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2017; 3(7):26–37

REVIEW ARTICLES

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Hydrogen Peroxide in Biology and Medicine: An Overview

 

Robert Z. Hopkins

AIMSCI Research Institute, P.O. Box 37504, Raleigh, NC 27626, USA

Correspondence: rzh@aimsci.com (R.Z.H.)

Hopkins RZ. Reactive Oxygen Species 3(7):26–37, 2017; ©2017 Cell Med Press

http://dx.doi.org/10.20455/ros.2017.809

(Received: December 23, 2016; Revised: January 6, 2017; Accepted: January 7, 2017)

ABSTRACT | Hydrogen peroxide (H2O2) is one of the most extensively studied reactive oxygen species  (ROS) in biology and medicine. It is generated constitutively from various cellular processes either directly via two-electron reduction of molecular oxygen indirectly via dismutation of superoxide. The notable direct cellular sources for H2O2 include xanthine oxidoreductase, monoamine oxidase, endoplasmic reticulum oxicireductin 1, oxidases in peroxisomes, and possibly certain members of the NOX/DUOX family. Because of the high activation energy, H2O2 reacts poorly with most cellular constituents. However, it may oxidize the thiol groups in certain proteins and enzymes, including these involved in cell signaling transduction. The potential of H2O2 to cause oxidative stress and tissue injury primarily results from its reactions with other molecules to form secondary reactive species, including hydroxyl radical and hypochlorous acid. While the tightly controlled production of H2O2 plays important roles in various physiological responses, overproduction of this ROS contributes to the pathophysiology of a variety of disease processes and related conditions, including cardiovascular diseases, diabetes, neurodegeneration, cancer, and aging, among many others.

KEYWORDS | Disease process; Hydrogen peroxide; Immunity; Redox signaling

ABBREVIATIONS | CYP, cytochrome P450; ERO1, endoplasmic reticulum oxidoreductin 1; MAO, monoamine oxidase; MPO, myeloperoxidase; NOX, NADPH oxidase; ROS, reactive oxygen species; SOD, superoxide dismutase


CONTENTS

1. Overview

2. Sources

2.1. Indirect Formation via Superoxide Dismutation

2.2. Direct Formation via Two-Electron Reduction

3. Chemistry and Biochemistry

3.1. General Chemical Properties

3.2. Oxidation of Protein Sulfhydryl Groups

3.3. Fenton Reaction to Form Hydroxyl Radical

3.4. Reaction with Chloride Ion Forming Hypochlorous Acid

3.5. Reaction with Other Molecules

3.6. Half-Life, Diffusion, and Membrane Permeability

4. Cell and Tissue Defenses

5. Biology and Medicine

5.1. Innate Immunity

5.2. Adaptive Immunity

5.3. Redox Signaling

5.4. Stem Cell Biology

5.5. Wound Healing

5.6. Circadian Rhythm

5.7. Disease Process


1. OVERVIEW

Hydrogen peroxide (H2O2) was discovered in 1818 by Louis Jacques Thénard (1777‒1857), a French chemist [1], and the biological catalyst of H2O2 was identified and named as catalase in 1900 by Oscar Loew (1844‒1941), a German chemist [2]. In the early 1970s, H2O2 was shown to be produced by animal cells and tissues [3‒5]. It is now known that formation of H2O2 occurs ubiquitously in both animal and plant cells, as well as microorganisms. The past two decades have witnessed the explosion of knowledge on H2O2 in biology and medicine, ranging from its well established ability to cause oxidative stress and tissue injury to its emerging roles in cell signaling and normal physiology. Indeed, H2O2 is also among the most extensively investigated reactive oxygen species (ROS) in biology and medicine.

In biological systems, H2O2 is the primary product of superoxide dismutation, which occurs either spontaneously or catalyzed by superoxide dismutase. Hence, the sources of production and the biological activities of these two ROS overlap significantly. Different from superoxide, H2O2 is a non-radical species with a relatively long half-life in biological milieu and is able to readily cross cell membranes and diffuse into different cellular compartments. As such, H2O2 may act as a novel second messenger in cell signal transduction. This review article considers the source, chemistry and biochemistry, as well as biology and medicine of this simple, but biologically unique molecule.

2. SOURCES

H2O2 is a major ROS formed in animal cells from various intracellular sources, which are discussed next. It is noteworthy that H2O2 is also formed in plant cells, with mitochondria and chloroplasts being the major sources [6]. In addition to the animal and plant kingdoms, H2O2 is found in Earth’s atmosphere as well as interstellar space [7]. Regarding the cellular production of H2O2 in animals, including humans both direct and indirect mechanisms have been identified (Figure 1).

