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Unraveling the Biological Roles of

Michael P. Murphy,1,* Arne Holmgren,2 Nils-Go¨ ran Larsson,3 Barry Halliwell,4 Christopher J. Chang,5,6 Balaraman Kalyanaraman,7,8 Sue Goo Rhee,9 Paul J. Thornalley,10 Linda Partridge,11 David Gems,11 Thomas Nystro¨ m,12 Vsevolod Belousov,13 Paul T. Schumacker,14 and Christine C. Winterbourn15 1MRC Mitochondrial Biology Unit, Hills Road, Cambridge CB2 0XY, UK 2Department of Medical Biophysics and Biochemistry, Karolinska Institutet, SE-171 77 Stockholm, Sweden 3Max Planck Institute for Biology of Ageing, Gleueler Straße 50a, 50931 Ko¨ ln, Germany 4National University of Singapore, University Hall, Lee Kong Chian Wing, UHL #05-02G, 21 Lower Kent Ridge Road, Singapore 119077, Republic of Singapore 5Department of Chemistry 6Howard Hughes Medical Institute University of California at Berkeley, Berkeley, CA 94720, USA 7Department of Biophysics 8Free Research Center Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA 9Division of Life and Pharmaceutical Sciences, Ewha Womans University, Science Building C, Room 103, 11-1 Daehyun-dong, Seodaemun-ku, Seoul 120-750, Korea 10Clinical Sciences Research Institute, University of Warwick, University Hospital, Coventry CV2 2DX, UK 11Department of Genetics, Evolution, and Environment, Institute of Healthy Ageing, University College London, Gower Street, London WC1E 6BT, UK 12Gothenburg University, 405 30 Go¨ teborg, Sweden 13Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997, Russia 14Northwestern University, Chicago, IL 60611, USA 15Department of Pathology, University of Otago, Christchurch 8011, New Zealand *Correspondence: [email protected] DOI 10.1016/j.cmet.2011.03.010

Reactive oxygen species are not only harmful agents that cause oxidative damage in pathologies, they also have important roles as regulatory agents in a range of biological phenomena. The relatively recent develop- ment of this more nuanced view presents a challenge to the biomedical research community on how best to assess the significance of reactive oxygen species and oxidative damage in biological systems. Consider- able progress is being made in addressing these issues, and here we survey some recent developments for those contemplating research in this area.

Introduction Consequently, results need to be assessed cautiously with a clear Reactive oxygen species (ROS), oxidative stress, and oxidative understanding of what the methods used do or do not measure. damage are increasingly assigned important roles in biomedical The multiple facets of this problem pose a challenge to those science as deleterious factors in pathologies and aging. There is studying the chemical and biophysical sides of ROS to explain also the growing recognition that many ROS are in addition better what is possible and what is not, and to develop and publi- important mediators in a range of biological processes such as cize more effective tools for investigating the impact of different signaling. However, this greater interest in ROS raises the con- ROS, particularly in vivo. cern that too often a certain biological phenomenon is ascribed To discuss current understanding of ROS in biology, and to to ROS or oxidative damage based on inadequate rationales, explore how challenges in the field could be addressed, a group technical approaches, or understanding of what is chemically of leading researchers with biological and chemical perspectives plausible. This tendency is surprising as there is considerable on ROS was brought together for an interdisciplinary confer- knowledge available on the detailed chemistry of individual ence, ‘‘The Chemistry and Biology of Reactive Oxygen Species,’’ ROS and the oxidative reactions that can occur within biological funded by the Wenner-Gren Foundations and held in Stockholm systems. However, this knowledge is often seen as technically from September 8–11, 2010. A number of common threads specialized or inaccessible to those in other areas of biomedical emerged from the meeting that are of general interest to the science whose research, perhaps unexpectedly, leads them to biomedical research community. suspect a role for ROS in their work. Consequently, there are many examples of otherwise well-conducted studies of consid- Making the Chemistry Explicit erable general interest that contain superficial or flawed conclu- A major recurrent theme from those working on the chemistry of sions about the involvement of ROS in the process investigated. ROS and oxidative damage was the importance of under- A corollary is that technical approaches to measuring and block- standing the nature of the particular ROS under consideration ing the actions of ROS and oxidative damage within biological in a biological context (Winterbourn, 2008). In other words, systems are often difficult to interpret and prone to artifact. ‘‘ROS’’ is often used imprecisely in the biomedical literature

