[doi 10.1016_j.cbi.2014.10.016] v. i. lushchak -- free radicals, reactive oxygen species, oxidative...

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Chemico-Biological Interactions xxx (2014) xxx–xxx

CBI 7166 No. of Pages 12, Model 5G

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Chemico-Biological Interactions

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Free radicals, reactive oxygen species, oxidative stress and itsclassification

http://dx.doi.org/10.1016/j.cbi.2014.10.0160009-2797/� 2014 Elsevier Ireland Ltd. All rights reserved.

Abbreviations: BOS, basal oxidative stress; ETC, electron transport chain; HOS, high intensity oxidative stress; 8-OHG, 8-hydroxyguanine; G6PDH, glucose-6-phdehydrogenase; GPx, glutathione-dependent peroxidase; GR, glutathione reductase; GSH, glutathione reduced; GSNO, S-nitrosoglutathione; GSSG, glutathione oxidizNADP+-isocitrate dehydrogenase; IOS, intermediate intensity oxidative stress; LOOH, lipid peroxides; LOS, low intensity oxidative stress; MDA, malonic dialdehydeME, NADP-malic enzyme; NOE, no observable effect point; �NO, nitric oxide radical; 6PGDH, 6-phosphogluconate dehydrogenase; 8-oxodG, 8-oxo-7,8-dihdeoxyguanosine; 8-oxoGua, 8-oxo-7,8-dihydroguanine; PPP, pentose phosphate pathway; RNS, reactive nitrogen species; ROS, reactive oxygen species; RS, reactiveO2��, superoxide anion radical; SOD, superoxide dismutase; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reactive substances; TRR, thioredoxin glutathione re⇑ Tel./fax: +380 342 714683.

E-mail address: [email protected]

Please cite this article in press as: V.I. Lushchak, Free radicals, reactive oxygen species, oxidative stress and its classification, Chemico-BioInteractions (2014), http://dx.doi.org/10.1016/j.cbi.2014.10.016

Volodymyr I. Lushchak ⇑Department of Biochemistry and Biotechnology, Precarpathian National University named after Vassyl Stefanyk, 57 Shevchenko Str., Ivano-Frankivsk 76025, Ukraine

a r t i c l e i n f o

282930313233343536373839

Article history:Received 15 June 2014Received in revised form 13 October 2014Accepted 17 October 2014Available online xxxx

Keywords:Reactive oxygen speciesHomeostasisCell functionOxidative stressClassification

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a b s t r a c t

Reactive oxygen species (ROS) initially considered as only damaging agents in living organisms furtherwere found to play positive roles also. This paper describes ROS homeostasis, principles of their investi-gation and technical approaches to investigate ROS-related processes. Especial attention is paid to com-plications related to experimental documentation of these processes, their diversity, spatiotemporaldistribution, relationships with physiological state of the organisms. Imbalance between ROS generationand elimination in favor of the first with certain consequences for cell physiology has been called ‘‘oxi-dative stress’’. Although almost 60 years passed since the first definition of oxidative stress was intro-duced by Helmut Sies, to date we have no accepted classification of oxidative stress. In order to fill upthis gape here classification of oxidative stress based on its intensity is proposed. Due to that oxidativestress may be classified as basal oxidative stress (BOS), low intensity oxidative stress (LOS), intermediateintensity oxidative stress (IOS), and high intensity oxidative stress (HOS). Another classification of poten-tial interest may differentiate three categories such as mild oxidative stress (MOS), temperate oxidativestress (TOS), and finally severe (strong) oxidative stress (SOS). Perspective directions of investigations inthe field include development of sophisticated classification of oxidative stresses, accurate identificationof cellular ROS targets and their arranged responses to ROS influence, real in situ functions and operationof so-called ‘‘antioxidants’’, intracellular spatiotemporal distribution and effects of ROS, deciphering ofmolecular mechanisms responsible for cellular response to ROS attacks, and ROS involvement in realiza-tion of normal cellular functions in cellular homeostasis.

� 2014 Elsevier Ireland Ltd. All rights reserved.

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Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002. Free radicals and reactive oxygen species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003. Generation, conversion, and elimination of ROS in living systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004. Oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 005. Regulation of antioxidant systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 006. Intensity-based classification of oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 007. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Conflict of Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

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osphateed; IDH,; NADP-ydro-20-species;ductase.

logical

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Fig. 1. Reduction of molecular oxygen via four- and one-electron schemes.Description in the text.

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1. Introduction

Free radicals were first described by Moses Gomberg more thana century ago [1]. For a long time they were not considered to pres-ent in biological systems due to high reactivity and consequentlyshort living time. More than 30 years later, Leonor Michaelis [2]proposed that all oxidation reactions involving organic moleculeswould be mediated by free radicals. Although that statement gen-erally was wrong, it stimulated interest to the role of free radicalsin biological processes. In 1950th, free radicals were found in bio-logical systems [3] and immediately supposed to be involved indiverse pathological processes [4] and aging [5,6]. Since that timeour knowledge on involvement of free radicals in living processesextended enormously. Clearly, long time and actually up to nowthey have been mainly suggested to play deleterious roles consid-ering mainly as damaging species. This point of view was strength-ened by discovery of McCord and Fridovich [7] who described thefirst protective enzyme against free radicals called superoxide dis-mutase. In 1970–1990th understanding of free radicals as onlydeleterious species for biological systems was substantially chal-lenged by several important discoveries. The first, free radicalswere found to be responsible for combating of infection agentsby immune system [8–11]. The second, in 1980th vascular endo-thelial cells were found to produce nitric oxide from L-arginine,and this accounted for the biological activities attributed to endo-thelium-derived relaxing factor (EDRF) [12–15]. That discoveryopened the second avenue for free radical research, their signalingfunction, initially for nitric oxide and further for other reactive spe-cies [16–19]. Finally, the level of free radicals was found to beregulated by hormones like insulin [20], and they were suggestedto be regulators of core metabolic pathways [21]. Therefore, it isabsolutely clear now that free radicals are active participants indiverse processes and they cannot be considered anymore as onlydamaging agents, but real players in many normal functions of liv-ing organisms.

The field of free radicals or more common reactive species (RS)research in biological systems is one of the most dynamic, but alsois among most complicated due to many reasons [22]. Main ofthem are: (i) low stability and high reactivity resulting in lowsteady-state concentrations; (ii) high diversity of reactions theycan participate in; (iii) complicated spatiotemporal distributionin cell space and extracellularly; (iv) dependence on physiologicalstate of the organism, and (v) absence of technical tools for reliableevaluation of their absolute and even relative levels. Because ofpresence of many complications in free radical studies and exten-sive involvement of new researchers from different fields in thisarea and due to obvious universality and importance of RS-relatedprocesses, this paper aims to clarify and highlight some basic termsused, processes they are involved in, describe the concept of oxida-tive stress and give general classifications of this sort of the stress.

