Superoxide Dismutase Mimics, Other Mimics, Antioxidants

Review pubs.acs.org/crt

Superoxide Dismutase Mimics, Other Mimics, Antioxidants, Prooxidants, and Related Matters Stefan I. Liochev* 8 Foxlair Court, Durham, North Carolina 27712, United States ABSTRACT: A significant number of low molecular weight metal complexes as well as metal-free compounds that are capable of scavenging superoxide and/or other radicals and reactive species in simple systems have been proposed to be used as potential drugs in the case of various diseases and/or as antiaging agents. Some have been used or suggested to be used as diagnostic tools for the involvement of such species in biological processes. In the present work, analysis of such claims indicates that their use as specific detectors of superoxide or of other reactive oxygen species is unsupported and might be confusing. Many of these compounds exert beneficial effects by counteracting the toxic effects of oxidative stress in a significant number of models of pathological processes. However, it is concluded that these actions are more likely due to other effects including prooxidant actions and that their beneficial effects also may be exerted in pathological processes that do not practically involve reactive oxygen species. Adaptation may be a common mode of action explaining a sizable portion of the beneficial effect of the so-called mimics and other compounds including prooxidants.

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CONTENTS

1. Introduction 2. Analysis and Discussion 2.1. Kinetic Considerations 2.1.1. General Considerations 2.1.2. Simplified Mathematical Analysis of the Kinetics of the Action of the so-Called SOD Mimics 2.2. The Case of MitoQ 2.3. Many Effects of the Mimics in Vitro Are Not Well Understood or Are Underestimated 2.3.1. Problems Due to Interactions with Flavoenzymes and Reductants 2.3.2. Complexity of the Reactions of the Mimics in Vitro Is Often Not Appreciated 2.4. Mimics Are Unlikely to Act As Scavengers of Free Radicals and ROS in Vivo 2.5. Inconsistencies and Misconceptions Concerning the Actions of the Mimics and Other Antioxidants 2.6. The Uniqueness of SOD 2.7. Could the Mimics Act as Such in Vivo after All? 2.8. What Could Be the Mode of Action of SODMimics in Vivo? © 2013 American Chemical Society

2.8.1. Cross-Protection Due to Adaptation 2.8.2. Scavenging or Adaptation? 3. Epilogue Author Information Corresponding Author Notes Abbreviations References

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1. INTRODUCTION Low-molecular weight compounds capable of scavenging certain reactive oxygen species (ROS) in vitro are broadly used for intracellular detection of ROS and are suggested as potential drugs in the treatment of various diseases and even aging. However, there are significant disagreements on their modes of action. This short review focuses mainly on a number of the best characterized in vitro superoxide dismutase (SOD) mimics; however, the conclusions are proposed to be valid for other mimics as well as for other compounds, including some originating from plants, that are known to exert beneficial effects.

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Received: May 12, 2013 Published: August 1, 2013

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dx.doi.org/10.1021/tx4001623 | Chem. Res. Toxicol. 2013, 26, 1312−1319

Chemical Research in Toxicology

Review

scavengers, the mimics must win the competition with SOD and abundant sinks such as GSH and other cellular targets for these ROS. These mimics are supposed also to work in cells (including human cells) with normal, increased, or somehow decreased [SOD]. Despite the extremely high rate constants for the reactions of the reduced Mn-porphyrins with the carbonate radical (CO3•−), for example, they have no chance to out compete other intracellular targets for this radical; which is only a slightly less potent oxidant than the hydroxyl radical (HO•).8 The rate constants for the reactions of the reduced Mnporphyrins with O2 (stated to be ∼105 M−1s−1) and probably with other intracellular components are lower than those for their reaction with most of the above named ROS. The catalytic rate constants for the reactions of the most promising mimics with O2•− are stated to be in the middle of the 107−108 M−1s−1 range. Nevertheless, the reduced form of the “mimics” should disappear mostly in reactions with O2 (producing O2•−) and other cellular components, under biologically relevant situation in cells containing normal, below normal, and even zero [SOD]. Rates matter, not just second order rate constants! According to Czapski, Goldstein, and coauthors, mimics whose rate constants for the oxidation of their reduced forms by O2 are relatively high are poor mimics in biologically relevant situations since intracellularily the oxygen/superoxide ratio is too high.9,10 The rate of reduction of their oxidized forms by O2•− will be a fraction of the sum of the rates of their reduction by other enzymes and other factors. These alleged mimics might preferentially act as mimics under the conditions of SOD assays, although some may interfere the assays by reacting with other electron donors and/ or acceptors in these assays.11 To what extent if any the alleged mimics act as SOD mimics or even are able to decrease the steady state level of superoxide in vivo is one of the main subjects of this review. 2.1.2. Simplified Mathematical Analysis of the Kinetics of the Action of the so-Called SOD Mimics. The purpose of the analysis is to consider if and to what extent the ability of a mimic to catalyze these reactions will affect its ability to dismute or scavenge superoxide. As discussed in section 1, the oxidized form of the mimic (Mox) is reduced by O2•− and by an electron donor (D) or donors. The reduced form (Mred) reacts with an electron acceptor (A) or acceptors, as well as with O2 and O2•−. At steady state, the rates of appearance and disappearance and the concentrations of these reactants do not change, and in particular, the rates of reduction of Mox is equal to the rate of oxidation of Mred (eq 1).

