidation of 2 mM ferrocyanide in the presence of 1 mM HX, 30 mU/ml XO, 2 mM TPL, 25 U/ml catalase, and 100 µM DTPA in 50 mM MOPS buffer pH 7.8 at 25°C were determined spectrophotometrically at 430 nm in the absence (V) and the presence (v) of various concentrations of SOD. The values of V/v are presented vs. the activity of SOD. Inset: linear dependence of V/v on [SOD] at relatively low doses of the enzyme obeys the expression: V/v = 1+ [SOD]·k3/{[TPL]·k1}

Opposing effects of SOD
On a further increase of [SOD] beyond 20 U/ml, the trend was reversed (See Fig. 6 ). Obviously an increase in the concentration of SOD resulted in a respective increase of the rate of ferrocyanide oxidation. It has been reported that inorganic phosphate, which is present in commercial Cu,Zn-SOD preparations, might be responsible for effects erroneously attributed to the activity of the enzyme (37) . In the present study, addition of phosphate to phosphate-free reaction system did not affect the rate of ferrocyanide oxidation, thus excluding an artifact due to phosphate contamination. The results thus indicate that the ratio of the antioxidant/pro-oxidant effects of SOD decreases as its concentration increases.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 NITROXIDE SOD MIMICS
 ADVERSE EFFECTS
 POTENTIAL MECHANISMS
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
The increased use of nitroxides as LMWA (4 , 6 , 9 , 38) is also based on the assumption that among their other advantages, including stability, nonimmunogenicity, catalytic activity, high permeability, and solubility, they exert little adverse effects (16) . In most studies of more complex test systems, such as cells, isolated rat heart, or whole animals, low concentrations of nitroxides were applied usually not exceeding a few micromoles. The present experimental model is sufficiently simple to enable distinction of the effects of O2·-/HO2· and H2O2 from those of other potentially deleterious oxidants and to elucidate the potential adverse effects of SOD or SOD mimics.

The deleterious species in the papain inactivation system
Papain can be oxidatively inactivated by O2·-/HO2· and H2O2 (32) . The rate of its inactivation and the relative individual contribution of O2·-/HO2· to the damage increase as the pH decreases. This implies that HO2·, which is a stronger oxidant than O2·-, is also more effective in oxidizing the papain Cys25SH. Oxidation by HO2· also explains the decrease in the relative protective effect of catalase and the increase in the protective activity of TPL or SOD as the pH is lowered.

Adverse effect of nitroxides
TPL provided only partial protection, even where H2O2 contribution toward papain inactivation was relatively small (Fig. 3) . Moreover, at concentrations greater than 15 mM, TPL lost much of its protective activity against papain oxidation (Fig. 3) , although it removes O2·- through reaction 2. This is because TPL+ can directly oxidize the Cys25SH at the papain catalytic site. When [TPL] increases, the [O2·- ]steady state decreases and the papain Cys25SH can better compete with O2·- for TPL+. Such an explanation is corroborated by the effect of TPL on ferrocyanide oxidation (Fig. 5) . In the absence of TPL, O2·-/HO2· hardly induced any oxidation of ferrocyanide (Fig. 5) . Because ferrocyanide is readily oxidized by TPL+, a maximal rate of oxidation could be established when TPL was added. Figure 5 indeed shows that TPL, even at 10 µM, induced maximal rate of ferrocyanide oxidation. Elevating the [ferrocyanide] facilitated the oxidation, yet although superoxide was essential for ferrocyanide oxidation, it was TPL+ (rather than O2·-/HO2.) that directly oxidized the ferrocyanide:

In other words, although TPL is an effective SOD mimic that catalytically removes O2·-/HO2·, oxidation of the detector molecule (in this case, ferrocyanide) reflects a pro-oxidative activity of the catalyst. In the case of papain, both the antioxidative (protection at low [TPL]) and the pro-oxidative (-SH oxidation at high [TPL]) activities of nitroxides are manifested.