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2. Free radicals and reactive oxygen species

In the most cases, the terms ‘‘free radicals’’ and ‘‘reactive oxy-gen species (ROS)’’ are used interchangeably. If in many cases thatis correct, in some ones it is wrong. Therefore, to clarify the issue Iwill provide below the information how to discriminate betweenthese two terms in order to provide understanding based on mod-ern knowledge in the field. Probably, analysis of ROS generation,interconversion and elimination is the easiest way to do that. Theseprocesses are schematically presented in Fig. 1. In living organismsunder aerobic conditions more than 90% of oxygen consumed isreduced directly to water by cytochrome oxidase in electron-transport chain (ETC) via four-electron mechanisms without ROSrelease [23,24]. In eukaryotes, the system is represented by ETC,

Please cite this article in press as: V.I. Lushchak, Free radicals, reactive oxyInteractions (2014), http://dx.doi.org/10.1016/j.cbi.2014.10.016

located in internal mitochondrial membrane, whereas in prokary-otes ETC components are located in plasmatic membrane. Opera-tion of ETC is coupled with oxidative phosphorylation to produceenergy in ATP form. Much less than 10% of oxygen consumed isreduced via one-electron successive pathways resulting in conver-sion of molecular oxygen to superoxide anion radical (O2

��) fol-lowed by one-electron reduction with concomitant accepting oftwo protons to yield hydrogen peroxide (H2O2) (Fig. 1). This com-pound is not a free radical, but is chemically more active thanmolecular oxygen due to which is included in the ROS group.Hydrogen peroxide molecule accepting one more electron is splitup to hydroxyl radical (HO�) and hydroxyl anion (OH�). Finally,HO� interacts with one more electron and proton resulting in for-mation of water molecule. In biological systems, this reaction ismainly realized through abstraction of hydrogen atom from differ-ent compounds such as proteins and lipids resulting frequently ininitiation of chain processes. Summarizing described above, O2

��,H2O2, and HO� collectively are called reactive oxygen species, butonly O2

�� and HO� are free radicals, whereas H2O2 is not. Reactiveoxygen species include not only mentioned above O2

��, H2O2, andHO�, but also diverse peroxides, like lipid peroxides, and peroxidesof proteins, and nucleic acids. Moreover, their homeostasis is clo-sely related to many other reactive species (RS), such as reactivecarbonyl species (glyoxal, methylgyoxal) [25]. There are also manyother RS of nitrogen (nitric oxide, peroxynitrite, and related com-pounds) [26–28], carbon [27,29,30], sulfur [31], halogens [32],etc. However, in this short review I will focus only on ROS.

3. Generation, conversion, and elimination of ROS in livingsystems

After ROS discovery in living organisms, the interest ofresearchers was focused on identification of mechanisms of theirgeneration. To date, many of them have been disclosed. It has beenestablished that in eukaryotic cells over 90% of ROS are producedby mitochondria [24]. Probably of that amount dependently onspecific situation most ROS are generated via escaping of electronsfrom mitochondrial ETC mainly from coenzyme Q to molecularoxygen. The electrons escaping ETC interact with molecular oxygento give O2

��. Further it spontaneously or enzymatically is convertedto H2O2 and HO�. The amount of electrons escaping ETC variesbroadly and depends on physiological state of organisms. Totalamount of ROS produced by mitochondria seems is poorly con-trolled by the cell at this stage and to maintain low ROS steady-state level cells possess finely regulated antioxidant systems. Inaddition, minor amounts of ROS are produced by ETC located in/at endoplasmic reticulum (ER), plasmatic, and nuclear membranesas well as some oxidases. Production of ROS in ER is mainly con-nected with operation of hydroxylation system represented bycytochrome P450 family enzymes. At oxidation of substrates, someportion of electrons escape ETC and similarly to mitochondria,joins O2 giving rise to O2

�� and products of its transformation. Theproducts formed during hydroxylation reaction also may

gen species, oxidative stress and its classification, Chemico-Biological


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themselves become ROS generators because they may enter autox-idation cycles.

Diverse oxidases are also rather powerful ROS producers. Theyoxidize carbohydrates, aldehydes, amino acids, heterocyclic com-pounds and others. The physiological role of xanthine oxidase intotal ROS balance, at least in animals, received substantial atten-tion. It is believed that under hypoxic conditions this enzymemay be the main ROS producer [33,34]. However, NADPH oxidasecomplex (Nox) is the best studied oxidase generating ROS in deli-cately controlled manner. The system was discovered in late1970th [35,36] and was found to be responsible for inactivationof bacteria and other inviders by immune system cells. Later Noxwas found also in nonimmune cells and was not necessary relatedto defense system where produces ROS in finely controlled mannerfor realization of specific functions in spatiotemporal manner[37,38].

There is also one more ROS source related to autoxidation of dif-ferent small molecules of endo- and exogenous origin. Epinephrine(adrenalin) and norepinephrine provide a good example of endog-enous molecules subjected to autoxidation coupled with ROS pro-duction [39,40], whereas many xenobiotics, especially differenthomo- and heterocyclic compounds, are clearly related to this sortof ROS generators [41,42].