Mn-porphyrins are proposed as SOD mimics and/or catalytic antioxidants and redox modulators.1−6 Early reports, discussed in refs 1−6, suggested that if Mn-porphyrin complexes could efficiently scavenge superoxide, be comparatively nontoxic, and were relatively specific for superoxide, then that they might be used as SOD mimics in vivo.6 One such porphyrin (Mn-TMPyP) had several orders of magnitude lower dismutase activity than SOD, yet it markedly improved the aerobic growth of cytoplasmic SOD-null E. coli. It was known that the low SOD activity of the mimic is due to the low rate constant of the reaction of superoxide (O2•−) with the oxidized form of the mimic, while the rate constant of the reduced form with O2•− is approximately that of SOD. We found that the mimic, which accumulated substantially in E. coli, is reduced by thioredoxin reductase and probably other cellular redox systems, and we proposed that it may act as an electron donor, O 2 •− oxidoreductase,6 which later became known as superoxide reductase (SOR) activity. Other mimics also improved the aerobic growth of a SOD-null E. coli mutant as reported in the reviews in refs 1, 2, and 4. Other excellent work led to significant improvements in the catalytic efficiency, stability, lipophilicity, and other properties that are required for compounds to serve as efficient SOD or SOR mimics.1−5 It was also found that in vitro, the reduced and in some cases the oxidized forms of these SOD mimics efficiently scavenge practically any ROS including, but not limited to, O2•−, NO, NO2•, CO3•−, ONOO−, ClO−, and the peroxyl radical.1,2,4,5 Many of these mimics were found to be reduced by biological agents such as glutathione (GSH), ascorbate, flavoenzymes, tetrahydrobiopterin, and many others.1,2,4−6 The reduced forms of these mimics are efficiently oxidized by species including, but not limited to, O2 and the ROS named above. A plethora of other types of mimics now are known with chemistry that is not more discriminative than the one of the Mn-porphyrins.1 As discussed in the above cited comprehensive reviews, protective effects of these mimics were reported in a significant number of models and usually were interpreted as being due to superoxide and in some cases to other reactive species scavenging. Yet, as a coauthor of papers that contributed to and to some extent even encouraged the research in the area of mimics, I feel the responsibility to point out that this research may not have been going entirely in the right direction for a significant period of time and that some problems were overlooked from the beginning. I am also concerned that this situation likely diverts the attention from possible significant findings, as Kalyanaraman et al. warned about in their paper, for the case of the fluorogenic probes for ROS,7 and even creates a self-propagating chain of confusion. Except when indicated otherwise, I discuss (in the text below) data, conclusions, compounds, and their structures and other characteristics described in a recent comprehensive review.1 The same review as well as others2−5 contain extensive, well systemized, and the most current information about the redoxpotentials of the discussed compounds, the rate constants for their reactions with superoxide and other species, and other useful data about the mimics

ka[Mox][O2•−] + k b[Mox][D] = kc[Mred][O2•−] + kd[Mred][O2 ] + ke[Mred][A] (1)

Here, ka to ke are second order rate constants. Equation 1 is complex, and the full analysis is very involved, but it is not necessary to show here since we can approach the problem by discussing simplified cases. Case 1: The mimic reacts practically only with superoxide, and the other reactions are so slow that they safely can be excluded in the present analysis (eq 2).

2. ANALYSIS AND DISCUSSION 2.1. Kinetic Considerations. 2.1.1. General Considerations. With so much more known now in the areas of both free radicals in general and mimics in particular, it is hard if not impossible to believe that significant effects of these compounds in vivo are to scavenge O2•− and/or other ROS. To act as

ka[Mox][O2•−] = kc[Mred][O2•−]

(2)

In this case, if ka is approximately equal to kc, the turnover (catalytical) rate constant (kcat) is equal to each of these rate constants, [Mox] = [Mred], and [Mox] + [Mred] = [M], where 1313



Review

decrease of [O2•−], but that decrease will slow down with the increase of the [O2]/[O2•−] ratio. It is not necessary to introduce more equations in order to conclude that the superoxide reductase mimic will become less and less efficient in proportion to an increase of kd and/or of the [O2]/[O2•−] ratio and that above a certain ratio it will be effectively producing superoxide rather than scavenging it. Thus, in the situation described by case 3, the mimic will cause as much production of superoxide as it generates when kc[Mred][O2•−]/kd[Mred][O2] ratio is 1 and hence when kc/kd = [O2]/[O2•−]. If the [O2]/[O2•−] in a given experiment is higher than the kc/kd ratio, then the mimic will be producing more superoxide than it generates. The kc/kd of SOD is approximately 1 billion,9 while in vivo the [O2]/[O2•−] ratio in the presence of SOD is generally less than a million. In contrast, the kc/kd ratio of the mimics is generally orders of magnitude lower than 1 million. The analysis of other possible situations could be continued. However, even if we succeed in analyzing the most complex situation represented by eq 1, we will still be far from fully appreciating how different the effects of an unspecific mimic are from those of SOD. For example, this analysis does not take in to account that the one electron oxidation or reduction of nonradical electron donors or acceptors creates radicals, many of which might react directly with oxygen or react with other compounds whose radicals react with oxygen and therefore lead to the production of superoxide. Nor does it take in to account the effects of oxidants and reductants on mimics such as mitoQ that can be involved in both one electron and two electron transfer reactions. All this leads to the conclusion that a mimic of nearly everything is not a good mimic of anything (at least in vivo). Factors such as binding to intracellular components or other factors are likely to change the reactivity of the mimics, but it is very unlikely that such changes will necessarily result in increased specificity and ability to scavenge superoxide. After all, the effectiveness and specificity of the enzymes have evolved over a long time and under the pressure of natural selection. The evolution of highly efficient and specific enzymes allows benefits such as the existence of complex self-regulating metabolic networks. In addition, the increase in specificity is likely important for achieving optimum efficiency. An argument for such a generalization is that an oxido-reductase which specifically oxidizes a given reductant and reduces a given oxidant should be more efficient in its specific function than it would be if its reduced and oxidized forms were able to also react in a variety of parasitic reactions including those with oxygen and superoxide. Some situations that may affect the activity of SODs to catalyze superoxide dismutation and/or some slow additional activities (mainly of Cu and Zn SOD), have been kinetically analyzed previously.12 That analysis may be even more relevant for the case of the mimics. 2.2. The Case of MitoQ. The proposed catalytic action of another proposed antioxidant MitoQ is described1 as follows: MitoQ is rapidly reduced by O2•− to MitoQ•, while the fully reduced form MitoQH2 is oxidized by peroxynitrite (ONOO−) to MitoQ•, in a reaction producing NO2, and finally the two mitoQ• dismute. This is certainly an elegant but unfortunately unlikely proposal for an in vivo situation since SOD that is already available in the cells, in cooperation with targets for NO and ONOO−, should severely limit both O2•− and ONOO− steady state levels. Furthermore, the dismutation of the two MitoQ• in a sea of other possible targets including O2 should be an extremely