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as a monolithic term, as if all ROS molecules were the same. Of Box 1. A Checklist for Assessing a Role for ROS in Biological course, there are many situations in which the use of ROS as Processes a generic description is appropriate. However, in some cases it d What is the specific ROS responsible? can be unhelpful and misleading because biologically important d Is the proposed reaction chemically plausible? ROS encompass a diverse range of species, including super- d Is the ROS or present at the appropriate location at oxide, , nitric oxide, peroxynitrite, hypochlo- a sufficient concentration to carry out the proposed reaction? rous acid, singlet oxygen, and the hydroxyl radical. Each of these d Does altering the amount of the particular ROS or type of oxida- molecules is a distinct chemical entity with its own reaction tive modification thought responsible impact on the pathology or preferences, kinetics, rate and site of production, and degrada- the signal? tion and diffusion characteristics in biological systems. Conse- quently the biological impacts of ‘‘ROS’’ depend critically on the particular molecule(s) involved and on the microenvironment and physiological or pathological context in which it is being Determining whether a Particular ROS Plays a Role generated. To group all ROS together as a single entity is in a Biological Phenomenon imprecise and can lead to vague and untestable hypotheses. To overcome the shortcomings outlined above, we recommend Therefore, wherever possible, the particular species thought that researchers (and reviewers) routinely go through a checklist to be responsible for the phenomenon under study should be of a few simple, common sense principles before assigning specified. a proposed redox mode of action of ROS and A similar error is a tendency to treat all antioxidants as if they (Box 1). This has been suggested before (Gutteridge and Halli- were alike, when in fact each has its own chemical properties well, 2010; Halliwell and Gutteridge, 2007), and is, if you like, and selective reactivity with particular types of ROS, as well equivalent to ‘‘Koch’s postulates’’ for ROS and oxidative as distinctive distribution, metabolism, recycling, and other damage. It will not be possible to satisfy all these criteria in every potential effects within a biological context. For example, case, for example the direct measurements of particular ROS vitamin E and interact with different ROS in vivo in suggested in Box 1 may not be feasible in vivo. Even so, these quite different ways because of their very different chemistry criteria are still useful to assess whether the conclusion reached and in addition because the former is present within lipids while or the hypothesis proposed is in principle plausible. Without the latter is found in the aqueous phase. Furthermore, neither such a heuristic approach it is difficult to determine whether they nor most other small molecule antioxidants react with a change in concentration of a particular ROS or the effect of a hydrogen peroxide. Enzymatic antioxidants such as superoxide certain antioxidant is indicative of a role for a specific ROS in dismutase, peroxidase, catalase, and peroxiredox- a pathology or redox signal. Alternative interpretations should ins are selective for particular ROS, and some, such as super- be considered; for example, oxidative damage can accumulate oxide dismutase, act by converting one type of ROS (super- as a secondary consequence of other events (e.g., changes in oxide) into another (hydrogen peroxide) and by preventing repair rates, since oxidative damage and its repair occur contin- formation of a further potent oxidant (peroxynitrite). Thus an ually in aerobes), and many of the molecules used as antioxi- antioxidant may block the activity of one type of ROS but leave dants have other pharmacological effects. another form unscathed. Furthermore, localization and catalytic The above considerations can