Going retro: Oxidative stress biomarkers in modern redox biology

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Going retro: Oxidative stress biomarkers in modern redox biology N.V. Margaritelis a,e, J.N. Cobley b, V. Paschalis c,d, A.S. Veskoukis a, A.A. Theodorou d, A. Kyparos a, M.G. Nikolaidis a,n a

Department of Physical Education and Sports Science at Serres, Aristotle University of Thessaloniki, Agios Ioannis, 62110 Serres, Greece Division of Sport and Exercise Sciences, Abertay University, Dundee, UK c Department of Physical Education and Sport Science, University of Thessaly, Karies, Trikala, Greece d Department of Health Sciences, School of Sciences, European University Cyprus, Nicosia, Cyprus e Intensive Care Unit, 424 General Military Hospital of Thessaloniki, Thessaloniki, Greece b

art ic l e i nf o

a b s t r a c t

Article history: Received 29 October 2015 Received in revised form 8 January 2016 Accepted 2 February 2016

The field of redox biology is inherently intertwined with oxidative stress biomarkers. Oxidative stress biomarkers have been utilized for many different objectives. Our analysis indicates that oxidative stress biomarkers have several salient applications: (1) diagnosing oxidative stress, (2) pinpointing likely redox components in a physiological or pathological process and (3) estimating the severity, progression and/or regression of a disease. On the contrary, oxidative stress biomarkers do not report on redox signaling. Alternative approaches to gain more mechanistic insights are: (1) measuring molecules that are integrated in pathways linking redox biochemistry with physiology, (2) using the exomarker approach and (3) exploiting -omics techniques. More sophisticated approaches and large trials are needed to establish oxidative stress biomarkers in the clinical setting. & 2016 Elsevier Inc. All rights reserved.

Keywords: Biomarkers Oxidative stress Redox signaling Physiology Pathology

1. Introduction Oxidative stress biomarkers have been used in the free radical field from its inception. For example, in their 1982 paper, Kelvin Davies and Lester Packer measured a biomarker of lipid peroxidation, in what is probably the most influential paper in the free radical and exercise field [1]. Even today, some of the most important exercise studies have used several oxidative stress biomarkers in order to substantiate the usefulness or uselessness of antioxidant supplementation (e.g., [2–4]). This supports the idea that the field of redox biology is inherently intertwined with oxidative stress biomarkers. In the first experiments, oxidative stress biomarkers were useful in establishing the presence or absence of oxidative stress in various physiological processes and diseases [5]. This is a task that several available oxidative stress biomarkers can certainly fulfill. As the field of redox biology was progressing, the experimental questions concerning the nature and the consequences of the redox perturbation accompanying exercise have been refined. For example, efforts are now being made for unraveling the exact redox mechanisms through which exercise can ameliorate the negative consequences of a disease (e.g., [6]). Now, and in the past, oxidative stress biomarkers have been often used as a tool to n

Corresponding author. E-mail address: [email protected] (M.G. Nikolaidis).

reveal redox mechanisms. This actually implies that the oxidative stress biomarkers feature the required biochemical characteristics to report on redox signaling. However, oxidative stress biomarkers do not provide mechanistic insights [7], because they were not made for that purpose. In fact, the molecular understanding of the role of redox biochemistry in health and disease requires the precise identification of the modifying species, the biomolecular targets involved, the type of modification, the specific residue (s) modified, the reversibility of the oxidative modification and the cellular/organelle compartment that this process takes place [8]. Therefore, an intriguing question is what might be the true value of oxidative stress biomarkers in the modern redox biology era. To answer this question and demonstrate the plausible areas of application of redox biomarkers towards linking reactive species with physiology and pathology, we draw examples from exercise physiology. The main aim of this review is to highlight the applications, misuses and limitations of oxidative stress markers in modern redox biology. A secondary aim is to emphasize currently available mechanistic biomarkers that look promising and suggest specific steps that have considerable potential to progress current understanding. Our analysis indicates that oxidative stress biomarkers have several salient applications: (1) diagnosing oxidative stress, (2) pinpointing likely redox components in a physiological or pathological process and (3) estimating the severity, progression and/or regression of a disease (Fig. 1).

http://dx.doi.org/10.1016/j.freeradbiomed.2016.02.005 0891-5849/& 2016 Elsevier Inc. All rights reserved.

Please cite this article as: N.V. Margaritelis, et al., Going retro: Oxidative stress biomarkers in modern redox biology, Free Radic. Biol. Med. (2016), http://dx.doi.org/10.1016/j.freeradbiomed.2016.02.005i

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Oxidative stress biomarkers

Mechanistic measurements

Oxidation of lipids, proteins and DNA

Redox signaling

Mechanistic information

Descriptive information

Physiology / Pathology Fig. 1. Applications of oxidative stress biomarkers. Oxidative stress biomarkers are advantageous and practical for confirming the presence of oxidative stress and for providing a descriptive view of redox-associated processes linked to physiology and pathology. This descriptive view includes the recognition of a potential redox component of the process and the estimation of the severity, progression and regression of a disease. On the contrary, oxidative stress biomarkers do not convey mechanistic information, since they lack the mandatory specificity and selectivity required for such a purpose. Thus, they cannot report on redox signaling processes.