The control of ROS steady-state level is provided not only viatheir production, but also via elimination. Living organisms possessmultilevel and complicated antioxidant system operating either toeliminate ROS, or minimize their negative effects. There are severalapproaches to classify these systems and here we will use themostly appreciated one based on molecular masses. According tothis system, antioxidants are placed in two groups: low molecularmass antioxidants (usually with molecular masses below one kilo-dalton) and high molecular mass antioxidants (with molecularmass higher than one or actually higher than ten kilodaltons).The group of low molecular mass antioxidants includes chemicallydifferent compounds usually well known to readers such as vita-mins C (ascorbic acid) and E (tocopherol), carotenoids, anthocya-nins, polyphenols, and uric acid. Most of them are received byhuman organism as food or supplement components. However,one very important antioxidant glutathione (tripeptide c-glutam-yl-cysteinyl-glycine, GSH) is synthesized by most living organismsand used to control ROS level either via direct interaction withthem, or serving as a cofactor for ROS-detoxifying enzymes [43].Glutathione level is finely adjusted by organisms to specific condi-tions via several regulatory pathways. Interestingly, GSH as otherthiols may interact with nitric oxide (�NO) to neutralize it and atthe same time providing additional regulatory mechanism forROS-related processes like s-nitrosylation [43–48]. This pathwaynot only decreases �NO level, but also creates buffer for this gaseoussignal transmitter and provides its transportation on relativelylong distances and protects thiol groups from irreversible oxida-tion during oxidative boots [43]. Although frequently notmentioned, antioxidant role of melatonin (N-acetyl-5-methoxy-tryptamine) should be highlighted here also [49–51]. Certainly,its health benefits like optimization of blood pressure [52], poten-tial beneficial effects on retinopathy in diabetic rats [53] and othereffects attracted attention and called for disclosing molecularmechanisms involved. Probably due to antioxidant activity melato-nin can improve glucose metabolism via correction of insulin pro-duction protecting pancreatic b-cells against ROS-induced damage[54]. Interestingly, last years a capability of melatonin to regulateexpression of certain genes via specific regulatory pathways wasdisclosed. For example, Kim and colleagues [55] found that melato-nin and its metabolite N1-acetyl-N2-formyl-5-methoxykynur-amine (AFMK) stimulated differentiation of human epidermis,and Sayyed with colleagues [56] disclosed melatonin protectiveeffects against inflammatory processes via inhibition of NF-kB


factor. It is absolutely clear, that all antioxidant defense systemsoperate in concert to provide optimal ROS level.

Last decades, most attention of ROS researches was concen-trated on investigation of operation of high molecular mass antiox-idants, primary antioxidant and associated enzymes. Description ofsuperoxide dismutase (SOD, EC 1.15.1.1) [7] was the first revolu-tionizing discovery in this field. The enzyme catalyses the reaction:

O��2 þ O��2 þ 2Hþ�!O2 þH2O2 ð1Þ

Formed H2O2 is further either dismutated by catalase (EC1.11.1.6):

2H2O2�!2H2Oþ O2 ð2Þ

or reduced by different peroxidases like glutathione-dependent per-oxidases (GPx, EC 1.11.1.9) which in addition to H2O2 (3) reducelipid peroxides (4):

H2O2 þ 2GSH�! H2Oþ GSSG ð3Þ

LOOHþ 2GSH�!LOHþ GSSG ð4Þ

Many other substrates can be also used for H2O2 reduction, likeguaiacol.

Certainly, if GPx are used to detoxify H2O2, the pool of GSHshould be maintained and for that GSSG reduction is provided byglutathione reductase (GR, EC 1.6.4.2):

GSSGþ NADPH�!2GSHþ NADPþ ð5Þ

In some organisms, such as Drosophila, thioredoxin glutathionereductase (Dm TrxR-1, or TRR, EC a1.8.1.B1) carries out GR functionfor the replenishment of GSH.

The pool of NADPH is maintained via reduction of NADP+ inseveral reactions catalyzed by the enzymes of pentose phosphatepathway glucose-6-phosphate dehydrogenase (G6PDH, EC1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH, EC1.1.1.43):

NADPþ þ glucose-6-phosphate�!NADPH

þ 6-phosphoglucolactone ð6Þ

NADPþ þ 6-phosphogluconate�!NADPH

þ ribuloso-5-phosphateþ CO2 ð7Þ

In some tissues, particularly, in brain, malate dehydrogenase(oxaloacetate-decarboxylating NADP+) (EC 1.1.1.40) or NADP-malicenzyme (NADP-ME) produces substantial NADPH amounts in thereaction:

ðSÞ-malateþ NADPþ ��!pyruvateþ CO2 þ NADPH ð8Þ

Finally, NADP+-dependent isocitrate dehydrogenase (IDH,threo-DS-isocitrate: NADP(+) oxidoreductase (decarboxylating, EC1.1.1.42) may serve as a supplier of some NADPH amounts:

Isocitrateþ NADPþ�!2-oxoglutarateþ CO2 þ NADPH ð9Þ

The described above high molecular mass systems of ROS elim-ination operate with O2

�� and H2O2, but not with HO�. Actually,there is no known enzymatic system to eliminate HO� formed,which is probably related with its high reactivity. Prevention ofhydroxyl radical production is the best way to protect living organ-isms from its deleterious effects. And seems living organisms havesolved this problem very successfully and enzymatic antioxidantsystem efficiently prevents HO� formation complementing in thisway by low molecular mass antioxidant system. However, smallamounts of ROS escape antioxidant defense and can be convertedto HO� and some portion of this the most chemically active ROSmay be scavenged by low molecular mass antioxidants like



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31 October 2014

ascorbate, tocopherol, GSH, etc. Tiny amounts of HO� and other ROSescaping protective systems cause damage to cellular componentswhich can be registered as signatures of ROS presence in livingorganisms.

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4. Oxidative stress

Above the generation and elimination of ROS were describedand it is clear that two processes are closely related. Living organ-isms possess finely regulated systems to maintain very low ROSlevels, i.e. their production and elimination are well balancedresulting in certain steady-state ROS level. However, under certaincircumstances this balance can be disturbed. There are several rea-sons for that: (i) increased level of endogenous and exogenouscompounds entering autoxidation coupled with ROS production;(ii) depletion of reserves of low molecular mass antioxidants; (iii)inactivation of antioxidant enzymes; (iv) decrease in productionof antioxidant enzymes and low molecular mass antioxidants;and, finally, (v) certain combinations of two or more of the listedabove factors. Certainly, increase in steady-state ROS level, whichresults from imbalance between generation and elimination pro-cesses, can affect many, if not all living processes. The conse-quences of this increase differ and depend on the level and placeof ROS generation, efficiency of antioxidant systems, availabilityof plastic and energetic resources, and cellular targets they interactwith. These processes are presented in Fig. 2 where I also demon-strate induction of oxidative stress the first definition of which wasintroduced by Helmut Sies [57,58]. Under normal conditions, ROSlevel fluctuates in certain range defined by concerted operationof systems of their generation and elimination. Due to some rea-sons, such as introduction of certain oxidants, ROS level may shar-ply increase and leave the range of control (rest) conditions. Ifantioxidant systems are capable adequately cope with enhancedROS amounts, this level would return into initial corridor. Theseevents may be called ‘‘acute oxidative stress’’. It should be under-lined here, that it is not enough to have enhanced ROS level for cer-tain period of time to develop oxidative stress. It should have somemore or less specific physiological consequences. Probably, thebest studied example here is provided by enhanced expression ofantioxidant and related enzymes like SOD, catalase, glutathionereductase, etc. [59–62]. In some cases, the cell cannot neutralizeenhanced ROS amounts and return ROS level into initial corridor.Even enhanced expression of antioxidant and related enzymeswould not be able to do that. Therefore, the ROS level may be