M is the total concentration of the mimic. In this case, we can write eq 2a as follows: kcat[M][O2•−] = 2ka[Mox][O2•−] = 2kc[Mred][O2•−] = rate of superoxide consumption

(2a)

Equation 2a describes (kinetically) the action of SOD, and hence in such a case, the mimic will be equally as efficient as SOD if its kcat is the same as that of SOD . Case 1a: However, ka is not always equal to kc for the mimics, and in fact, these rate constants can be very different in both directions. The catalytic efficiency of such a mimic is determined by the rate-limiting step, and such a mimic is a less efficient catalyst of superoxide dismutation than SOD. This leads, as shown by Goldstein, Czapski, and colleagues,9,10 to a more complex equation describing the relationship betveen ka and kc and the relevant turnover rate constant (kcat). Thus, if at a given rate of superoxide production, SOD is replaced by a mimic with the same concentration as SOD but with lower ka and/or kc and therefore kcat, the [O2•−] in the presence of the mimic will be higher than that in the presence of the same [SOD]. Case 2: Let us suppose that Mox reacts practically only with superoxide but that Mred reacts with oxygen as well (eq 3): ka[Mox][O2•−] = kc[Mred][O2•−] + kd[Mred][O2 ]

(3)

As shown,9,10 in this case the equation for the overall rate constant is even more involved. Furthermore, when the rate of Mred oxidation by oxygen is significant, due to high kd or high [O2], the overall turnover rate constant of a mimic that reacts with oxygen as well is lower than that for SOD. This is the case even if both the mimic and SOD are used at the same concentration and have equal ka and kc. The same authors present many examples of how big (orders of magnitude!) the decrease of the efficiency of the mimic to diminish [O2•−] could be since the [O2]/[O2•−] ratio in vivo is at least 100000, especially in SOD proficient cells. The mimic becomes proportionally less effective in decreasing the concentration of superoxide with increases in the [O2]/[O2•−] ratio and of kd. Case 3: In this case, a compound is involved significantly only in the following reactions. Its oxidized form is reduced by a reductant, while the reduced form reacts with both superoxide and with oxygen. k b[Mox][D] = kc[Mred][O2•−] + kd[Mred][O2 ]

(4)

When kd[Mred][O2] approaches zero, such a compound acts as a mimic of superoxide reductase. When kc[Mred][O2•−] approaches zero, the compound acts as an oxygen-reducing oxidoreductase, such as xanthine oxidase, for example. The following reasoning explains how the increase of kd and of the [O2]/[O2•−] ratios compromises the efficiency of a superoxide reductase mimic. Imagine that in the absence of a superoxide source we add a mimic, whose reduced form reacts with both superoxide and oxygen, to a reductant whose concentration is so high that for some time it does not decrease significantly. In this case, immediately after the addition of the mimic the concentration of superoxide will start rising and will rise until the rate of its production approaches the rate of its disappearance in a reaction with Mred and in a reaction of spontaneous dismutation. The addition of the same compound as before but in the presence of an efficient flux of superoxide so that [O2]/[O2•−] is sufficiently low will result in an initial fast 1314