2. Applications of oxidative stress biomarkers In biomedical research, a biomarker is defined as “a characteristic that can be objectively measured and evaluated as an indicator of normal biological or pathogenic processes, or pharmacological responses to a therapeutic intervention” [9]. The diagnostic potential of a biomarker depends, therefore, on the accurate distinction of the alterations that correspond to the onset, progression, regression, and ideally, to the prediction of a specific biological process at the time of specimen collection. On this basis, biomarkers are regarded as an integral part of molecular epidemiology aiming to fulfill the largely unmet need to integrate biochemical with translational and clinical research [10–12]. To this aim, emerging technologies, including proteomics, lipidomics, metabolomics, transcriptomics, genomics and real-time imaging have been implemented and have widely facilitated the identification of such biomarkers [13–16]. Along with these analytical advances, detailed criteria for an ideal biomarker have been proposed, briefly summarized as follows: (1) high sensitivity and accuracy, (2) high reproducibility, (3) biological plausibility with the investigated phenotype, (4) opposite responsiveness after a therapeutic treatment and (5) non-invasive collection and easy handling [16–22]. A noteworthy observation is that the potential to disclose the exact underlying mechanism of the investigated condition is not included in the proposed criteria of the ideal biomarker. This emphasizes that the biomarkers were not developed for unraveling mechanisms, but instead, for providing an integrative view of the biological process/disease under study. Correspondingly, to date several oxidative stress biomarkers reliably reflect the exposure to an oxidant insult. On behalf of this objective, great analytical advances have been made, epitomized by the improvement in lipid peroxidation and protein oxidation assessment. In particular, and with regard to lipid peroxidation, less reliable and outdated approaches (e.g., the TBA test) have been replaced by sophisticated state-of-the-art techniques (e.g., MSbased methods), which infer non-enzymatic free radical-mediated lipid peroxidation in a more accurate way [23,24]. Likewise, great progress has been achieved in protein oxidation assessment. More specifically, the crude measurement of carbonyl groups that may also derive from diverse redox-unrelated processes (i.e., protein glycation by sugars or binding of aldehydes to proteins) is gradually being displaced by improved techniques that identify specific oxidized/nitrated amino acids, such as 3-nitrotyrosine and

oxidized tryptophan. These techniques predominately involve mass spectrometry, in combination with gas chromatography (GC– MS, GC–MS/MS) or liquid chromatography (LC–MS/MS) [24,25]. When referring to redox biomarkers it is important to distinguish the biomarkers that offer a descriptive view of the general redox state of the organism from those that provide a more mechanistic read-out. By definition, oxidative stress biomarkers belong to the former category, providing a global snapshot of lipid peroxidation, protein oxidation or DNA oxidation according to the biomarker. On the contrary, they cannot be regarded as mechanistic biomarkers, because they are not integrated into a specific redox pathway and do not regulate redox signaling in a canonical manner (see Section 2.2). Below, we present selected examples of how oxidative stress biomarkers may be applied to exercise and clinical exercise physiology and pathology. 2.1. Physiology Exercise is probably one of the most characteristic examples demonstrating that reactive species and oxidative stress are not necessarily “harmful” entities. In fact, regular exercise leads to many beneficial redox-related and redox-mediated adaptations, which are accompanied by repeated episodes of reactive species production [26,27]. With regard to the redox-related adaptations, it is well-established that exercise training results in: (i) increased levels of non-enzymatic antioxidants (e.g., glutathione), (ii) upregulated gene expression and increased activity of the antioxidant enzymes (e.g., superoxide dismutase), (iii) increased molecular chaperone content (e.g., heat shock proteins), and (iv) decreased amount of oxidative stress biomarkers at rest (e.g., F2-isoprostanes) [28–32]. Remarkably, when vitamin C was used along with a chronic exercise protocol, hampered redox-related adaptations were found in muscle (e.g., decreased expression of superoxide dismutase and glutathione peroxidase; [33]). From a functional perspective, the aforementioned changes increase protection against exercise-induced oxidative stress. From a molecular perspective, these changes are regulated by several redox sensitive transcription factors, such as NF-κB, AP-1, HIF-α, PGC-1α, p53, HSF1 and Nrf2, which activate cyto-protective, angiogenic, metabolic and mitochondrial gene clusters [34–37]. The repeated induction of the aforementioned gene clusters underpins several exercise adaptations, such as mitochondrial biogenesis, angiogenesis, muscle hypertrophy, O2 uptake and insulin

Please cite this article as: N.V. Margaritelis, et al., Going retro: Oxidative stress biomarkers in modern redox biology, Free Radic. Biol. Med. (2016), http://dx.doi.org/10.1016/j.freeradbiomed.2016.02.005i