Fig. 2. Dynamic of ROS level under control and stressful conditions in biologicalsystems. Steady-state levels of reactive oxygen species fluctuate over a certainrange under normal conditions. However, under stress ROS levels may increase ordecrease beyond the normal range resulting in acute or chronic oxidative orreductive stress. Under some conditions, ROS levels may not return to their initialrange and stabilize at a new quasistationary level. Modified from [43].


slightly enhanced or initial corridor may be extended. Due to thatincreased ROS level can be stabilized and enhance modification ofdifferent cellular components, substantially disturbing homeosta-sis. This state can be called ‘‘chronic oxidative stress’’. Finally,one more scenario may take place after oxidative boots, or due tochange in physiological state of organisms – ROS level may notreturn into initial corridor and stabilize at new, so-called ‘‘quasi-stationary level’’ [61,62]. This state needs substantial reorganiza-tion of whole homeostasis, including ROS one. Several pathologiessuch as cancer [63,47], diabetes mellitus [64,65], cardiovascular[66,67] and neurodegenerative [68–70] diseases clearly exemplifythe chronic oxidative stress. The situations must be carefully inves-tigated in order to understand if oxidative stress causes the dis-eases or opposite. At least in many cases they are registeredsimultaneously and frequently it is not clear if that is coincidenceor not. Also these pathological states may be analyzed from pointsof view of chronic oxidative stress and quasi-stationary ROS levels.

Currently the stress is defined as ‘‘Oxidative stress is a situationwhen steady-state ROS concentration is transiently or chronicallyenhanced, disturbing cellular metabolism and its regulation anddamaging cellular constituents’’ [62]. This definition includes allmentioned above features and underlines dynamics of the pro-cesses involved which is reflected as steady-state (stationary)ROS levels.

Oxidative stress may be induced not only by externally addedoxidants and compounds either stimulating ROS production orweakening antioxidant defense. Although if it could be expectedthat increase of external level of oxygen causes oxidative stresswith potential tissue injury [71,72], and ischemia/reperfusionmight affect biological systems similarly [73–76], hypoxia-inducedoxidative stress was somewhat unexpected but well experimen-tally supported to date [74,77–79].

In conclusion, after oxidative boots, organisms can be subjectedto acute or chronic oxidative stress, or reach new quasi-stationarystate. In many cases, development of such events depends on thepossibility of living organisms to adjust their defense systems toenhanced ROS levels. These systems usually operate at the levelof expression of specific genes encoding antioxidant and associatedenzymes, or enzymes responsible for production of antioxidants[80]. Therefore, in the next section we will briefly provide theinformation on described to date systems of ROS sensing, transduc-tion of signals to certain targets and operation of adaptive machin-ery to cope with ROS excess.

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5. Regulation of antioxidant systems

Under oxidative stress, if it is mild or intermediate, organismsusually block general programs of their life cycle such as reproduc-tion or extensive biosynthesis, to develop responses to prevent orneutralize negative ROS effects. However, under these conditionssome mechanisms providing surviving under oxidative stress areactivated as mentioned above. These mechanisms are mainlybased on up-regulation of antioxidant and related enzymes. Con-trol principles of realization of this reprogramming include ROSsensing, transduction of signals through specific pathways andup-regulation of target genes to enhance level of their products.To the end, up-regulation of antioxidant systems increases theircapability to eliminate ROS creating in this way autoregulated neg-ative feedback control loop. Further I will briefly describe wellestablished to date systems realizing this processes. Interestingly,in bacteria functions of sensing and signal transduction may beprovided by the same proteins.

The first systems assisting organisms to adapt to oxidativestress were described in bacteria. To beginning of 1990th, opera-tion of two such systems was deciphered. They were OxyR and



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SoxRS ones [59,62,81,82]. For the first time the system OxyRregulating expression of genes consisting OxyR regulon wasdescribed in Salmonella typhimurium [17,18,83] and further detailswere disclosed with Escherichia coli [18,84,85]. The system OxyRregulates expression of genes consisting OxyR regulon in responseto bacteria exposure to H2O2. In this case, sensor protein OxyR notonly senses oxidative signal, but also up-regulates target genesproducts many of which have clearly protective properties likeE. coli peroxidase HPI [59,81,85]. This type of response was calledone cycle response in order to discriminate from so-called twocycle response found in SoxRS system consisting soxRS regulon.In the latter case, the protein SoxR senses O2

��, further up-regulatesSoxR gene expression, and finally synthesized SoxR protein up-regulates the expression of target genes, many of which encodeantioxidant enzymes like Mn-SOD. SoxRS system is also responsi-ble for the bacterial adaptive response to nitric oxide, peroxynitriteand other oxidants [59,61,81]. It is clearly that regulation of soxRSregulon needs concerted operation of two regulatory proteins SoxRand SoxS which act together to enhance cellular potential to com-bat or minimize negative ROS effects on the cell. Finally, at thebeginning of 2000th the interplay between two regulons, oxyRand soxRS was described, when H2O2 was found, at least partially,up-regulate expression of genes of SoxRS regulon [82,86–89].Interestingly, OxyR protein, a key player of OxyR regulon responsi-ble for bacterial response to exposure to hydrogen peroxide, oper-ates owing to reversible oxidation of its cysteine residues[59,61,81]. SoxR protein sences O2

�� and other oxidants owing toreversible oxidation of [4Fe-4S] clusters [59,61,81].

Eukaryotes possess more complicated than prokaryotes systemof adaptive response to ROS exposure. Usually, they have severalcomponents. Sensors are located in the cytosol and after oxidationeither themselves or modified by them components migrate intonucleus where they up-regulate expression of certain target genes.Each group of eukaryotes possesses several more or less specificsystems providing directed and finely regulated adaptive responseto ROS treatment. Therefore, here I briefly will describe these sys-tems for three different eukaryotic kingdoms, but interested read-ers are addressed for details to the previous our review paper [61]or other publications.