help identify shortcomings in activities of antioxidant enzymes may be regulated via post- ROS-related investigations, some of which occur regularly in translational modification (such as acetylation, phosphorylation, the biomedical literature. For example, there is a tendency to and cysteine oxidation) and interaction with other proteins. For automatically link mitochondrial dysfunction with increased example, the mechanisms by which peroxiredoxins remove production of superoxide or hydrogen peroxide. However, the peroxides and peroxynitrite require disulfide reduction by thio- experimental support for this link is weak as there are many redoxin. Moreover, mammalian thioredoxins are themselves examples of mouse models with severe respiratory chain regulated by reversible inactivation via two-disulfide formation dysfunction that do not have any major increase in hydrogen or nitrosylation involving structural cysteine residues (Hashemy peroxide or superoxide production or oxidative damage (Wang and Holmgren, 2008). Such regulation may provide a means of et al., 2001; Trifunovic et al., 2005; Kujoth et al., 2005). In addi- generating favorable gradients of hydrogen peroxide around the tion, studies of isolated mitochondria suggest that the link appropriate location at the right time for signaling in response between hydrogen peroxide or superoxide production and mito- to stimulation of cell surface receptors (Woo et al., 2010). In chondrial dysfunction is complex (Murphy, 2009). Finally, it addition, aquaporins and related channels may provide a mech- should be mentioned that the role for oxidative damage in aging anism to regulate local hydrogen peroxide fluxes (Miller et al., as originally proposed (Harman, 1956) is still actively debated. 2010). For example, experimental studies imply that moderately in- In summary, it would be helpful if researchers specified the creased mitochondrial oxidative damage is well tolerated and particular ROS and the reactions thought to be responsible for does not affect life span in the mouse (Jang and Remmen, 2009). given biological effects and, wherever possible and appropriate, Another area that can be problematic is interpreting the effects avoided vague usage of generic terms like ROS and antioxidant. of antioxidants. Consider the use of superoxide dismutase (SOD) Likewise, researchers should show that an antioxidant actually mimetics to test whether superoxide is responsible for a partic- reacts with the particular ROS under discussion and can, at least ular effect in a biological system. The rate constants for SOD in principle, lower the concentration of that ROS sufficiently mimetics are generally 100-fold lower than that of enzymatic in vivo to explain its action. SOD itself, which is often present at concentrations greater

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than 10 mM in vivo. Consequently, it is important to show that the significant ways without necessarily resulting in oxidative SOD mimetic increases tissue SOD activity significantly over damage accumulation. endogenous levels (e.g., Keaney et al., 2004) before concluding Validation of better biomarkers for the assessment of oxidative that its biological activity is due to depleting superoxide. Another damage in vivo, particularly in human samples, should be consideration is that it is most unlikely that the biological mode of a priority. A great deal of research effort is directed toward linking action of any antioxidant is through hydroxyl radical scavenging the detailed chemistry of oxidative damage to protein, lipid, and (Halliwell and Whiteman, 2004). This is because the hydroxyl DNA with the production of measurable biomarkers (Jacobson radical reacts at a diffusion limited rate with most biological et al., 2010; Portero-Otı´n et al., 2004). The current best available molecules, so no exogenous molecule could achieve the biomarker for lipid peroxidation seems to be isoprostanes. concentration required to compete with this process (Halliwell However, work (e.g., Xue et al., 2009) on the detailed measure- and Whiteman, 2004). A further point is the widespread use of ment of oxidative damage to proteins and nucleic acids by N-acetylcysteine (NAC) as a generic antioxidant in biological mass spectrometry suggests that many more biomarkers are situations (Atkuri et al., 2007). As a membrane-permeant likely to be developed and our understanding of the link between cysteine precursor, many of its