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sensitivity [4, 38–40]. It is, however, not always clear how and which reactive species regulate redox-sensitive transcription factors. For example, whether the modification of critical residues proceeds in a direct or indirect (i.e. via intermediates) fashion is not known [7,41]. In light of the above, and applied to an exercise setting, assessing oxidative stress biomarkers for monitoring the redox state of athletes may be a helpful tool to supervise the progression of athletes’ adaptations. On the contrary, athletes who do not exhibit the anticipated redox adaptations may also experience blunted physiological adaptations regarding phenotypes that contain a redox component. In the same manner, the monitoring of oxidative stress biomarkers could be an effective way to identify those participants who fall into the hotly debated category of “non-responders to exercise” [42,43]. 2.2. Pathology Despite the fact that their use in everyday clinical practice is not established yet, oxidative stress biomarkers have been widely utilized in clinical research [44]. For instance, they have been effectively employed to monitor the stages of numerous diseases, with the most investigated being neurodegenerative diseases (e.g., Alzheimer and Parkinson diseases), type II diabetes, cardiovascular diseases, several types of cancer, ischemia/reperfusion injury and rheumatoid arthritis [20,45,46]. They have been also used for screening the potential efficacy of therapeutic treatments, including exercise training and/or antioxidant supplementation [5,47–51]. Towards these goals, oxidative stress biomarkers have been measured in human studies at tissue and systemic level, such as skeletal muscle, blood plasma or cells, urine and saliva [52–56]. Based on this, the measurement of oxidative stress biomarkers represents a promising approach for predicting the onset of a disease, or assessing the progression of a disease, and for evaluating the effect of exercise as a treatment. In an exercise setting, oxidative stress biomarkers may be useful for monitoring the “overtraining syndrome” (referred hereafter as overtraining [57]). Overtraining is a condition predominately experienced by elite athletes [58] and is characterized by an unanticipated decline in performance, despite an extended rest period [59,60]. Nowadays, no single diagnostic biomarker of overtraining is universally accepted, with the declined physical performance and the mood disturbances being ascribed as the most fundamental features. In a recent joint consensus statement of the European College of Sport Science and the American College of Sports Medicine [61], oxidative stress biomarkers were not included in the proposed set of overtraining biomarkers. Considering that a series of both human and animal studies have reported aberrant redox status during overtraining, oxidative stress biomarkers may be potentially incorporated in a battery of overtraining indicators in the near future [62–68]. 2.3. Subject/patient stratification and enrollment Our group has documented large inter-individual variability in redox biomarker levels both at rest [69] and in response to an exercise session [70]. In addition, we have demonstrated that the initial value of a biomarker represents an important determinant of the degree of the post-exercise alteration [70,71]. That is, individuals with higher initial values in oxidative stress biomarkers tend to exhibit smaller relevant increases after exercise, and vice versa. Considering that reactive species are important regulators of exercise adaptations [7,35,72,73], it could be hypothesized that individuals with lower resting values in oxidative stress biomarkers would exhibit greater increases in response to exercise. Therefore, they would presumably experience greater alterations

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in exercise adaptations. Indeed, we have found that vitamin C-deficient participants experienced greater redox and performance benefits compared to vitamin C-sufficient individuals, after vitamin C administration [69]. This could partially explain the controversy in the literature regarding the true effectiveness of antioxidants to change redox homeostasis [7]. Indeed, several studies have reported that antioxidant supplementation (e.g., vitamin C) failed to reduce oxidative stress [74] and to affect health and disease (e.g., mortality, cancer, or physical performance) [75–77]. A likely explanation for this failure could be that the antioxidant supplements have been administered in humans with normal levels of oxidative stress biomarkers. This possibility has already been stressed by other authors [78,79] and has been experimentally verified [69,80,81]. In particular, data show that the beneficial effects of antioxidant supplementation are restricted to individuals with high baseline levels of oxidative stress and/or low antioxidants (Fig. 2). On this basis, the potential to use redox biomarkers for identifying individuals that are more likely to benefit in response to a given redox stimulus (i.e., treatment personalization) may represent an innovative approach for the development of new redox therapeutics.1