Fungi, particularly budding bakery’s yeast Saccharomyces cerevi-siae, were the first eukaryotes where these mechanisms were deci-phered. Reversible oxidation of cysteine residues in this case playsa pivotal role. In Yap1-based signaling of S. cerevisiae, oxidativeboots are sensed by specific glutathione peroxidase GPx3 [82,90].Oxidation of cysteine residues of this peroxidase initiates cascadeof oxidative signals, and protein Yap1 in oxidized form is tran-siently accumulated in the nucleus. Here it binds to regulatoryregions of target genes products of many of them are responsiblefor increase of yeast antioxidant potential. Similar systems weredescribed in other yeasts (Candida albicans, Schizosaccharomycespombe) and filamentous fungi [90,91]. Recently we found that inS. cerevisiae Yap1-based signaling also played pivotal role in adap-tive response to nitric oxide [61,92]. Although Yap1-based systemseems play a central role in yeast defense against ROS, it operatesin concert with other systems like Msn2/4p and MAP kinases.However if Yap1-based system mainly regulates rather specificresponse to enhance antioxidant potential, both Msn2/4 and MAPkinase systems provide adjustment of other cellular functions tooxidative stress, including reprogramming of life cycle, core cellu-lar pathways, etc.

Although we do not know a lot about regulation of adaptiveresponse to ROS exposure in plants, at least two such systemsdemonstrate that they also exploit cysteine-centered sensinginvolving reversible oxidation. They have been called NPR1/TGAand Rap2.4a pathways [61]. In both cases, sensor proteins are cyto-plasmic residents which after oxidation are translocated into the


nucleus where up-regulate expression of target genes. Interest-ingly, plants also adopted ROS as signaling molecules in hormonalsignaling. At least it is believed that realization of hormonal effectsof jasmonic acid and ethylene includes H2O2 as intermediate[61,93–96].

Finally, animals possess plural systems responsible for adapta-tive response to oxidative stress. They form some cascadesresponding to oxidative stress of different intensity. Probably, thelowest (mild) intensity oxidative stress is sensed by Nrf2/Keap1system (Fig. 3) which is known to be activated by minute amountsof ROS. NF-E2-related factor 2 (Nrf2) is a transcription factor of theleucine zipper family which operates in concert with Kelch-likeECH-associated Protein 1 (Keap1). These two proteins cooperateto regulate cellular response to oxidants and electrophilic xenobi-otics as well as dithiolethiones, isothiocyanates, and triterpenoids[61,97,98]. Under normal conditions, Nrf2 protein interacts withKeap1, a substrate adapter protein for the E3 ubiquitin ligase com-plex formed by CUL3 and RBX1. Ubiquitination targets Nrf2 fordegradation by the proteasome, thus resulting in the suppressionof its transcriptional activity. In response to increased ROS levelsthiol groups of protein Keap1 are oxidized making impossible itsinteraction with Nrf2. Due to that the latter is moved into nucleuswhere it is transiently accumulated and interacts with so-calledantioxidant response element (ARE) in promoters of target genesencoding defense proteins/enzymes. The up-regulated genesinclude ones which encode either antioxidant enzymes (SOD, cat-alase, peroxidases, GST-transferases), or enzymes involved inbiosynthesis of antioxidants like gamma-glutamylcysteinesynthetase, a key enzyme of glutathione biosynthesis, productionof reducing equivalents, proteasome function and other [43,61].Therefore this Keap1/Nrf2 pathway is suggested to be a key playerin coordinating of adaptive response of the cell to the previouslymentioned compounds.

Animal responses to intermediate intensity oxidative stress arecoordinated by at least three regulatory systems called NF-kB, AP-1(animal homologue of yeast YAP1), and MAP kinases. These sys-tems also up-regulate expression of antioxidant enzymes, but inaddition also stimulate expression of certain genes of inflammationand reprogramme general cellular functions. To cope with highlyintensive oxidative stress, when operation of Nrf2/Keap1, NF-kB,AP-1, and MAP kinases cannot provide surviving of oxidativeinsults, there is a need to limit substantially cellular ROS produc-tion. Since bulk ROS amounts are produced by mitochondria, ani-mals adopted mechanism substantially decreasing their potentialto generate ROS. Production of ROS by mitochondria to big extentdepends on transmembrane electrochemical gradient on innermitochondrial membrane. Therefore, induction of permeabilitytransition pore (PT) is very efficient way to decrease intracellularROS production. Again it seems that PT formation includes oxida-tion of cysteine residues of membrane proteins which at oxidationform this pore [99,100]. To the end, if the cell cannot cope withhighly intensive oxidative stress, the resulting fatal consequencesmay be minimized by entering apoptosis. The latter let organismprevent negative effects of cellular components released into envi-ronment at cell death like it takes place at noncontrolled scenariocalled necrosis. Fig. 4 demonstrates graphically how intensity ofoxidative stress affects specific signaling pathways and lead finallyto physiological responses. As one can see, multilevel animal sys-tem of response to oxidative stress of different intensity let ani-mals adequately response to these challenges in dose-dependentmanner.

Molecular mechanisms of regulation of redox signaling havebeen delineated to date. There are two principally distinct systemsand in both reversible oxidation of active sites has been implicated.In bacterial SoxRS regulatory system reversible oxidation of[4Fe-4S] cluster was found to be a critical player [59,61,81]. Such



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Fig. 3. Operation of the Nrf2/Keap1system during response to oxidative stress in animals. Under nonstressed conditions the transcription factor Nrf2 binds to the Keap1homodimer. The resulting protein complex can then further complex with Cullin 3 leading to ubiquitination of Nrf2 followed by proteasomal degradation. Following anoxidative insult or electrophilic attack, Keap1 cannot bind Nrf2 which allows Nrf2 to diffuse into the nucleus and, in concert with small Maf proteins (sMaf), Map and others,Nrf2 binds to the ARE/EpRE elements of regulatory regions in genes encoding antioxidant or phase 2 detoxification enzymes. Nrf2 migration into the nucleus is promoted byat least three different mechanisms: oxidation of Keap thiol groups to form disulfides, modification of Keap1 cysteine residues by electrophiles, or phosphorylation of Nrf2 byprotein kinases that, in turn, may be activated by oxidants. Modified from [43].

Fig. 4. Graphical presentation of hierarchy of oxidative stress responses in animals. At low intensity oxidative stress, Keap1/Nrf2 system up-regulates genes encodingantioxidant enzymes. Intermediate intensity oxidative stress up-regulates antioxidant enzymes and induces inflammation proteins and heat shock proteins via NF-jB, AP1,MAP, kinases and HSF. At high intensity oxidative stress perturbations of mitochondrial PT pore, activation of apoptosis cascade, destruction of electron transporters takereplace which may culminate in apoptosis and/or necrosis. Modified from [43].