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rare event. The rate constant for the disappearance of MitoQ• with O2 is stated to be 2.7 × 107 M−1s−1, and that reaction produces O2•−. Rates are what matter, and NO2• is a reactive radical.8 Meanwhile, MitoQ is a quinone and should be viewed as a quinone or a mimic of another quinone yet is discussed in a review entitled MnSOD and Its Mimics.1 Moreover, in the same review1 the authors seriously consider papers claiming that in the presence of increasing concentrations of Mn SOD CoQ is involved in dramatic superoxide and hydrogen peroxide production. 2.3. Many Effects of the Mimics in Vitro Are Not Well Understood or Are Underestimated. 2.3.1. Problems Due to Interactions with Flavoenzymes and Reductants. The SOD activity of the various mimics is often measured by indirect methods, and one of the most reliable and most often used is a method in which xanthine plus xanthine oxidase serve as a source of superoxide and SOD, and supposedly, the mimics compete with cytochrome c for O2•−. A control, to document that the mimics do not interfere with the enzyme, is often described to be the inability of most of the Mn-porphyrins, except MnTBAP, for example,6 to inhibit urate production. However, what we first and then the others that followed failed to consider is the following: Xanthine oxidase is a complex enzyme, and the electrons from xanthine are transferred via iron−sulfur centers to the flavin (FAD) center, which then reduces NAD to NADH or O2 to H2O2 and O2•−, and it also reduces a significant number of other electron acceptors. When NADH is used as a substrate for xanthine oxidase, its oxidation is increased by paraquat.13 Clearly, oxidation of the reduced flavin will not result in decreased urate production. Concerning the inhibition of urate production by MnTBAP, plausible explanations could be that probably it does not act as an oxidant of the reduced flavin but acts prior to this step, or maybe it binds to xanthine oxidase but is not able to be reduced due to thermodynamic or mechanistic limitations. However, as mentioned above and in the above cited reviews, all flavin containing enzymes studied so far, including thioredoxin reductase, NO synthase, and importantly glucose oxidase and xanthine oxidase, readily reduce MnTE2PyP, MnTMPyP, and other mimics. That Mn-porphyrins are efficiently reduced by flavoenzymes and reoxidized by O2 is illustrated by the work of Day and Kariya.14 The efficiency of the studied compounds differed broadly, yet all had significant activity, and one of them was more active than paraquat. These authors proposed that part of the beneficial as well as nonbeneficial actions of the mimics might at least in part be due to the interference with the functions of the flavoenzymes. Glucose oxidase itself does not produce O2•− but produces it when it reduces a compound capable of autoxidation.15 In the case of the glucose oxidase-dependent reduction of MnTE2PyP, Ferrer-Sueta et al.11 observed accumulation of the reduced form of MnTE2PyP only after the oxygen was exhausted.11 Since they had catalase in their medium, this likely can be interpreted as a confirmation of the estimated high rate constants of the reaction of at least some Mn-porphyrins with O2. Xanthine oxidase also readily reduces MnTE2PyP.11 What exactly happens in the latter case is difficult to judge, without further experiments, especially since reactions of the fully as well as the half-reduced and the fully oxidized forms of the flavin with the mimic also need to be considered. An action of the mimic due to the increase in the rate of one electron transferat the expense of two electron transfer (that generates directly hydrogen peroxide) cannot be excluded. Some eventual contribution of urate in the reduction of some of

the mimics also cannot be excluded and certainly needs to be studied. In our original paper, we considered the possibility of our mimics being reduced by components of the assay but noticed that the spectrum of our mimic did not change under the conditions of the assay.6 However, at that time, we failed to consider the possibility that the reduced mimic may rapidly react with O2. This became clear later when we noticed that MnTMPyP oxidizes ascorbate producing hydrogen peroxide (H2O2), while SOD did not inhibit this process.16 In future experiments, one should also consider a recommendation made approximately 30 years ago that in the case of using that method for measuring the dismutase activity of Mn-complexes, acetaldehyde should be used as a substrate rather than xanthine.17 Therefore, in order to answer the question as to what extent the catalytic rate constants for the Mn-porphyrins and other metal complexes catalyzed O2•− dismutation has been correctly estimated with the xanthine oxidase method, more control experiments may be needed. This is important since, for example, many other reductants and oxidants are available in vivo in addition to oxygen, superoxide, and the components of an assay for SOD. 2.3.2. Complexity of the Reactions of the Mimics in Vitro Is Often Not Appreciated. The reactions of many Mn-complexes with H2O2 and with O2•− are much more complex than they appears even in in vitro systems and involve the formation of complexes of these species with the Mn−ligand complex.18−20 In the case of O2•−, it reacts with Mn2+ (ligand) producing MnOO+ (ligand).19,20 According to one group, the main pathway of disappearance of the latter species in the case of their Mn−ligand complex is through dismutation.19 According to another, the complex of MnOO+ with a different ligand dissociates, and/or upon protonation, H2O2 and the Mn3+ (ligand) are produced.20 In fact, for different Mn complexes (different ligands) some of these pathways will be more or less favored in vitro, but consider how these mechanisms will be profoundly changed in vivo. Mn3+(ligand) will likely be preferentially reduced by reductants different from O2•−. Similarly, Mn2+(ligand) will be preferentially oxidized by other oxidants. MnOO+, to the extent that it is formed during the competition of O2•− and other reactants for the Mn2+(ligand), will likely disappear in reactions with both cellular reductants and oxidants. The mechanism of dismutation of O2•− by MnSOD also involves the formation of SOD bound MnOO+, which through protonation regenerates the fully oxidized form and releases H2O2.21 However, within the active center competing reactions are avoided. The point is that even in a simple in vitro situation the mechanism behind the effects of the supposed mimics is less than satisfactorily understood, although this should be a priority. 2.4. Mimics Are Unlikely to Act As Scavengers of Free Radicals and ROS in Vivo. In a mouse study, designed to investigate the accumulation of one of these mimics (MnTnHex2-PyP5+), it was given at the maximally tolerable dose of 2 mg per kg of body weight. In this situation, its concentration in the murine brain or heart cytosol was 5 times lower than that in the mitochondria, and in mitochondria, its concentration was 21 μM.1 Even if its reactivity is not limited at all by binding, given the estimated rate constants of MnSOD and of MnTnHex-2-PyP5+ for their reactions with O2•− (or more correctly their turnover rate constants) and their concentrations, the intramitochondrial rate of the enzyme catalyzed dismutation will be at least 20 times faster than the Mn-porphyrin catalyzed one. Yet, in some of the models reported in the review,1 MnTnHex-2-PyP5+ was given at 0.1 or even 0.05 mg/kg body weight, and its action was 1315