3. Misuse and limitations of oxidative stress biomarkers 3.1. Oxidative stress biomarkers measured in tissues do not report on redox signaling Redox regulated biological processes are generally characterized by a delicate spatiotemporal specificity [83]. This is best exemplified by the complex nature of reactive species production and metabolism. For instance, several differentially localized NADPH oxidase (NOX) isoforms are expressed in skeletal muscle, including mitochondria (NOX4), plasma membrane (NOX2 and NOX4), and t-tubules (NOX2 and NOX4) [84]. The NOX isoforms transfer electrons from NADPH to molecular oxygen, thereby generating superoxide. The NOX derived superoxide may subsequently be enzymatically dismutated to hydrogen peroxide by superoxide dismutase (SOD) isoforms localized in different cell fractions (i.e., MnSOD and CuZnSOD in mitochondria, cytosol or extracellularly; [20]). In turn, the hydrogen peroxide generated can be metabolized by specific and highly redundant antioxidant enzymes, dictated by the magnitude and/or the site of the hydrogen peroxide production and its subsequent diffusion. It is evident, therefore, that antioxidants also exhibit spatial and kinetic specificity, adding a further layer of complexity. Another aspect of the strict specificity that characterizes redox biology processes is that redox signaling is defined by regulated biochemical reactions [7,83,85–87]. These reactions include selective and reversible compartmentalized post-translational modifications of target cysteine and/or methionine residues, which fine-tune redox signaling processes (i.e., via regulation of kinases, phosphatases and/or transcription factors; [88]). Interestingly, the subtle procedure of signal transmission is cooperatively regulated by antioxidant enzymes (i.e., peroxiredoxins, 1 An important threat when selecting individuals based on their initial values for a given variable and assigned into ‘high’ and ‘low’ categories is the “regression to the mean” statistical artifact [82]. According to this phenomenon, a group of participants with an extreme mean value for a given variable during the first evaluation tends to obtain a less extreme value on a subsequent follow-up test (even in absence of any intervention). Therefore, the use of a parallel placebo control group or performing more than one measurement prior to the main experimental intervention is essential to minimize the effect of regression to the mean [82].

Please cite this article as: N.V. Margaritelis, et al., Going retro: Oxidative stress biomarkers in modern redox biology, Free Radic. Biol. Med. (2016), http://dx.doi.org/10.1016/j.freeradbiomed.2016.02.005i

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A ESR signal (arb. units)

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Low resting ESR High resting ESR

20

15 10 5 0 Pre supplementation

B

Low F2 -Isop High F2 -Isop

100 F2 -Isop (μg/mL)

Post supplementation

80 60

3.2. Oxidative stress biomarkers measured in blood reflect redox alterations in muscle, but are derived from diverse tissues

40 20 0 Pre supplementation

Post supplementation

C Low vitamin C High vitamin C

60 VO2 max (ml/kg/min)

neither mechanistic information (e.g., type and site of reactive species production), nor in-depth insights into the redox-regulated posttranslational modifications and their functional consequences. This highlights the sheer inadequacy of global oxidative macromolecular adducts to report on mechanistic and compartmentalized regulatory processes, which is required to unravel the molecular origin of redoxregulated exercise/disease phenotypes. These limitations apply regardless of the oxidized macromolecule adduct, even to F2-isoprostanes, which are generally regarded as the gold standard of oxidative stress biomarkers [32]. In particular, arachidonate (the precursor molecule of F2-isoprostanes) is one of the most abundant polyunsaturated fatty acids and can be found in various locations in muscle cell, such as in plasma membrane, endoplasmic reticulum and mitochondria [92]. Thus, limited spatial and/or mechanistic evidence can be drawn from arachidonate oxidation, without subcellular fractionation. Yet, the most common practice includes measurements in whole homogenized muscle. The generic and descriptive character of the oxidative stress biomarkers is in stark contrast to the firm spatiotemporally controlled redox signaling processes. Hence, from a mechanistic perspective, the measurement of such surrogate biomarkers of reactive species production is not recommended.

50

40

30 Pre supplementation

Post supplementation

Fig. 2. Studies showing the dependence of redox homeostasis changes on the initial values and the opportunity to stratify subjects based on resting values of redox biomarkers: (A) individual responses have been separated into two groups, those with low and high basal free radical levels. The antioxidant cocktail (vitamin C, vitamin E and alpha-lipoic acid) decreased the level of ESR signal only in those individuals with high basal free radical levels (the figure is based on data from Rossman et al. [81]). (B) Vitamin C supplementation reduced lipid peroxidation levels in the group with baseline F2-isoprostane 450 μg/mL (high F2-Isop group), whereas did not change the levels of lipid peroxidation in the group with baseline F2-isoprostane o50 μg/mL (low F2-Isop group) (the figure is based on data from Block et al. [80]). (C) Vitamin C supplementation improved exercise performance only in individuals with poor initial vitamin C status (the figure is based on data from Paschalis et al. [69]). ESR, electron spin resonance; F2-Isop, F2-isoprostanes; VO2max, maximal oxygen consumption.

thioredoxins, glutathione peroxidases and catalase) through diverse proposed mechanisms, such as the “floodgate” and “redox relay” hypotheses for peroxiredoxins [89–91]. In contrast to the aforementioned complexity and specificity of the redox network, the measurement of oxidative stress biomarkers provides only a generic “snapshot” of the global redox state of an organism. Subsequently, the use of these descriptive biomarkers conveys