31 October 2014

signaling system was found only in prokaryotes. All other regula-tory systems in bacteria, fungi, plants, and animals demonstrateutilization of universal mechanism – reversible oxidation of


cysteine residues of specific sensor and signal-transducing proteins[61]. Interested in detains readers are addressed to our previousreviews [43,61,81,90] and here I will only describe the chemical



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Fig. 5. Reversile oxidation of protein cystein residues involved in redox signaling. The residues may be oxidized to sulfenic, sulfinic, or sulfonic derivatives and also involvedin the formation of glutathionylated proteins. In biological systems, sulfenic and sulfinic derivatives may be reduced by thioredoxin (TR) and sulfiredoxin (Srx), respectively,whereas sulfonic moieties cannot be reduced. Glutathionylated proteins are formed by direct interaction of GSH with sulfenic acid derivatives, exchange between cysteineresidues and GSSG, or interaction with oxidized glutathione forms. Modified from [43].



31 October 2014

aspects of the problem (Fig. 5). Exposure of cysteine residues ofregulatory proteins to ROS results in oxidation with the conse-quent formation of stable sulfenic, sulfinic, or sulfonic acid deriva-tives and several unstable transient forms. Sulfenic acid may bereduced to the original cysteine form by several reductases[101,102]. Sulfinic acid can be reduced only by the specific actionof sulfiredoxin [103–105]. Finally, it is believed that sulfonic acidcannot be reduced in living organisms. It is well established thatcysteine oxidation to sulfenic acid is used for ROS signaling. Inaddition, sulfenic acid residues may interact with reduced glutathi-one to form mixed disulfides adapted to signaling and/or protec-tion of thiol groups from further oxidation [43,47,48]. Sinceglutathione residue can be removed from the proteins it is sug-gested to be protective mechanism under oxidative boots whichmeans that at recovery initial functional activity of glutathionylat-ed proteins may be restored.

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Fig. 6. Schematic classification of oxidative stress based on its intensity. This figureshows four zones for ‘‘Endpoint‘‘ vs. ‘‘Dose/concentration of inducer’’ which areproposed to be called: I – basal oxidative stress zone (BOS); II – low intensityoxidative stress (LOS); III – intermediate intensity oxidative stress (IOS); andIV – high intensity oxidative stress (HOS).

6. Intensity-based classification of oxidative stress

Although since 1985 year when H. Sies proposed the first defi-nition of oxidative stress [57], almost 30 years passed and veryintensive works in this field were carried out, actually we haveno classification of this biological phenomenon. Long term per-sonal interest, own experimental experience, and detail inspectionof the literature has stimulated me to develop classification ofoxidative stress based on its intensity.

Usually researchers say about oxidative stress when one or bet-ter several parameters reflecting balance of free radical processes isdisturbed to increase steady-state ROS level which affects manyvital processes. In most cases, several parameters are typically usedas markers of oxidative stress. These parameters usually includeoxidatively modified lipids [106–108], proteins [109–112], nucleicacids [113–115], and glutathione status [43,116,117] whereaschanges in the activities of antioxidant and associated enzymesprovide additional issues. Main problems with the activities ofthe mentioned enzymes are mainly related with complexity oftheir response because dependently on the intensity of oxidativestress the activities may behave differently: increase, decrease ornot change. At least several principal processes affect the activity– they may be increased due to up-regulation of their biosynthesisalong with maturation, and ROS-induced inactivation. The balance


between these two groups of the effects results in the net activity.Therefore, usually increased level of oxidatively modified cellularcomponents is commonly accepted marker of oxidative stresswhich can be used for classification, whereas the activities of thementioned above enzymes may be questioned. The only way offin this case is related with application of several parameters whichinclude levels of at least couple oxidatively modified cellular com-ponents and activities of antioxidant and related enzymes. It isclear that the situation is rather complicated to be described insome scheme or model. Probably that was the main reason whyto date there is no widely accepted system for classification of oxi-dative stress. Obviously, there is a need for this classificationbecause in many cases researchers have to compare effects of dif-ferent inducers or their doses/concentrations which are needed torange effectiveness of the inducers. In order to fill up the gape, inthis paper I propose couple classification systems presentedschematically in the ‘‘idealized’’ form (Fig. 6).

Curve 1 in Fig. 6 shows the relationship between the dose orconcentration of effector inducing oxidative stress and endpointparameter measured. The measured end point parameter is someROS-induced ROS-sensitive integral characteristics of the system.Certainly, it differs dependently on study character and may be



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31 October 2014

such as cell survival, activation of ROS-sensitive regulatory path-ways, activities of antioxidant and associated enzymes. For conve-nience and simplification in this paper we will analyze someindices which are increased above control under effect of lowdoses/concentrations of stress inducer and which are at the sametime sensitive to ROS. Curve 2 in Fig. 6 shows the relationshipbetween dose/concentrations of the oxidant and the level of oxida-tively modified components. Those can be different products ofoxidation of proteins, peptides, lipids, nucleic acids, carbohydrates,etc. The parameters collectively reflect ROS-induced modificationof cellular components.

Theoretically, oxidative stress may be divided for four ranges:basal, low intensity, intermediate and high intensity oxidativestress which can be abbreviated as BOS, LOS, IOS and HOS, respec-tively. In the BOS area, there is no actually way to register observa-ble effects – they are so negligible that cannot be observed by themethods routinely applied. Indeed, it is well known that ROS arealways generated in living organisms and they successfully copewith them and even more – they prevent registered effects of smalladditional amounts of extra added ROS or other inducers of oxida-tive stress. Due to that, we may not say about any observable oxi-dative stress. Higher ROS doses/concentrations may induce LOSwhen we can register both: increased level of oxidatively modifiedcomponents (Fig. 6, curve 2) and, at the same time, increased end-point parameter measured – i.e. ROS-induced ROS-sensitiveparameter (Fig. 6, curve 1). They usually can be registered by con-ventional analytical techniques and involve described to datemechanisms of up-regulation of ROS-regulated parameters, suchas activity of antioxidant or associated enzymes briefly discussedabove. Curve 1 in the zone LOS may be divided for two components– increasing (IIA) and decreasing (IIB) after it passes maximum.Finally, this curve decreases to the point when no observable effect(NOE) is seen in ROS-sensitive ROS-dependent parameter, but thelevel of oxidatively modified component at the NOE point is sub-stantially higher relatively to control. At further increase of dose/concentration of inducer, curve 1 passes NOE point and the systementers IOS range. At this zone two curves clearly demonstratedevelopment of oxidative stress: level of oxidatively modifiedcomponents is rather high, whereas ROS-induced ROS-sensitivecomponents clearly show decrease. If at Fig. 6 behavior of curve2 is easy to accept from logical point of view, one of curve 1 needsespecial comment. At this range, intensity of oxidative stress is sohigh, that up-regulation of the monitored parameter if even takesplace is counterbalanced by its inactivation or may be related alsowith effects on other parameters related to some extent with eval-uated parameter. For example, it can be ROS-promoted inactiva-tion of antioxidant and associated enzymes, which could be up-regulated in LOS area. At the same time, curve 1 behavior in LOSarea could reflect regular degradation of the enzymes accompaniedby ROS-inactivated de novo biosynthesis (actually inhibited bio-synthesis). Finally, in the HOS range both measured functions con-verge to some plateau – i.e. virtually all available potentialsubstrates are oxidized in this situation which results in the devel-opment of a near maximum response.