effects may be due to increasing the levels of biomarkers such as protein carbonyls, 8-hydroxy- cellular thiol (e.g., GSH) levels, but often this is not examined and deoxyguanosine, F2-isoprostanes, and reactive aldehydes that many other effects may contribute (Zafarullah et al., 2003). For can be measured in biological fluids, and the reactions that example, an increase in the free thiol content within the cell, or lead to their accumulation may assist this. A corollary of this is perhaps just in the cell culture medium (Xu and Thornalley, that it is unlikely that a single biomarker will ever give a complete 2001; Mi, et al., 2010), may explain many of the observed effects. picture of oxidative damage in vivo as different types of damage However, direct scavenging of hydrogen peroxide by NAC is to lipids, the proteome or the genome will lead to distinct unlikely as the reaction between these species is very slow. In patterns of accumulation of biomarkers, while interventions addition, NAC might exert its effect by causing structural such as antioxidants will also affect the accumulation of changes in cell-surface proteins (Hayakawa et al., 2003), biomarkers differentially. Hence, whatever biomarkers are used because the extracellular domain of many cell surface receptors they are not necessarily a simple single marker of overall oxida- contains disulfide bridges, which can be reduced by NAC (Hay- tive damage but will each respond differently to alternative types akawa et al., 2003). Thus, the action of NAC may be more as of damage and interventions. a redox modulator, and it should not be described as an antiox- Research into new approaches in the technically difficult area idant unless there is specific evidence that it is acting in this way. of measuring the levels of specific ROS in biological systems is Similarly, many other compounds such as dietary polyphenolics gaining momentum. Often these experiments are done with act as antioxidants in simple in vitro systems, but it is often redox-sensitive probes that are very susceptible to artifactual unclear whether the compound is present at a sufficient concen- side reactions. For example, dichlorodihydrofluorescein tration in vivo to lower the levels of the relevant ROS. Conse- (DCFH) oxidation to the fluorescent dichlorofluorescein (DCF) quently, many ‘‘antioxidant’’ effects in vivo may be due to is widely employed and is often described as measuring ROS some other pharmacological interaction distinct from a decrease production. It is frequently inferred that this is synonymous in a particular ROS. Therefore, proposed lowering effects of with hydrogen peroxide production, but all too often evidence specified ROS by a particular antioxidant should, if possible, for this link is not made. Furthermore, there are many issues be confirmed by measuring levels of the particular ROS suppos- with respect to the specificity of this assay, as discussed in edly scavenged by the compound. more detail elsewhere (Wardman 2007). A major consideration is that DCFH does not react directly with hydrogen peroxide Measuring ROS Production and Oxidative Damage but requires a peroxidase or transition metal catalyst. The signal A major theme that emerged from the meeting is the urgent need also depends on the extent of uptake of DCFH, so there are for better approaches to measuring the different types of ROS many ways in which it can vary without any change in hydrogen and forms of oxidative damage that occur in biological systems, peroxide production (Wardman, 2007). In addition, DCFH is particularly in vivo. It is important to distinguish between the readily photosensitized and can also catalyze the production of measurement of particular ROS themselves and the assessment superoxide. Finally, the relationship between the intensity of of the damage that these ROS cause. Biomarkers of oxidative DCF fluorescence and the concentration of the ROS under damage such as protein carbonyls, lipid peroxidation products, consideration is often assumed to be linear, in that a doubling or breakdown products of damaged DNA are often used, partic- of fluorescence indicates a doubling of the ROS concentration, ularly in vivo, to infer production of ROS such as the hydroxyl however the relationship between DCF fluorescence and partic- radical. However, the link between the accumulation of a marker ular ROS levels is often nonlinear (Wang and Joseph, 1999), for oxidative damage and production of a