In most studies, oxidative stress biomarkers are evaluated at the systemic level in various blood compartments [93]. Although clinical studies should be also performed in the tissue of interest, this option requires the complicated and costly extraction of tissue samples, while inflammatory responses due to serial biopsies may artificially amplify the signal. Further, the collection of biopsies is a difficult (e.g., skeletal muscle, adipose depots), or even an impossible (e.g., brain, liver) task in human studies. It is, therefore, highly unlikely that human tissues will be routinely analyzed in large populations in an experimental or clinical setting. On the contrary, blood is easily accessible via venipuncture and is, thus, the most widely investigated specimen. This is probably the most important reason why the ultimate objective of the biomarker discovery research still remains to be the development of a simple blood test that will predict and/or confirm the malfunction of a certain organ or tissue [94,95]. Towards this goal, promising advances have been made, such as in the case of monitoring the progression of cardiovascular diseases by measuring circulating levels of oxidized LDL [96,97]. Our group has previously shown that blood adequately reflects tissue oxidative stress for most redox biomarkers, which is particularly useful in a clinical setting [98,99]. However, blood oxidative stress biomarkers cannot be mechanistically informative for two reasons. First, the blood and the respective tissue under study do not share common redox components and/or pathways (e.g., lack of mitochondria in erythrocytes and plasma). This prevents the use of blood for deriving mechanistic information about a process that takes place in a distant tissue. Second, both plasma and blood cells can autonomously produce significant amounts of reactive species [93]. At the same time, blood interacts with all organs and tissues, and consequently, with many possible sources of reactive species and oxidatively modified products [93,100–102]. Hence, blood, and especially plasma (since blood cells maintain a semi-autonomy), do not exclusively transport their own redox load, but instead they afford a comprehensive systemic redox perspective of all organs and tissues. Accordingly, it cannot be assumed that the oxidative stress biomarkers found in blood plasma necessarily originate from the tissue of interest (Fig. 3). In conclusion, the adequate reflection of tissue oxidative stress in blood [98,99] does not equate to a causal relationship. Therefore, it is recommended that mechanistic studies should be performed in the tissue of interest using appropriate analytical approaches (see Fig. 3).

Please cite this article as: N.V. Margaritelis, et al., Going retro: Oxidative stress biomarkers in modern redox biology, Free Radic. Biol. Med. (2016), http://dx.doi.org/10.1016/j.freeradbiomed.2016.02.005i

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and highly selective probes for reactive species identification have been developed and coupled with state-of-the-art analytical techniques, namely electron spin resonance (ESR), fluorescence, magnetic resonance imaging, mass spectrometry and chemiluminescence [103–113]. Unfortunately, some of these techniques are only suitable for use in cell cultures or ex vivo set-ups. Next, we present novel approaches that may yield informative mechanistic information. 4.1. Molecules integrated in pathways linking redox biochemistry to physiology

Fig. 3. Superoxide/hydrogen peroxide and nitric oxide are generated by all tissues (e.g., skeletal muscle, smooth muscle, endothelium) and blood cells (i.e., erythrocytes, leukocytes, platelets) neighboring blood plasma. The superoxide/hydrogen peroxide and nitric oxide generated from multiple sources can oxidize tissue substrates and/or diffuse into blood plasma and oxidize plasma substrates. Therefore, it is virtually impossible to detect the origin of oxidative stress biomarkers measured in blood plasma. This is probably the main reason why oxidative stress biomarkers measured in plasma cannot provide mechanistic insights of any physiological/pathological process in a distant organ/tissue such as skeletal muscle. H2O2, hydrogen peroxide; NADPH, nicotinamide adenine dinucleotide phosphate; NO
, nitric oxide; O2
 , superoxide.

4. Mechanistic redox biomarkers Taking into account the shift towards a more mechanistic description of the biological roles of reactive species, molecular proxies of redox signaling should provide information on its spatiotemporal regulation. During the last decade, great advances have been made towards this aim. In particular, organelle targeted

A potential approach to find mechanistic redox biomarkers is to “deconstruct” the oxidative pathway that results in a specific biological outcome and to select those inherent molecules that plausibly play a crucial role in this pathway. Ideally, these molecules should be able to provide key space (e.g., compartmentalization) and identification (e.g., reactive species implicated) insights into the occurring redox reaction. Ideally, mechanistic biomarkers should also minimally contribute to other key signaling pathways. Having these biochemical features distinguishes mechanistic from descriptive redox biomarkers. A representative approach consistent with this idea is the measurement of the activity of a specific malfunctioning NADPH oxidase subunit in skeletal muscle (e.g., overexpression) coupled with the determination of oxidized myosin content. In fact, NADPH oxidases are regarded as a major source of superoxide production in skeletal muscle [84]. Despite that low-level superoxide production is essential for normal muscle function [114], the steadystate overproduction of superoxide impairs force production (via aberrant contractility like in muscle dystrophy; [115]). In order to obtain more mechanistic insights, the identification of the precise NADPH subunit that is responsible for the continuous overproduction of superoxide becomes obligatory (e.g., gp91phox, p67phox or rac 1; [116]). In addition, equally important to source

NADPH oxidase complex

Aberrantly expressed subunit

Oxidized actin-binding site

Actin Increased production of O2•−

Myosin

CuZnSOD

H2O2

Actin M line

REDUCED MUSCLE FORCE PRODUCTION

Z line

Fig. 4. A simplified exemplar approach showing how molecular measurements (expression of a specific NADPH oxidase subunit and the degree of myosin oxidation) can be linked with a physiological outcome (muscle force production). CuZnSOD, copper–zinc superoxide dismutase; H2O2, hydrogen peroxide; NADPH, nicotinamide adenine dinucleotide phosphate; O2
 , superoxide.