Fig. 6 also can be used to propose a bit different from thedescribed above classification of oxidative stress. In this case, thecurves 1 and 2 maintain biological meaning as previously. We willignore zone BOS and call LOS range as ‘‘mild oxidative stress’’(MOS), zone IOS – as ‘‘temperate oxidative stress’’ (TOS), and finallythe most intensive oxidative stress may be called ‘‘severe (strong)’’oxidative stress’’ (SOS).

The question left from the above two paragraphs – how at Fig. 6to discriminate zones III and IV (IOS vs. HOS, or TOS vs. SOS)? It isreally rather complicated issue without any clues to provide someobvious rationale here. However, we can apply here Hill equation.Using experimental data used to build curve 2 at Fig. 6 we can


calculate maximum of the function formally described by thisequation. In this case, ‘‘maximum saturation’’ reflects maximumamount of ROS-modified component. Then we can use dose/con-centration at which curve 2 reaches 90% of its maximum as a bor-der between zones of interest, i.e. zones III and IV.

Certainly, it is clear that proposed approach is not so simple tobe routinely applied. Due to that we can simplify the system pro-posing to operate only with two zones which are relatively easyto be discriminated: mild oxidative stress (MOS) correspondingto zone II and strong oxidative stress (SOS) which correspondszone III combined with zone IV at Fig. 6.

The application of proposed classification is complicated also bythe presence of different compounds and processes which can sub-stantially modify responses of systems to inducers of the stress. Forexample, increase in the level of antioxidant GSH, preconditioning,physical training etc. may shift curves 1 and 2 at Fig. 6 to the rightdue to its high potential to neutralize ROS and in this mannerweakening cellular responses [43]. Further modification of thecurves may be associated with presence of nitric oxide. From onehand it can operate as an antioxidant, but on the other hand – asa prooxidant with consequent response of the system. And evenmore, both mentioned above compounds may interact betweenthem with the formation of GSNO [43] and the final effect of nitricoxide on the system may be even less predictable than at operationof single compound.

It is possible to combine our knowledge on the involvement ofdifferent systems in response to oxidative stress of different inten-sity. Especial question is related with the parameters which can beused to characterize experimentally theoretically developed classi-fication. Although the parameters were listed briefly above, there isa need to give some clues for experimental investigation. Probably,viability is the most integral parameter. If we deal with unicellularorganisms, classic biological approaches can be applied herebecause they are well developed to date and provide the most inte-gral parameter. However, when we deal with multicellular organ-isms the situation is principally different. Tissue- and organ-specific response complicates research substantially. Two princi-pally different approaches can be used in this case: (1) identifica-tion and characterization of organ/tissue specific responses, and(2) identification of target or critical organ/tissue responsible forsurvival of all organisms. If the first approach is usually appliedin most studies [118,119], the second one is not commonly appliedin the field of oxidative stress. I suggest for the second case, thestudies carried out in radiobiology can be used with the focus onorgans/tissues determining survival or wellbeing of all organismswhich were called target ones [120,121]. It would be much easierto follow one end point than several and which would be the mostcomplicated – the results dependently on the selected end pointsmay differ. When we will look at less integral parameters whichcan be used as end points for classification of oxidative stressintensity, the situation is not easy to be solved also. It looks thatparameters regulated by ROS and in some way involved in antiox-idant defense may be most relevant for these purposes. Due to ourexperience, the activity of antioxidant enzymes such as superoxidedismutase and catalase as well as other related enzymes could beamong the best candidates [122]. That is because their activity isup-regulated at low doses/concentrations of oxidants anddecreased at higher ones (the enzymes seems are simply inacti-vated by ROS). Glutathione is one more potential candidate herebecause its biosynthesis is stimulated under mild oxidative stressowing to increased expression of glutamate cysteine ligase (EC6.3.2.2), a key enzyme for its biosynthesis, whereas at highly inten-sive stress glutathione is consumed in several ways – at least viaoxidation, conjugation and extrusion from the cell [43].

The situation with oxidatively modified components also seemsis not simple. Usually levels of ROS-oxidized lipids, proteins, and



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31 October 2014

nucleic acids and/or their components/products are monitored. Insome cases, the level of oxidized glutathione is also registered.Essential techniques for evaluation or lipid peroxidation includemeasurement of so-called thiobarbituric acid reactive substances(TBARS) – products of interaction between thiobarbituric acidand as commonly believed malonic dialdehyde. However, thisreaction is rather non-specific and therefore is not recommendedfor extensive usage [123]. Last time at least in our practice wereplaced this parameter be measurement of lipid peroxides whichis much more specific and due to which it usually gets less critiquefrom colleagues and reviewers [123,124]. Certainly, adaptation ofhigh-performance liquid chromatography and gas-chromatogra-phy especially in combination with mass spectroscopy and othermodern techniques may be very helpful here [125–127]. Oxidationof proteins by ROS may be registered by different techniques, butto date seems assays of formed additional carbonyl groups pro-posed by R. Levine and colleagues is most commonly applied one[123,128]. Oxidative modification of nucleic acids is rarely usedparameter to monitor ROS-induced processes due to big array offormed products and technical reasons. In this case, several prod-ucts are usually monitored and those are mainly oxidativelymodified guanine derivatives, of which 8-hydroxyguanine(8-OHG) is the most commonly used marker [129,130], but8-oxo-7,8-dihydro-20-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanine (8-oxoGua) [131] can also be measured. Finally,glutathione level and usually more important ratio between oxi-dized and reduced (or total) one is a good indicator of ROS-relatedprocesses [43]. Several approaches are used to monitor glutathionehomeostasis such as HPLC, chemical techniques, etc. However, forroutine measurements of glutathione level not expensive, butspecific enzymatic method with the use of glutathione reductasecoupled with glucose-6-phosphate dehydrogenase may be recom-mended [132–135].