particular ROS is perhaps due to changes in levels of transition metals that convert indirect, because a change in clearance can dramatically alter more H2O2 into hydroxyl radical or to the artifactual generation of the level of the marker with no change in production of a superoxide and hydrogen peroxide by this probe (Halliwell and given ROS. For example, protein carbonylation is elevated by Gutteridge, 2007; Wardman 2007). Caveats also apply to the mistranslation of proteins, even when the levels of ROS are interpretation of results obtained with other probes such as unaltered, due to an increased susceptibility of misfolded hydroethidine, its mitochondria-targeted derivative MitoSOX, proteins to oxidative attack (Dukan et al., 2000). Conversely, as and Amplex Red which also have side reactions and a range of hydrogen peroxide can act as a redox signal without causing associated artifacts (Zhao et al., 2005; Selivanov et al., 2008). significant oxidative damage, its levels can change in biologically There is no simple solution to the difficulty of measuring a

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particular ROS accurately within biological systems but a more to study the redox changes within organelles in specific cell cautious use of probes and an effort to corroborate the measure- types; for example, by using a tyrosine hydroxylase-promoter- ment using orthogonal techniques is needed. driven mitochondrial roGFP, it was possible to explore how In developing the next generation of probes for specific ROS, DJ-1 affects mitochondrial redox state within dopaminergic a few simple principles must be observed: the location of the neurons (Guzman et al., 2010). Finally, the midpoint potential of probe molecule within the cell or organism must be understood, the roGFP sensors can be manipulated by inserting an extra the chemistry and rates of the reaction of the probe with different amino acid between the cysteine residues in the sensor, making types of ROS must be clear so that the particular ROS respon- it more difficult to oxidize. This approach has already been sible for the response can be established, and the subsequent reported to be effective in assessing the redox state of the metabolism or decomposition of the oxidized probe must be endoplasmic reticulum, where the more oxidizing environment understood (Zhao et al., 2005; Winterbourn, 2008). In doing so, renders the original sensor ineffective because it remains almost the parallel assessment of the probes to correlate the end points fully oxidized under basal conditions (Meyer and Dick, 2010). of their reactions with the ROS of interest to the changes in However, as with all probes, there are limitations and caveats signals such as fluorescence and an assessment of the sensi- in using this class of protein probe. For example, for some of tivity of the probe to particular ROS, as has been done to char- them the effect of pH on their sensitivity in different cell compart- acterize the superoxide-selective hydroethidine probe (Zhao ments can complicate analysis and the dynamic range of their et al., 2005; Zielonka et al., 2008), will be essential. Using these response is generally lower than that of small molecule fluores- approaches to measure particular ROS in simple systems such cent probes (Meyer and Dick, 2010). as isolated enzymes or mitochondria helps us to better under- An alternative to the use of fluorescent proteins is to improve stand the basic processes involved. However, these findings the selectivity of small-molecule fluorescent probes. A good should be extrapolated to the in vivo situation with great caution, example of this process is the use of the selective reactivity of if at all, because often the factors determining the production of alkylboronates with hydrogen peroxide (Miller et al., 2007; Dick- a particular ROS in vivo are quite different from those in vitro inson et al., 2011) and their even faster reaction with peroxynitrite (Murphy, 2009). Therefore, there is a great need for methods to (Sikora et al., 2009; Zielonka, et al., 2010) to generate novel fluo- measure ROS in vivo, and considerable progress has been rescent products. This enables the construction of probes that made with probe development for live imaging, although some appear to be selective for these species and that avoid many of this is still in the chemical literature and has yet to be widely of the limitations of earlier ROS probes such as DCFH. These