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2.6. The Uniqueness of SOD. SOD is unique because of its ability to efficiently catalyze dismutation and because due to its relatively high intracellular concentration, it vastly outcompetes all other cellular targets of O2•−. Just as importantly, SOD is unique due to the very low rate of oxidation of its reduced form by O2 or by other oxidants and the low rate of reduction of its oxidized form by reductants other than O2•−. SODs have activities different from dismutation that may or may not be biologically relevant, but they are very slow.12 When choosing to study Mn-complexes and not complexes of Fe or Cu, as possible SOD mimics, the ability of the latter two to cause increased production of HO• or similarly reactive species by reducing H2O2 was an important consideration.6 However, later it became clear that manganese complexes are also involved in the production of CO3•−.18 Moreover, metal complexes can exert effects including toxic ones by a variety of Fenton chemistry independent ways. Low molecular copper complexes, for example, may not be good as SOD mimics not just because of concerns about possible Fenton chemistry dependent toxicity but in addition because they have indiscriminate reactivity and interfere in various ways even in simple SOD activity assays.24 However, evolution has found a way to limit the indiscriminate reactivity of such metal ions and complexes and meanwhile to use their reactivity for beneficial purposes, as exemplified by Fe-, Cu-, Mn-, and Ni-SODs. Recently, the ability of low nontoxic concentrations of Feporphyrins having substantial SOD activity as measured in vitro to improve the growth of cytosolic SOD-null E. coli was studied.25 The growth was improved; however, the effect was due not to their SOD activity but simply because they served as a source of iron for the iron starved bacteria. Indeed, the medium used in this study is absolutely minimal and deficient in not only iron but of many other ingredients that if added would have increased the growth rate as well. As the authors comment,25 these findings have to be considered when similar observations made in studies with animals are concerned. In addition, the importance of eating well-balanced and nearly optimal diets cannot be overemphasized. Moreover, as far as humans are concerned, the benefits of proposed drugs need to be in addition to the benefits due just to eating such a diet or to eating a somehow suboptimal diet and in addition taking vitamin and/or mineral supplements. Much higher concentrations of manganese or Mn-porphyrins were required in order to improve the growth of the SOD-null bacteria.25 This is said not for the purpose of marveling about the uniqueness of SODs one more time but to suggest that part of the effort might be devoted to the creation or eventually finding natural ones of real mimics having not just satisfactory activity but matching specificity. In principle, such mimics do not need to be necessarily Mn dependent but could be Ni, Fe, Cu, and possibly other transition metals based. 2.7. Could the Mimics Act as Such in Vivo after All? Recently, Liang et al. reported that a Mn-porphyrin used as a SOD mimic attenuated indices of oxidative stress, such as mitochondrial aconitase inactivation and increased 3-nitrotyrosine seen in MnSOD-deficient mice, but did not cause a full reversion to the wild type phenotype.26 The lifespan of the MnSOD-deficient mice was severely shortened. Again, the mimic extended the lifespan of the MnSOD-null mice but fell very short of extending it to that of wild type mice. While, mechanisms such as inhibition of the NO production or an adaptation caused by the mimic may explain at least part of the effects, it is apparent that the possibility that at least part of the

protective! Since in such doses and concentrations only very selective drugs are effective, the effect seen cannot be due to superoxide scavenging or to some general radical-scavenging activity. Hence, considering the information about the effects of such complexes plus the information about the phenomenology and mechanism of the given pathological condition might reveal the real mechanism involved and the steps most susceptible to intervention and thus allow the design of specific drugs. This will likely be preferable to injecting MnTnHex-2-PyP5+ at 0.05 mg/ kg body weight 24 h before ischemia is supposed to happen. 2.5. Inconsistencies and Misconceptions Concerning the Actions of the Mimics and Other Antioxidants. The reasoning that even the 5 μM mitochondrial concentration of Mn-porphyrin based SOD mimic will be enough to protect in vivo from the toxicity of ONOO− since even 3 μM of it protected submitochondrial particles from the toxicity of ONOO−1 seems unconvincing for reasons discussed in the points above. In addition, these mimics are reduced by nitric oxide synthase and tetrahydrobiopterin, which might limit both NO and ONOO− formation and steady state concentrations, and such effects may mimic effects due to peroxinitrite scavenging. Similarly, reactions such as self-dismutation of O2•−, reactions of ascorbyl radical with O2•−, and mimic dependent dismutation of O2•− are unlikely to play a perceptible role in the production of H2O2 in vivo. The increase of H2O2 by the mimics when that occurs should be mostly a consequence of oxidation by the mimics of cellular components, including but not limited to ascorbate and flavoenzymes. The oxidation of such components by the oxidized forms of the mimics will generate radicals and the reduced forms of the mimics. In turn, their auto-oxidation may generate syperoxide, which will be dismuted by SOD to oxygen and hydrogen peroxide. Furthermore, in agreement with other papers that they cite, the authors of ref 1 express the opinion that when overexpressed, MnSOD plays a role as an “oncogene.” The interpretation is that this effect is due to increased intracellular H2O2 production caused by the overproduced SOD. In fact, no such significant increase in hydrogen peroxide production due to overproduction of MnSOD should be expected to occur since MnSOD scavenges most of the superoxide in the mitochondrial matrix at normal levels and due to the direct formation of H2O2 by numerous oxidases and by dismutation of superoxide by Cu and Zn SOD and due to other previously discussed reasons.22 The increased [SOD] in some cancers is rather likely an adaptation, allowing the cancer cells to withstand the attempts of the defensive systems of the organism to eliminate them. MnTMPyP and MnTBAP are criticized for different reasons (including for not being good SOD mimics!).1,4 Nevertheless, their beneficial effects have been described in significantly more papers than those cited in ref 4. Therefore, it makes sense that since MnTBAP is not a good SOD mimic even in vitro to use the already existent significant phenomenological information and eventually new experiments in order to pinpoint why MnTBAP is protective. Of course, this is valid not just for MnTBAP and will help one to design or find more effective pharmaceuticals. Some SOD mimics such as Mn porphyrins are sometimes promoted as catalytic antioxidants being compared to presumably stoichiometric antioxidants such as ascorbate and GSH. The idea that GSH, ascorbate, and vitamin E are stoichiometric antioxidants rather than catalytic ones is a misconception, which has already caused significant confusion.23 The oxidized forms of both the natural antioxidants and the mimics are being recycled back to their reduced forms. 1316