Please cite this article as: N.V. Margaritelis, et al., Going retro: Oxidative stress biomarkers in modern redox biology, Free Radic. Biol. Med. (2016), http://dx.doi.org/10.1016/j.freeradbiomed.2016.02.005i

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Implementation of redox – omics tools after an experimental treatment (e.g., acute exercise)

Selection of the responsive-redox related molecules (candidate redox biomarkers)

Separation of redox biomarkers based on the reversibility of the redox modification Reversible modification

Candidate redox signaling biomarkers (e.g., specific cysteines in target proteins)

Biomarker validation (large trials in physiological/pathological populations)

Identification of the valid and reliable redox signaling biomarkers

Irreversible modification

Candidate oxidative stress biomarkers (e.g., F2 -isoprostanes)

Biomarker validation (large trials in physiological/pathological populations)

Identification of the valid and reliable oxidative stress biomarkers

Fig. 5. Process flow for the development of novel redox biomarkers using -omics tools. The biomarker pipeline is composed of the following essential processes: (i) candidate discovery, (ii) selection of the most responsive molecules, (iii) separation based on the reversibility of oxidative modification, and (iv) validation of the candidate biomarkers in large trials.

identification, is to reveal how reactive species may affect muscle function. Several mechanisms have been proposed, including alteration of Ca2 þ homeostasis, reduction in Na þ –K þ -pump activity, (in)activation of transcription factors and oxidative modifications of the contractile proteins [117,118]. With regard to the latter case, hydrogen peroxide has been reported to react with redox sensitive amino acid residues in the actomyosin interface (i.e., cysteine and methionine) [119,120]. These post-translation modifications reduce the ability of myosin to bind to the actin filament, leading subsequently to a loss of muscle force production (the physiological outcome) (Fig. 4). Thus, the measurement of the oxidized myosin and the evaluation of the expression of the “flopping” NADPH oxidase subunit represent a realistic redox mechanistic couple. 4.2. Exomarkers An appealing approach for acquiring mechanistic insights is the use of artificial exogenously administered biomarker molecules, also known as “exomarkers” [121]. The exomarkers approach is a combination of the spin trap strategy (used in the ESR technique) with the determination of oxidatively modified products (e.g., F2-isoprostanes, protein carbonyls, 8-hydroxy-2′-deoxyguanosine). In fact, a selective and targeted probe is administered directly to an organism and the reaction of this probe with a specific reactive species produces a reporter molecule, the “exomarker”, which is subsequently measured [121]. Changes in the levels of this exomarker proportionally infer changes in the production of the corresponding reactive species. It is important that the probes used as exomarkers also exhibit specific cellular localization (e.g., in mitochondria). The most representative example of this approach is MitoB, which is used for the measurement of the mitochondrial hydrogen peroxide levels in vivo [122]. MitoB incorporates a lipophilic cationic triphenylphosphonium (TPP), which facilitates the transfer of the probe through the inherently negative mitochondrial membrane, driving thereby its accumulation to mitochondria (up to a thousand fold) [112,123]. Within mitochondria, the arylboronic acid, which is bounded to the TPP moiety of MitoB, selectively and irreversibly reacts with hydrogen peroxide and forms a phenol, called MitoP. After a period of incubation, the MitoP/ MitoB ratio is evaluated via mass spectrometry and infers the

mean production of hydrogen peroxide in mitochondria over time [124]. Nevertheless, even this approach is not without caveats. In particular, pre-analytical (e.g., handling of the probe and route of administration), analytical (e.g., methods for accurate quantification of the exomarker) and metabolic (e.g., possible fates of the probe after administration) concerns have been raised [113,121]. However, the use of exomarkers for in vivo exercise redox biology studies holds promise. Yet, there is a need to develop a greater array of exomarkers, particularly for non-mitochondrial cellular compartments. 4.3. Proteomics and lipidomics Another approach that will facilitate the identification of both descriptive and mechanistic redox biomarkers is the use of –omics techniques [7,41]. These techniques provide the opportunity to pinpoint specific molecules, out of a plethora of other options, which change in response to a redox perturbation, and thus can be regarded as candidate redox biomarkers. However, the main goal of the –omics approach is not just the high throughput and global detection of redox-responsive molecules, but also the identification, validation and optimization of key biomolecules that can serve as robust and accurate biomarkers of a redox-related biological process. For instance, redox proteomic analysis of tissue biopsies can identify not only the proteins that undergo oxidative modification, but also the site and extent of oxidation, affording qualitative and quantitative insights [125] (Fig. 5). Due to the broad implication of proteins and lipids in redox reactions and their importance in cell function and structure, proteomics and lipidomics are probably the two most widely investigated types of –omics in redox biology [126–133]. It is important to identify the proteins that become oxidized in response to a redox stimulus (e.g., exercise), since redox signaling typically occurs via reversible oxidation of methionine and cysteine residues [41,85,134]. Beyond signaling, proteomics have been utilized to reveal the biology of irreversible post-translational modifications, which have been proven to contribute to human disorders (e.g., carbonylation of the contractile proteins in muscle dystrophies, chronic obstructive pulmonary disease, cancer cachexia and ageing) [135–137]. Correspondingly, oxidized lipids exert a wide spectrum of bioactive effects associated with physiology and pathology, depending on the nature and content of the