SOD pro-oxidant effect
The same reasoning might be extrapolated to native SOD, which at low doses blocks superoxide-induced nitroxide-catalyzed oxidation of ferrocyanide. The rate of oxidation was lowered by low [SOD] (Fig. 6 , inset). However, ferrocyanide oxidation was resumed when high [SOD] was included, implying that under those conditions SOD was acting as the ultimate oxidant.

Reaction 5 is expected to predominate at low [O2·-]steady state if ferrocyanide competes better than O2·- for Cu(II),Zn-SOD. This is not necessarily the case with papain. Whereas SOD can readily oxidize ferrocyanide (39) , its access to the thiol at the active site of papain might be limited due to steric hindrance. This may account for the lack of bell-shaped dose response of SOD in protecting papain. SOD provided less than 50% protection even in cases where the relative damage due to H2O2 was negligible (Fig. 4) . However, on increasing [SOD] beyond 50 U/ml, its protective activity did not diminish.

Potential mechanisms
The present results agree with previous findings that high [SOD] might be a less effective antioxidant and even an exacerbatory reagent (19) . Several explanations have been presented to account for the adverse effect of high [SOD]. 1) The possibility that overexpression of SOD could increase [NO] and hence a consequent edema has previously been considered for an animal model (18) since O2·- radicals facilitate the decay of NO. 2) Liochev and Fridovich (40) suggest that overproduction of SOD competes for ATP and raw materials, thus limiting the biosynthesis of other essential proteins. Such an inhibitory effect does not depend on the catalytic activity of SOD. Furthermore, higher [SOD] lowers the [O2·- ]steady state and diminishes the induction of soxR regulation. Certainly, explanations 1 and 2 are not applicable to the present cell-free experimental model.

3) The failure of SOD at high concentration to protect hearts subjected to ischemia-reperfusion injury (20) , the increased susceptibility of SOD-overproducing cells to oxidative stress (27) , and the potential adverse effect of SOD in Down’s syndrome (28) were all attributed to an increased level of H2O2 produced by SOD-catalyzed dismutation of O2·-. Normally, however, low levels of SOD are sufficient to dismutate most of the superoxide radicals to yield H2O2. Hence, an elevation of [SOD] cannot significantly increase [H2O2], as previously explained (18) . Moreover, this explanation is not applicable to the present experimental model since H2O2 had a small effect on papain inactivation and the added DTPA pre-empted the Fenton reaction. 4) The lack of protection by Cu,Zn-SOD at high doses was also attributed to its weak but nonspecific peroxidase activity toward several cellular components (41) . Liochev and Fridovich have also observed O2·--dependent peroxidative activity of an SOD variant associated with familial amyotrophic lateral sclerosis (42) . However, Mn-SOD, which lacks peroxidase activity, exhibited a bell-shaped dose-response in its protection of postischemic isolated rabbit heart (19) . This shows that the exacerbating effect is unlikely to be due merely to a peroxidative activity (19) . 5) According to another mechanism, H2O2 could mediate the toxicity of SOD by reacting with the catalytic site of the enzyme, producing ·OH radicals (25) . However, H2O2 is not ultimately required for the oxidation of ferrocyanide by high level of SOD because catalase was included in the reaction system (Fig. 6) . 5) The adverse effect of SOD has also been ascribed to the ‘schizophrenic behavior’ of O2·- (43) . McCord assumes that O2·- plays a dual role as modulator of cell division vs. malignant transformation and apoptosis (44) or in initiating radical chain reaction of lipid peroxidation, as well as terminating the chain (43) . According to this explanation an adequate level of SOD is required to maintain an optimal [O2·-]steady state (44) . However, the results obtained with the present cell-free experimental system could neither support nor exclude this hypothesis. Most likely, depending on the specific system tested, more than a single mechanism might be underlying the bell-shaped dose response behavior of SOD.