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above). Therefore, although it cannot be excluded, it has not been established, even in a mitochondrial SOD deficient background, that a significant part of the effects of the mimic seen in the discussed study was due to decreasing the intracellular level of superoxide in competition with intracellular targets. The same can be concluded based on the discussion concerning the Feporphyrins in section 2.6. This is stated not just to dampen the general enthusiasm about the mimics. Rather, it is an attempt to initiate careful (re)considerations and/or debate about the existing problems which are obviously more serious than so far thought. As previously pointed out,30 the accumulation of interesting and suggestive but inconclusive observations in otherwise solid papers might lead to the impression that the matter is solved. As Goldstein et al. predicted quarter of a century ago,10 the search for good SOD mimics is not (going to be) easy. 2.8. What Could Be the Mode of Action of SOD-Mimics in Vivo? While other mechanisms should by no means be excluded and some have been indeed discussed or at least suggested,1−5,14,25,26 triggering of beneficial adaptations may be a unifying explanation for the effect of many if not all SOD mimics as well as other compounds. How are natural and artificial compounds, so different in regard to their structure, reactivity, redox potential, metal content etc., able to protect in many models? The mimics are usually administered at nontoxic dose prior to the toxic intervention that models a given pathological process. Hence, it is reasonable to suppose that the mimics by exerting a mostly prooxidative effect, although some very specific effects cannot be excluded, preadapt the cells and organisms toward both oxidative and nonoxidative challenges. Should the mimics be thought to be the most effective, practical, cheaper, and nontoxic agents that act in this way is not an issue here. In many cases, this adaptation phenomenon may work even after the pathological condition was already triggered since adaptations caused by the mimic or other agents may cause adaptations that the pathologic process itself did not trigger or enhance adaptations triggered by the pathologic process itself. In fact, Miriyala et al.1 themselves propose that adaptation or signaling might have been involved in the protective effect of mimics in several of the cases they discuss. Thus, the point here is not to rediscover the phenomenon of adaptation. Rather, it is to emphasize that the protective effect seen in so many different models is likely to be in a significant number of cases due to a common cause, which likely is an adaptation. Another point is that many of the pathological conditions found to be ameliorated by mimics might not be or at least not be entirely due to oxidative stress. The following two cases illustrate such concepts. 2.8.1. Cross-Protection Due to Adaptation. Preincubation with paraquat adapts wild type E. coli to the toxicity of paraquat and to that of other compounds.31−34 A significant part of the adaptation of E. coli toward paraquat and other oxidants is due to the induction of the SoxRS regulon that controls over 100 members including antioxidant enzymes such as Mn-SOD and glucose-6-phosphate dehydrogenase.33,34 Induction of SoxRS regulon by one agent provides protection not only from the toxicity of that agent but from the toxicity of many other agents. Even more interestingly, the induction of SoxRS regulon, provides protection against the toxicity of antibiotics some of which are not acting as redox-cycling agents or oxidants in general.34 Some antibiotics induce another regulon (marA), and this induction protects against the toxicity of oxidants that are able to induce SoxRS. This is so because the members of these regulons overlap.34 Hence, some but not all enzymes that protect

effect of the mimic was due to superoxide scavenging cannot be excluded as well. Similar studies using cytosolic SOD-null E. coli as a model, like those described in our early papers cited here and those cited in refs 1−5 and more extensive studies in the laboratory of L. Benov, lead to a similar conclusion. Thus, in this model the effect of a number of Mn-porphyrins was beneficial. It should be concluded that at least some of the compounds found to be SOD-mimics in vitro might be effective in alleviating pathological conditions that are due to complete inactivation or inhibition of SOD, at least in part due to superoxide scavenging. However, superoxide is extremely toxic, and natural selection will rapidly eliminate the unfortunate organisms that lack SOD. However, the damage seen both in the presence and in the absence of agents causing additional oxidative stress in organisms that have about half of the normal level of SOD, although higher than the one seen in the wild type, is much lower than the one observed in SOD-null organisms.27−29 It seems reasonable to conclude that the protection provided even by half of the normal level of SOD is much higher than that provided by mimics and specifically the one used by Liang et al.26 Thus, mice that have only about half of the wild type level of MnSOD live as long as the wild type, while still having higher incidents of cancer or are otherwise more impaired.27−29 Certainly, such organisms are still at a disadvantage compared to the wild type. Moderate overproduction of SOD over the wild type level often does not provide additional protection toward oxidative stress or at least significant protection, especially in mammals.27 In fact, significant overproduction of the cytosolic SODs in E. coli increases the sensitivity of the organisms to that stress in some situations, and possible explanations for these latter observations have been evaluated in refs 30 and 31. Considering studies on the effects of overproduction and of the level of SODs in general, it should be kept in mind that they involve laboratory animals on a standard diet and that such animals neither are subjected to external stress that will certainly damage them nor are they adapted to such a stress. Therefore, in the wild it may be more beneficial to have the wild type level of SOD than half of it. Similarly, a modest to moderate increase over the wild type level caused by induction, post-transcriptional regulation, or by genetic manipulations may prove to be more beneficial than what is apparent from studies involving laboratory animals with artificially altered levels of SOD. As speculated,23 the lifespan of wild type animals in the wild might be shorter than that of animals kept in the laboratory due to the damage caused by various types of stress and be even shorter in the case of animals with compromised adaptation or having less than the wild type level of SOD. All this being said, it should be noted that the extension of the life span of the MnSOD deficient mice caused by the mimic employed by Liang et al. amounted to days. All of the treated and untreated MnSOD-deficient animals were dead in less than a month after birth,26 while most of the laboratory mice having wild type level of MnSOD or even half of it were alive at least two years after birth and many lived for more than three years. While different explanations can be entertained, the most apparent one based on what has been discussed so far is that the SOD activity supplemented by the mimic used by Liang et al. (if any) was tiny compared to the one supplemented by even half of the wild type level of MnSOD. As they discussed, that mimic or catalytic antioxidant was not an especially good mimic of SOD even in vitro (see the discussion about the meaning of catalytic or turnover rate constant in the case of mimics in section 2.1.2 1317