FIGURE 1. Cellular sources of hydrogen peroxide (H2O2). Cellular H2O2 production may result from either dismutation of superoxide or directly via two-electron reduction of molecular oxygen. Enzymes that directly catalyze two-electron reduction of molecular oxygen to form H2O2 include xanthine oxidoreductase (XOR), monoamine oxidase (MAO), endoplasmic reticulum (ER) oxidoreductin 1 (ERO1), and oxidases in the peroxisome, as well as possibly p66SHC and some members of the NADPH oxidase (NOX)/DUOX family (e.g., NOX4, DUOX1, DUOX2). XOR is also capable of catalyzing one-electron reduction of molecular oxygen to superoxide. METC, mitochondrial electron transport chain; CYP, cytochrome P450 system.

2.1. Indirect Formation via Superoxide Dismutation

H2O2 is formed through either the spontaneous or superoxide dismutase (SOD)-catalyzed dismutation of superoxide. Therefore, the chief sources for superoxide formation are also the main ones for H2O2. In this regard, NADPH oxidases (also known as NOXs) and mitochondrial electron transport chain are widely considered the primary sources of superoxide-derived H2O2 in mammalian cells. Other sources of superoxide-derived H2O2 include xanthine oxidoreductase, cytochrome P450 enzyme system, and uncoupled endothelial nitric oxide synthase (eNOS), as well as the redox cycling of environmental chemicals and drugs by cellular one-electron reduction systems (e.g., cytochrome P450 reductase).

2.2. Direct Formation via Two-Electron Reduction

Some enzymes in mammals including humans may directly catalyze the two-electron reduction of molecular oxygen to form H2O2 or predominately produce H2O2 via an unclear mechanism. These include xanthine oxidoreductase, monoamine oxidase, some members of the NOX/DUOX family (e.g., NOX4, DUOX1, DUOX2), and multiple oxidases in peroxisomes, among others.

2.2.1. Xanthine Oxidoreductase

As noted above, xanthine oxidoreductase catalyzes the one-electron reduction of molecular oxygen to form superoxide. This enzyme also directly catalyzes the two electron-reduction of molecular oxygen to form H2O2. Hence, xanthine oxidoreductase is capable of causing both one- and two-electron reduction of molecular oxygen to form superoxide and H2O2, respectively [8].

2.2.2. Monoamine Oxidase

Another enzyme capable of directly producing H2O2 is the flavin-dependent monoamine oxidase (MAO). This enzyme catalyzes deamination of dopamine through a two-electron reduction of molecular oxygen to H2O2 [9]. There are two types of MAO, namely, MAOA and MAOB, and both are located in (or bound to) the outer membrane of mitochondria in most cell types in the body.

2.2.3. NOX/DUOX Family

While superoxide is the primary product of most NOX enzymes, NOX4 may predominantly produce H2O2 rather than superoxide [10, 11]. Dual oxidases 1 and 2 (DUOX1 and DUOX 2), members of the NOX/DUOX family, may also primarily generate H2O2 [12]. However, it remains unclear whether these enzymes can directly catalyze the two-electron reduction of oxygen to H2O2 or they produce H2O2 via a possible superoxide intermediate that may not be detected by current techniques due to rapid intramolecular dismutation or inaccessibility to the superoxide-detecting probes [12, 13].

2.2.4. Endoplasmic Reticulum

Endoplasmic reticulum (ER) is a significant source of cellular H2O2 due to the presence of various oxidoreductases in this organelle. While the cytochrome P450 enzyme (CYP) system associated with ER is a major indirect source of H2O2 (from dismutation of CYP-derived superoxide), oxidoreductases present in the ER lumen can directly reduce oxygen to form H2O2. For instance, the endoplasmic reticulum oxidoreductin 1 (ERO1), also known as endoplasmic reticulum oxidase 1, is a major source of H2O2 formed in the ER lumen [14]. The H2O2 produced by ERO1 plays an important role in oxidative protein folding in the ER. However, in cells lacking ERO1, H2O2 is also formed in the ER lumen and fuels peroxiredoxin 4-mediated oxidative protein folding, suggesting the existence of an unrecognized luminal source of H2O2 [15].

2.2.5. Oxidases in Peroxisomes

Peroxisomes contain various enzymes that produce H2O2 as part of their normal catalytic cycle. These enzymes include acyl-CoA oxidases, urate oxidase, D-amino acid oxidase, D-aspartate oxidase, L-pipecolic acid oxidase, L-α-hydroxyacid oxidase, polyamine oxidase, and xanthine oxidase [16].

2.2.6. Others

The mitochondria-associated redox protein p66SHC is a genetic determinant of lifespan in mammals [17]. This redox protein may reduce oxygen to H2O2 by utilizing the reducing equivalents of the mitochondrial electron transport chain via oxidation of cytochrome c [18].

Although not naturally occurring in mammalian tissues, glucose oxidase, an enzyme expressed in certain fungal species, is perhaps among the best know enzymes for producing H2O2. Glucose oxidase catalyzes the oxidation of beta-D-glucose to gluconic acid, by utilizing molecular oxygen as an electron acceptor with simultaneous production of H2O2 [19, 20]. This enzyme has a number of industrial and biotechnological applications, with its use in measurement of blood glucose being most notable [19, 20].