Despite its putative importance, quantification of the absolutechanges used for classification is not the key question. Moreover,it is very difficult to find some rationale to solve the issue. Forexample, the question is: how to discriminate MOS, TOS, andSOS? To our best understanding, it is impossible to give absolutedegree of changes to categorize the types of the stress. Only rela-tive parameters can be applied here. Indeed, relative changes incurves 1 and 2 at Fig. 6 would provide enough arguments to cate-gorize oxidative stress for classes. Potentially, some clues may beborrowed from hormesis research. Hormetic relationship in princi-ple virtually follows curve 1 at Fig. 6 of this paper [136]. After anal-ysis of thousands papers Calabrese and Baldwin [137] found that inactivated region increase in the parameter usually ranges between30 and 60%. With yeasts S. cerevisiae the increase of viability underlow intensity oxidative stress may reach up to 20–30% [138,139].However, if activity of some antioxidant enzyme is followed, atlow intensity oxidative stress it may be increased several timesand decrease to 30–60% [122,140]. It is virtually impossible toget higher decrease because cells are damaged due to which bio-logical reasons may be questioned.

Biological effects of oxidative stress depend on the intensity.Basal intensity oxidative stress (BOS) is usually not observable,but it may result in preadaptation of the organisms to higher inten-sity stresses [59,138–140]. Indeed, if basal or low intensity oxida-tive stress (LOS) is applied to the organisms, it can result inincreased tolerance to followed higher intensity stress even inthe case when the stresses were induced by different stressorswhich was called-cross adaptation [139,140]. It is important tonote that cross-adaptation may take place when two stresses ofinterest possess some common mechanism of their realization,for instance, oxidative stress. Low intensity oxidative stress, espe-cially at phase IIA (Fig. 6) may be beneficial due to strengthening ofprotective potential of organism as it was described above. In the


phase IIB, mainly positive effects of LOS may be overlapped withnegative ones because of increased intensity. In this region,negative effects may be even masked or exceeded by intensifiedROS-induced oxidation. The state IIB may be associated with path-ological or prepathological (prodromal) states and if not fixed maydevelop in pathologies. Further, intermediate intensity oxidativestress (IOS) already results in extensive oxidation of componentsof living organisms and weakening of antioxidant defense andfrequently is associated with certain pathologies such as obesity,metabolic syndrome, cancer, cardiovascular and neurodegenera-tive diseases. If the organism does not possess enough recoursesand/or capability to prevent further deterioration scenario, it candevelop high intensity oxidative stress (HOS) which if not fixedmay lead to death. It should be noted that in multicellular organ-isms it is very important to select the organ/tissue to monitorthe state of all organism because not all organs/tissues are equallyresponsible for surviving and state of all organism. Damage to crit-ical organ/tissue may result in fatal way off, whereas changes insome parts may be not so critical. Selection of noncritical organ/tis-sue can lead to overlooking general response of the organism.

Obviously, the proposed oxidative stress classifications andapproaches to characterize and get in would not be always applica-ble to real experimental works and therefore suggest that they canbe used just for previous description of the systems and needfurther development.

7. Conclusions and perspectives

The processes involving ROS in living organisms have beenextensively studied over six decades. Initial descriptive studieswith focus on their negative effects prevail up to now. However,the discoveries in the field, related to ROS roles in operation ofimmune system, signal transduction and finally regulation of nor-mal functions of the organisms opened new horizons for research.Technical capability to manipulate by genes along with site-directed mutagenesis and recombinant DNA technologies providedfurther progression in our understanding of ROS-related processes.However, if someone would be so optimistic in understanding ofthose processes in living organisms he/she would be disappointed.Use of traditional and modern approaches along with sophisticatedtools actually opened our eyes on complexity of the subjects ofinterest.

In my opinion, several hot and perspective points in the field ofROS research have to be mentioned here: (1) Since even inductionof oxidative stress is frequently debated, it seems that its classifica-tion is needed and it may be based on intensity (like described inthis paper). (2) Accurate description of cellular ROS targets mayhelp to develop corresponding tools for predictive investigations.The key question here is: why in situ some targets are reachedand modified faster than others, whereas in vitro the situationmay be opposite? (3) Do really low molecular mass antioxidants(such as tocopherols, ascorbate, uric acid and melatonin) possessantioxidant properties in vivo? Frequently their intracellular con-centrations are not high enough to demonstrate antioxidant prop-erties usually measured in artificial conditions in vitro. (4) Whichare real intracellular concentrations of ROS and how they are spa-tiotemporally distributed in the cellular and extracellular milieu,particularly in multicellular organisms. (5) How different defensemechanisms cooperate to combat ROS attacks? (6) Which is ROSrole in providing of normal organisms’ functions? (7) Are ROSreally involved in pathogenesis of certain diseases and accompanythem or really there is no relationships between pathologies anddescribed experimental facts are coincidences? Probably eachreader may extend this list dependently on own interests andexperience and this clearly warrants new discoveries in this excit-ing and poorly predictable field.



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Conflict of Interest

The author declares that there are no conflicts of interest.
959960961962963964965966967968969970971972
Acknowledgements

The author is grateful for long-term communication andinteresting discussions with colleagues in the field H. Sies,H. Semchyshyn, O. Lushchak, D. Abele, D. Gospodaryov, M. Bayliak,O. Kubrak, K. Storey, J. Storey, R. Levine, M. Nikinmaa, A. Boldyrev,A. Jha, V. Skulachev, M. Hermes-Lima, and many other who stimu-lated the author’s interest to the field of reactive species andoxidative/nitrosative stress.

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http://dx.doi.org/10.1038/eye.2014.127

http://dx.doi.org/10.1016/j.bbadis.2014.05.005

http://dx.doi.org/10.1016/j.bbadis.2014.05.005

http://dx.doi.org/10.1016/j.freeradbiomed.2014.06.004

http://dx.doi.org/10.1016/j.freeradbiomed.2014.06.004

http://dx.doi.org/10.1007/s12013-014-0006-5

http://dx.doi.org/10.1007/s12013-014-0006-5


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[doi 10.1016_j.cbi.2014.10.016] v. i. lushchak -- free radicals, reactive oxygen species, oxidative...

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