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biomolecules, and effects on cellular processes. Such information should be reevaluated, especially where participation of specific ROS or radicals was inferred. The mimics and especially the relatively well-studied Mnporphyrins have very diverse functions as described by BatinicHaberle et al.5 It seems reasonable to reconsider calling all of these diverse compounds SOD mimics, at least in vivo, and consider other implications. It may be concluded that the mimics even when and if acting as prooxidants, as well as other redoxcycling agents and prooxidants, could have beneficial effects and even paradoxically exert overall antioxidant effects in the treatment of a variety of pathological conditions. The efficacy and the mechanism of action of the agents used for such treatments should be expected to depend on the mechanisms of action of the given prooxidant and on the mechanism of the pathological process. Certainly, caution should be exercised when using prooxidants since while they are likely to exert beneficial effects due to triggering of adaptations, the long-term effects of and/or treatment with such agents may not be that beneficial and might be even toxic. Finally, an interesting recent critical review of some peroxidase mimics questioned to some extent even the need for development of such mimics, among other interesting matters.38 This author has similar doubts concerning the SOD mimics. Thus, if a nearly perfect SOD mimic is created one day, it will probably be beneficial in a small number of cases such as damage due to SOD insufficiency or impaired regulation. Although superoxide is likely the most diabolical radical and ROS, a number of other radicals, ROS, and nonradical oxidants are capable of causing toxicity by a variety of mechanisms. Furthermore, hydroxyl radical and other very reactive oxidants could be generated in superoxide-independent ways Improving the reductive repair of the damage caused by oxidative stress, including by triggering beneficial adaptations, might prove to be the easiest and most efficient approach to counteract the toxicity of oxidative stress as well as of other stresses, at least in the near future.

against one type of oxidative stress play an important role in protection against other types of stress, whether oxidative or not and vice versa. These and other regulons of E. coli interact in a mind-boggling way with each other, while maintaining the cell homeostasis and redox balance.33 2.8.2. Scavenging or Adaptation? Treatment with a combination of five medicinal plants was found to protect cardiomyocytes against hydrogen peroxide induced apoptosis due to activation of the transcriptional regulator Nrf2.35 The mechanism of the Nrf2 dependent effects exerted by phytochemicals and other biomolecules has been very recently reviewed.36 The relevant points here are not to propose that the SOD mimics act by way of Nrf2 dependent adaptation (signaling) nor even to support the likely correct view that the beneficial effects of phytochemicals in vivo are more or less due to a Nrf2 dependent mechanism. Rather, the point is to support an emerging paradigm that the effects of agents such as the alleged mimics and phytochemicals previously thought to act as direct scavengers of radicals and ROS, like catalytic antioxidants such as SOD, GSH, vitamin E, and ascorbate, may rather act by the way of causing (cross)protection by triggering adaptations. It has been proposed, that at least some phytochemicals and other compounds might exert beneficial effects by promoting adaptation and that promoting adaptation might be more useful strategy than oversupplementation with vitamins and antioxidants enough of which we obtain with a balanced diet.37 In agreement, a point has been made that the repairs (including reductive reactivations) of oxidized targets such as nucleic acids, iron−sulfur clusters, glutathione, and other antioxidants are likely rate limiting steps especially under oxidative stress.23 Indeed, what we observe during oxidative stress and what constitutes oxidative stress is the increase in the ratio of oxidized/ reduced forms of the targets including glutathione, ascorbate, and other natural scavengers. Furthermore, it was pointed out that an important part of the adaptation toward oxidative stress is achieved by increasing the rates of such repairs.23 In fact, oxidative stress impairs the effectiveness of the repair systems themselves in part because most of that repair is reductive, and the levels or the ratios of the reduced versus oxidized forms of reductants such as NADPH, ascorbate, and glutathione (which in addition to being major scavengers play important roles in that repair) are decreased under oxidative stress. Naturally, changes in the redox-status of major natural scavengers such as glutathione should trigger in turn adaptations aimed at maintaining the cellular redox-balance and homeostasis.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 919-382-8713. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS ROS, reactive oxygen species; SOD, superoxide dismutase; SOR, superoxide reductase; Nrf2, nuclear factor; MnTMPyP, Mn(III)tetrakis(N-methylpyridinum)porphyrin; MnTBAP, Mn(III)tetrakis(4-benzoic acid)porphyrin; MnTE2PyP, Mn(III) meso-tetrakis(N-ethylpyridinum-2-yl)porphyrin; MnTnHex-2PyP 5+ , Mn(III) meso-tetrakis(N-n-hexylpyridinum-2-yl)porphyrin; MitoQ (MitoQ10), ubiquinone analogue coupled to a cationic triphenylphosphonium ion via a lipophilic alkyl chain