The role of H2O2
The oxidation of ferrocyanide by TPL+ and by Cu(II),Zn-SOD (Fig. 6) took place in the presence of catalase and reflected H2O2-independent pro-oxidative effect of TPL and SOD. However, where papain served as target for oxidation, the effects of H2O2 and superoxide appeared to be synergistic. The observation that catalase, when added together with high doses of either SOD or TPL, fully prevented papain inactivation is intriguing. The control experiments excluded nonspecific protection of papain by the polypeptide or some contaminant of catalase. The explanation that catalase protects by preventing the H2O2-induced inactivation of SOD (30) has been ruled out as well. Instead, the results indicated that accumulating H2O2, though in the micromolar range, plays with superoxide some synergistic role. Our explanation, which correlates the lowering of [O2·-]steady state with the pro-oxidative effects of TPL and SOD, does not account for this observation.

Physiological relevance
Since nitroxides can protect from oxidative stress (1 2 3 4 5 6 7 , 33 , 38 , 45 , 46) , they are being considered for antioxidative treatment in various biological systems. On the other hand, the mechanism proposed above implies that pro-oxidant effect of high doses of nitroxides might be anticipated in cases where oxo-ammonium intermediate is involved in their reaction. Analogously, it is possible that similar argumentation holds also for the adverse effect of native SOD when the Cu(II),Zn-SOD is involved accordingly. Although O2·- generally acts as a reducing agent, there are many cases in which HO2· per se acts as an oxidant that can attack critical cellular targets (30 , 47) . SH groups, which are particularly prone to oxidation by HO2·, are essential in the catalytic activity of many enzymes and are involved in important biological functions, such as protein subunit association and membrane structure (48) . Cysteine proteases play key roles in human physiology and pathology, including protein processing and turnover, apoptosis, platelet aggregation, etc. (49) , or aberrant processing of the amyloid precursor in Alzheimer’s disease (50) . The lack of bell-shaped dose response of SOD in protecting papain from oxidative inactivation does not necessarily imply that high [SOD] would not exert a similar adverse effect, as was seen with high [TPL], toward other essential cellular targets.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 NITROXIDE SOD MIMICS
 ADVERSE EFFECTS
 POTENTIAL MECHANISMS
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
The present results provide additional insight at the potential adverse effects associated with overdoses of native SOD and SOD mimics. The results show that 1) nitroxide SOD mimics exhibit a bell-shaped dose-response behavior in protecting against oxidative stress, where TPL at > 20 mM fail to protect papain from oxidative inactivation; 2) O2·- (particularly HO2·) or H2O2 alone is capable of inactivating papain, whereas in combination they demonstrate a synergistic activity; 3) at high concentration, SOD loses its ability to block O2·-/HO2·-induced oxidation of ferrocyanide. The present findings offer a simple explanation for the pro-oxidant activity of SOD mimics and possibly of SOD, without a need to invoke dual effect of O2·-/HO2·. Obviously the most significant outcome of higher SOD concentration is a respective decrease in [O2·- ]steady state rather than any significant elevation of [H2O2]steady state. As a result, the reaction kinetics of the catalyst is altered. At high [SOD] and therefore ultra-low [O2·- ]steady state, the SOD or the SOD mimic in its higher oxidation state (Cu(II),Zn-SOD and oxo-ammonium cation, respectively) not only reacts with O2·-, but also attacks and oxidizes the target molecule for which it was supposed to provide protection.


   ACKNOWLEDGMENTS
 
This research was supported by grant 95–00287 from the USA-Israel Binational Science Foundation and from the Israel Science Foundation of the Israel Academy of Sciences. We gratefully acknowledge the critical comments and advice of Prof. Sandra L. Jewett, Chemistry Department, California State University, Northridge, California.


   FOOTNOTES
 
Received for publication June 3, 1999. Revised for publication February 3, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 NITROXIDE SOD MIMICS
 ADVERSE EFFECTS
 POTENTIAL MECHANISMS
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 

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