Please cite this article as: N.V. Margaritelis, et al., Going retro: Oxidative stress biomarkers in modern redox biology, Free Radic. Biol. Med. (2016), http://dx.doi.org/10.1016/j.freeradbiomed.2016.02.005i

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oxidized lipid [132,138]. In particular, these effects include the regulation of intracellular signaling cascades through the modulation of signal-transducing receptors, second messengers and transcription factors as well as through the self-transformation of the lipid to an antigen or ligand [139,140]. In addition, oxidized phospholipids have been found to exert Janus-faced effects during inflammatory conditions. For instance, oxidized phospholipidoriginated cyclopentenone-containing compounds contribute to the improvement of the endothelial barrier function, while oxidized phospholipids enhance pro-inflammatory cytokine production, especially in vascular endothelial cells [141,142]. Based on the above, the identification of the exact protein and/or lipid moieties that become oxidized will facilitate the uncovering of the precise mechanistic redox pathways that govern biological processes (e.g., exercise responses and adaptations; [8]). Interestingly, there is high interaction between oxidized proteins and lipids, a fact that further emphasizes the need for their clear-cut identification. Specifically, reactive species-mediated lipid peroxidation is a complicated and generally uncontrolled process (if not terminated by a proper agent), which results in the production of various reactive lipid species [143]. The latter molecules are strong electrophiles and, thus, can readily react with the nucleophilic groups of regional protein thiol residues, modifying thereby their functionality and reactivity [144] (Fig. 6). 4hydroxy-2-nonenal (4-HNE) is a representative example of these lipid species, which can cause posttranslational cysteine modifications via Michael addition (forming lipid–protein adducts) modulating their cellular function [145]. For instance, it was demonstrated that the Nrf2 activation is regulated by the Michael addition of 4-HNE to Kelch-like ECH-associated protein 1 [146]. The recognition of the relationship between the oxidized proteins and lipids has led to the development of assays focused on the identification of lipid-modified cysteines, including highthroughput analyses and large-scale profiling [147–149].

5. The future steps of biomarkers to "survive" validation In order to overcome the various methodological and interpretative difficulties emanated predominately from the global nature of oxidative stress biomarkers, we summarize five desirable methodological directions that could advance their utility in future research:

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(i) Despite the valid spectrophotometric assays available for routine use [150], oxidative stress biomarkers need to be much improved and developed in analytical terms. In fact, almost all methods, techniques and assays used for the quantitative determination of these biomarkers in biological samples have limitations [20]. These limitations relate to pre-analytical and analytical confounding factors and validation issues (e.g., accuracy, reproducibility, robustness) [19,46,151,152]. This concern applies to all oxidative stress biomarkers, even to F2-isoprostanes, which are regarded as the gold standard [32]. For instance, most of the commercially available kits used for the F2-isoprostane measurement do not exclusively measure 8-isoprostaglandin-F2a, but also measure other isomers and/or relevant metabolites (e.g., the 2,3-dinor-8-isoprostaglandin-F2a metabolite) [153]. Moreover, most techniques are not able to distinguish enzymatic from free radical-mediated F2-isoprostane formation [154], whereas it is also unclear if free and/or esterified F2-isoprostanes are finally measured [153]. Additionally, an inter-laboratory validation study found that F2-isoprostane measurement yields different results depending on the technique used (i.e., ELISA, GC–MS or LC-MS; [155]). Evidently, for each biomarker, some techniques are better than others and these techniques are advocated. On the contrary, less accurate methods (e.g., the TBA test for the determination of lipid peroxidation) should be discarded from future studies [156]. (ii) It is important to conduct large-scale inter-laboratory studies, in order to overcome the complications arising from the methodological uniqueness of each study. This uniqueness refers to age, sex, and physical characteristics of the participants, the use of generic antioxidants, the specimen collected, the time point of sample collection, the choice of redox biomarker and the technique used for the determination of oxidative stress [157]. Largescale studies would offer the opportunity for systematization and standardization of both the methodological and analytical practices among different laboratories. Another advantage of this approach would be the production of large data sets, which subsequently, would enable the development of redox biomarker biobanks. The mapping of specific redox signatures with the use of oxidative stress biomarkers (e.g., at rest and in response to acute exercise in healthy and diseased populations) would support the establishment of reference values for each biomarker and enhance their effectiveness in translational research (e.g., novel read-outs for the prevention of a plethora of pathologies). Three representative large-scale collaborative efforts are the European