adopted by the biomedical community. probes can also be coupled to functionalities that direct them One increasingly important approach is through the engi- to particular parts of the cell, such as mitochondria, in order to neering of green fluorescent protein (GFP) and its variants to report on both the nature and location of intracellular production produce redox-sensitive fluorescent probes, such as roGFP of particular forms of ROS (Dickinson and Chang, 2008; Robin- (Hanson et al., 2004) and HyPER (Belousov et al., 2006). These son et al., 2006). proteins incorporate redox-sensitive cysteines into the b barrel One potential limitation to all optical methods for ROS and of GFP (Hanson et al., 2004) or integrate GFP and related deriv- redox measurements is that they will not be applicable to most atives into redox-regulated protein motifs, such as the hydrogen animal models in vivo and there is the further problem of the quan- peroxide sensor from OxyR (Belousov et al., 2006) or the yeast tification of the levels of a particular ROS. Therefore to comple- peroxiredoxin Orp1 (Gutscher et al., 2009). These procedures ment these approaches, nonoptical methods to detect ROS are generate probes that react selectively with a particular ROS, also required. Among those that can be used now to gain some such as hydrogen peroxide, and then undergo changes in fluo- insight into ROS production in vivo is measurement of changes rescence with a high degree of sensitivity. There are a number in the transcription of genes that are sensitive to the levels of of advantages of these fluorescent protein probes: the signal is superoxide or hydrogen peroxide (e.g., Landriscina et al., reversible, allowing its calibration in the cell thereby permitting 2009). While these approaches provide useful information on a comparison of basal levels of particular ROS or the status of changes in some ROS in vivo, they do not indicate the level of particular redox couples among different cell types, or in ROS present in vivo and a number of other approaches have response to various stimuli, something that is not possible with been used to provide this information. Among these has been probes such as DCFH (Meyer and Dick, 2010). Another important the use of spin traps in vivo that react selectively with free radicals advantage is that these proteins can be easily modified with to generate a relatively stable free radical that can then be targeting sequences so as to be expressed at particular intracel- detected by electron paramagnetic resonance or by mass spec- lular locations, such as the mitochondria or endoplasmic retic- trometry (Reis et al., 2009; Yue Qian et al., 2005). However, this ulum, and thereby report on localized redox changes within the approach can be limited by its low sensitivity and the metabolism cell (Enyedi et al., 2010). Finally, using cell-specific promoters, of the spin adduct. This general approach is being extended by these sensors can be introduced into the genome to generate methods under development that greatly enhance the sensitivity transgenic animals that demonstrate cell- and region-specific of detection with the ratiometric analysis of a probe that reacts expression of the sensor (Guzman et al., 2010). Such methods with particular ROS and its stable reaction product relative to their can be readily translated to in vivo models in transgenic animals deuterated internal standards by liquid chromatography and expressing these redox-sensitive fluorescent proteins, such as tandem mass spectrometry. The first application of this com- zebrafish or worms, or in conjunction with two photon confocal bined the alkylboronate chemistry discussed above with mito- microscopy techniques to probe deeper beneath the surface in chondrial targeting to quantify the levels of hydrogen peroxide nontransparent animals. These methods can also be combined within mitochondria inside living fruit flies (Cocheme´ et al. 2011).