3. EPILOGUE There are important lessons to be learned from the successes and even more from the failures of the search for mimics. The expanding practice to support conclusions based on the usage of fluorogenic and luminogenic probes for the measurement of ROS with conclusions based on the usage of SOD mimics to scavenge radicals and ROS, often in one and the same paper and/or studying the same phenomenon, seems in need of reconsideration. In fact, it seems to this author that the usage of SOD mimics for the detection of ROS and free radicals in cell cultures has created a little more than significant confusion and controversy. This is especially the case when combined with the usage of other potential prooxidants such as fluorogenic and luminogenic probes, allegedly for the same purpose. On the positive side, significant potentially useful mechanistic and empirical information has been accumulated about the properties of the alleged mimics, their interaction with enzymes,

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REFERENCES

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effects of Mn porphyrin-based redox modulators. Med. Princ. Pract. 22, 103−130. (3) Keir, S. T., Dewhirst, M. W., Kirkpatrick, J. P., Bigner, D. D., and Batinic-Haberle, I. (2011) Cellular redox modulator, ortho Mn(III) meso-tetrakis(N-n-he5xylpyridinium-2-yl)porphyrin, MnTnHex-2PyP(5+) in the treatment of brain tumors. Anticancer Agents Med. Chem. 11, 202−212. (4) Batinic-Haberle, I., Reboucas, J. S., and Spasojevic, I. (2010) Superoxide dismutase mimics: chemistry, pharmacology, and therapeutic potential. Antioxid. Redox Signaling 13, 877−918. (5) Batinic-Haberle, I., Rajic, Z., Tovmasyan, A., Reboucas, J. S., Ye, X., Leong, K. W., Dewhirst, M. W., Vujaskovic, Z., Benov, L., and Spasojevic, I. (2011) Diverse functions of cationic Mn(III) Nsubstituted pyridylporphyrins, recognized as SOD mimics. Free Radical Biol. Med. 51, 1035−1053. (6) Faulkner, K. M., Liochev, S. I., and Fridovich, I. (1994) Stable Mn(III) porphyrins mimic superoxide dismutase in vitro and substitute for it in vivo. J. Biol. Chem. 269, 23471−23476. (7) Kalyanaraman, B., Darley-Usmar, V., Davies, K. J. A., Dennery, P. A., Forman, J. A., Grisham, M. B., Mann, G. E., Moore, K., Roberts, L. J., and Ischiropoulos, H. (2012) Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations. Free Radical Biol. Med. 52, 1−6. (8) Augusto, O., Bonini, M. G., Amanso, A. M., Linares, E., Santos, C. C., and De Menezes, S. L. (2002) Nitrogen dioxide and carbonate radical anion: two emerging radicals in biology. Free Radical Biol. Med. 32, 841−859. (9) Czapski, G., and Goldstein, S. (1988) The uniqueness of superoxide dismutase (SOD) - Why cannot most copper compounds substitute SOD in vivo? Free Radical Res. Commun. 4, 225−229. (10) Goldstein, S., Michel, C., Bors, W., Saran, M., and Czapski, G. (1988) A critical reevaluation of some assay methods for superoxide dismutase activity. Free Radical Biol. Med. 4, 295−303. (11) Ferrer-Sueta, G., Hannibal, L., Batinic-Haberle, I., and Radi, R. (2006) Reduction of manganese porphyrins by flavoenzymes and submitochondrial particles: a catalytic cycle for the reduction of peroxynitrite. Free Radical Biol. Med. 41, 503−512. (12) Liochev, S. I., and Fridovich, I. (2003) Mutant Cu,Zn superoxide dismutases and familial amyotrophic lateral sclerosis: evaluation of oxidative hypotheses. Free Radical Biol. Med. 34, 1383−1389. (13) Liochev, S. I., Ivancheva, E., and Fridovich, I. (1989) Effects of vanadate on the oxidation of NADH by xanthine oxidase. Arch. Biochem. Biophys. 269, 188−193. (14) Day, B. J., and Kariya, C. (2005) A novel class of cytochrome P450 reductase redox cyclers: Cationic manganoporphyrins. Toxicol. Sci. 85, 713−719. (15) Liochev, S. I., and Fridovich, I. (1995) Superoxide from glucose oxidase or from nitroblue tetrazolium? Arch. Biochem. Biophys. 318, 408−410. (16) Liochev, S. I., and Fridovich, I. (1995) A cationic manganic porphyrin inhibits uptake of paraquat by Escherichia coli. Arch. Biochem. Biophys. 321, 271−275. (17) Archibald, F. S., and Fridovich, I. (1982) The scavenging of superoxide radical by manganous complexes: in vitro. Arch. Biochem. Biophys. 214, 452−463. (18) Liochev, S. I., and Fridovich, I. (2004) Carbon dioxide mediates Mn(II)-catalyzed decomposition of hydrogen peroxide and peroxidation reactions. Proc. Natl. Acad. Sci. U.S.A. 101, 12485−12490. (19) Barnese, K., Gralla, E. B., Valentine, J. S., and Cabelli, D. E. (2012) Biologically relevant mechanism for catalytic superoxide removal by simple manganese compounds. Proc. Natl. Acad. Sci. U.S.A. 109, 6892− 6897. (20) Durot, S., Lambert, F. L., Renault, J.-P., and Policar, C. (2005) A pulse radiolysis study of catalytic superoxide radical dismutation by a manganese(II) complex with an N-tripodal ligand. Eur. J. Inorg. Chem. 14, 2789−2793. (21) Barnese, K., Sheng, Y., Stich, T. A., Gralla, E. B, Britt, R. D., Cabelli, D. E., and Valentine, J. S. (2010) Investigation of the highly

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