A Polyunsaturated fatty acid

Oxidation

Lipid peroxidation product / Electrophilic reactive lipid species Michael addition

Lipid-protein adduct

Nucleophilic thiol residue of a regional protein

B COOH Oxidation CH3 (arachidonic acid)

OH

OH

O (4-hydroxy-2-nonenal)

O Michael addition S

SH (cysteine residue)

Protein

Protein

Fig. 6. Lipid-mediated post-translational modification of proteins: (A) a representative mechanism describing how a lipid peroxidation product (a reactive lipid species) may induce a post-translational modification to a protein via the Michael addition reaction. (B) A characteristic example of the process, using arachidonic acid as the primary polyunsaturated fatty acid and 4-hydroxy-2-nonenal as the reactive lipid species.

Please cite this article as: N.V. Margaritelis, et al., Going retro: Oxidative stress biomarkers in modern redox biology, Free Radic. Biol. Med. (2016), http://dx.doi.org/10.1016/j.freeradbiomed.2016.02.005i

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8

Standards Committee on Oxidative DNA Damage (ESCODD), the Biomarkers of Oxidative Stress Study (BOSS Study) and the COST Action B35-‘Lipid Peroxidation Associated Disorders’ [19,155,158– 168]. These international laboratory consortia have addressed methodological and analytical issues and have proposed appropriate oxidative stress biomarker protocols. Similar future collaborations will enhance the development and dissemination of good laboratory practices in the field. (iii) There is a wide spectrum of oxidative stress biomarkers, which are biochemically heterogeneous, and sometimes noncomparable [169]. However, this heterogeneity might actually be advantageous. Indeed, it has already been proposed that different biomarkers do not measure identical aspects of oxidative stress [170,171]. Thus, it is important to unravel the exact redox-regulated biochemical characteristics of each biomarker to improve its utility. Answering the following dichotomous questions may assist in the development of valid biomarkers: (i) Does the biomarker better reflect acute or chronic alterations of redox homeostasis? (ii) Is the biomarker more suitable for assessing redox homeostasis at rest (i.e., steady state) or after an insult? (iii) Does the biomarker better serve as a predictor or as a monitor of a particular situation? (iv) Can the biomarker distinguish alterations in redox homeostasis inferred by an oxidant or an antioxidant? A comprehensive description of the oxidative stress biomarker responses after a redox treatment would facilitate the establishment of biomarker sets, including only the most suitable biomarkers according to the experimental question. (iv) Oxidative stress biomarkers, either as intermediary or end products, are frequently regarded as inert. However, this is not always the case. For example, F2-isoprostanes, apart from being indicators of lipid peroxidation and being risk/progression factor for coronary heart disease [172], they also serve many additional biological purposes. In particular, it has been proposed that F2-isoprostanes exert cardiovascular effects by inhibiting angiogenesis through activation of the thromboxane A2 receptor [173]. Moreover, F2-isoprostanes have been implicated in the production of hepatic collagen, in the contraction of human bronchial smooth muscle, in platelet activation, in mitogenesis of vascular smooth muscle cells and in the vasoconstriction of the renal glomerular arterioles [174,175]. Such bioactivity is not confined to F2-isporstanes, but it is also evident in several other biomarkers, including but not limited to 3-nitrotyrosine [176], 4-HNE [145] and malondialdehyde [177,178]. Consequently, it is very important to acknowledge the broader biological roles of the oxidative stress biomarkers and to reveal whether the outcomes assessed are a direct or indirect effect of reactive species generation and/or of differential biomarker metabolism. (v) It is important to elucidate some underestimated issues that may affect the results and the interpretation of studies using oxidative stress biomarkers. For example, diurnal variation [179– 182] and the dependence of the responses on the initial values [70,78,80,81,183] are two factors usually overlooked. For instance, if acute exercise is performed during a period of the day naturally characterized by “high values” of oxidative stress, or if the participants exhibit high values already before the exercise session (e.g., smokers or individuals with vitamin C deficiency; [71,184]), then, the post-exercise increase will be probably lower. It follows that it is significant to clearly establish the effects of the aforementioned parameters when interpreting study results.

6. Conclusion "Many a free radical biologist has dreamed of the day when a simple blood or urine test would divulge the oxidative stress status of an individual and the prediction that some organ or

tissue might need closer examination." Despite the fact that this piece was written by William Pryor in 1999, we believe that the dream described back then is still unrealized [79]. This is not meant to imply that no significant progress has been attained in the development of oxidative stress biomarkers. On the contrary, our analysis demonstrates that oxidative stress biomarkers have the following notable applications: (1) diagnosing oxidative stress, (2) pinpointing likely redox components in a physiological or pathological process, and (3) estimating the severity, progression and/or regression of a disease. Still, there is a long way to go until the ultimate goal of any oxidative stress biomarker being achieved: to be established as a clinical diagnostic.

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