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Consequently, there is cautious optimism that ‘‘next generation’’- Dickinson, B.C., and Chang, C.J. (2008). J. Am. Chem. Soc. 130, 9638–9639. specific ROS probes will replace the widely used, but prob- Dickinson, B.C., Peltier, J., Stone, D., Schaffer, D.V., and Chang, C.J. (2011). lematic, current generation of reagents and facilitate the Nat. Chem. Biol. 7, 106–112. measurement of particular ROS selectively in vivo. Dukan, S., Farewell, A., Ballesteros, M., Taddei, F., Radman, M., and Nystro¨ m, T. (2000). Proc. Natl. Acad. Sci. USA 97, 5746–5749. Conclusions and Future Perspectives 13 The difficulty of measuring oxidative damage and the levels of Enyedi, B., Va´ rnai, P., and Geiszt, M. (2010). Antioxid. Redox Signal. , 721–729. particular ROS in vivo, together with the uncertainty of interpre- tation, mean that much is still unknown about the roles of ROS Gems, D., and Doonan, R. (2009). Cell Cycle 8, 1681–1687. and oxidative damage in many fundamental biological pro- Gutscher, M., Sobotta, M.C., Wabnitz, G.H., Ballikaya, S., Meyer, A.J., cesses. For example, despite extensive investigation, most trials Samstag, Y., and Dick, T.P. (2009). J. Biol. Chem. 284, 31532–31540. of antioxidants in humans have proven disappointing (Bjelakovic Gutteridge, J.M., and Halliwell, B. (2010). Biochem. Biophys. Res. Commun. et al., 2008). However, without accompanying biomarker and 393, 561–564. ROS measurements, most of these studies are inconclusive. We cannot be certain that the lack of efficacy was due to the Guzman, J.N., Sanchez-Padilla, J., Wokosin, D., Kondapalli, J., Ilijic, E., Schumacker, P.T., and Surmeier, D.J. (2010). Nature 468, 696–700. antioxidants failing to affect the oxidative damage involved in the pathology (Gutteridge and Halliwell, 2010). Alternatively, Halliwell, B., and Gutteridge, J.M.C. (2007). Free Radicals in Biology and Medicine, Fourth Edition (Oxford: Oxford University Press). the compounds may have lowered or abolished oxidative damage but still had no impact on outcome, implying that oxida- Halliwell, B., and Whiteman, M. (2004). Br. J. Pharmacol. 142, 231–255. tive damage merely correlates with the pathology and is not Hanson, G.T., Aggeler, R., Oglesbee, D., Cannon, M., Capaldi, R.A., Tsien, causative (Cocheme´ and Murphy, 2010). R.Y., and Remington, S.J. (2004). J. Biol. Chem. 279, 13044–13053. Looking forward, this meeting of researchers in the chemistry 11 and biology of ROS revealed that two significant challenges must Harman, D. (1956). J. Gerontol. , 298–300. be met to help progress our understanding of ROS and oxidative Hashemy, S.I., and Holmgren, A. (2008). J. Biol. Chem. 283, 21890–21898. damage in living systems. The first challenge is to transmit to Hayakawa, M., Miyashita, H., Sakamoto, I., Kitagawa, M., Tanaka, H., Yasuda, a wider audience the knowledge we have already assembled H., Karin, M., and Kikugawa, K. (2003). EMBO J. 22, 3356–3366. so that it can be used to formulate more precise and testable hypotheses about the role of specific ROS and antioxidants in Jacobson, J., Lambert, A.J., Portero-Otı´n, M., Pamplona, R., Magwere, T., Miwa, S., Driege, Y., Brand, M.D., and Partridge, L. (2010). Aging Cell 9, the laboratory and clinic. The second challenge is to develop 466–477. better approaches for detecting and quantifying different types 44 of ROS and markers of a range of forms of oxidative damage Jang, Y.C., and Remmen, V.H. (2009). Exp. Gerontol. , 256–260. in biological systems, particularly in vivo. There was cautious Keaney, M., Matthijssens, F., Sharpe, M., Vanfleteren, J., and Gems, D. (2004). optimism among the group that a more rigorous approach would Free Radic. Biol. Med. 37, 239–250. enable faster progress in the field. Although the results may ulti- Kujoth, G.C., Hiona, A., Pugh, T.D., Someya, S., Panzer, K., Wohlgemuth, S.E., mately show that changes in ROS and oxidative damage in some Hofer, T., Seo, A.Y., Sullivan, R., Jobling, W.A., et al. (2005). Science 309, situations are merely epiphenomena, possibly (for example) 481–484. during aging (Gems and Doonan, 2009), it is also likely that it Landriscina, M., Maddalena, F., Laudiero, G., and Esposito, F. (2009). Antioxid. will reveal unexpected new roles for ROS at the heart of other Redox Signal. 11, 2701–2716. major biological phenomena both as damaging agents and as Meyer, A.J., and Dick, T.P. (2010). Antioxid. Redox Signal. 13, 621–650. important mediators of redox signaling and other cellular processes. Mi, L., Sirajuddin, P., Gan, N., and Wang, X. (2010). Anal. Biochem. 405, 269–271.

ACKNOWLEDGMENTS Miller, E.W., Tulyathan, O., Isacoff, E.Y., and Chang, C.J. (2007). Nat. Chem. Biol. 3, 263–267. We are grateful to the Wenner-Gren Foundations for supporting the meeting Miller, E.W., Dickinson, B.C., and Chang, C.J. (2010). Proc. Natl. Acad. Sci. that was the genesis of this Perspective. USA 107, 15681–15686.

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