3. CHEMISTRY AND BIOCHEMISTRY

3.1. General Chemical Properties

H2O2 is a strong two-electron oxidant, with a standard reduction potential of 1.32 V at pH 7.0 (H2O2/H2O). It is therefore more oxidizing than hypochlorous acid (OClˉ/Clˉ) and peroxynitrite (ONOOˉ /NO2ˉ), for which the standard reduction potentials are 1.28 and 1.20 V, respectively. However, in contrast to the above two oxidants, H2O2 reacts poorly or not at all with most biological molecules, including proteins, nucleic acids, and lipids, as well as low-molecular-weight antioxidants. This is because a high activation energy barrier must be overcome to release its oxidizing power, or in other words, the reactions of H2O2 are kinetically rather than thermodynamically driven [21].

Nevertheless, as discussed below, via two-electron oxidation, H2O2 reacts readily with certain biological molecules especially protein thiols to account for much of its signaling function. On the other hand, H2O2 is a weak one-electron oxidant with the standard reduction potential of 0.32 V (H2O2/ OH˙). But, its reaction with transition metals (e.g., iron and copper) generates the highly reactive hydroxyl radical which may account for much of the detrimental effects of H2O2 in biological systems.

3.2. Oxidation of Protein Sulfhydryl Groups

Although in general the reaction between H2O2 and proteins is much limited, the cysteine thiol groups (also known as sulfhydryl groups) in certain proteins are readily oxidized by H2O2. These proteins include antioxidant enzymes (e.g., peroxiredoxins) and cell signaling molecules (e.g., certain transcription factors) [22‒24]. Protein thiol oxidation is now recognized as a major chemical basis behind H2O2 sensing and signaling [25, 26] (see section below). However, extensive oxidation of protein thiols by large amounts of H2O2 causes irreversible oxidative protein damage, resulting in cell injury. Figure 2 depicts the redox modifications of protein thiols by H2O2.

FIGURE 2. Chemical and biochemical reactivity of hydrogen peroxide (H2O2). As illustrated, H2O2 may directly react with the thiol groups of the cysteine residues in certain proteins, resulting in the formation of protein sulfenic acid (protein-SOH), sulfinic acid (protein-SO2H), and sulfonic acid (protein-SO3H). These modifications may cause altered protein function and oxidative protein damage depending on the levels and duration of H2O2 exposure (also see the legend of Figure 9.3). H2O2 reacts with transition metal ions, such as ferrous iron ion (Fe2+), producing the highly reactive hydroxyl radical (OH˙). Likewise, reaction between H2O2 and chloride ion (Clˉ) in the presence of myeloperoxidase (MPO) results in the formation of hypochlorous acid (HOCl), another potent oxidant. Production of these secondary reactive species is largely responsible for H2O2-induced oxidative damage in biological systems. Although hydrogen peroxide at high levels causes damage to cells and tissues, under certain circumstances, at lower levels it can act as a signaling molecule to participate in cell signal transduction.

As illustrated in the above figure as well as Figure 3, mild oxidation of protein thiols by H2O2 results in the formation of protein sulfenic acid, which is unstable and readily reacts with an adjacent protein thiol group (either on the same protein or another protein) to form protein disulfides or with reduced form of glutathione (GSH) to become glutathionylated. The above redox modifications of proteins are reversible via the actions of antioxidant enzymes, including thioredoxin and glutaredoxin systems. Such a reversible nature is instrumental in H2O2-mediated redox signaling.

FIGURE 3. Thiol-dependent redox mechanisms of cell signaling mediated by hydrogen peroxide (H2O2). Oxidation of protein cysteine thiol groups by H2O2 is a major chemical basis for this ROS-mediated redox signaling. In this regard, a moderate and transient increase in the levels of H2O2 may cause oxidation of the cysteine thiols in certain signaling proteins, resulting in the formation of protein sulfenic acid (protein-SOH). Due to its high reactivity, the protein-SOH reacts with another cysteine thiol either on the same or another protein, forming protein disulfides (protein-S-S-protein). These reactions are reversible via the action of thioredoxin (Trx) system. The reversibility of the above reactions makes it possible for H2O2 to transiently alter the functionality of the protein (e.g., a protein kinase or a transcription factor), ensuring redox signaling. On the other hand, high levels and prolonged duration of H2O2 exposure may cause further oxidation of protein sulfenic acid to form protein sulfinic acid (protein-SO2H) and sulfonic acid (protein-SO3H). These hyperoxidative reactions are generally irreversible and thereby cause protein dysfunction and oxidative damage.

On the other hand, prolonged exposure to large amounts of H2O2 can cause further oxidation of the protein sulfenic acid to form sulfinic acid and oxidation of sulfinic acid to form sulfonic acid. Such hyperoxidative modifications of protein thiols typically result in irreversible damage to the protein (Figure 3). Hence, H2O2 serves as a signaling molecule only when its formation is tightly regulated. In this context, multiple families of enzymes are involved in the decomposition of H2O2 (see Section 4).

3.3. Fenton Reaction to Form Hydroxyl Radical

Reaction of H2O2 with transition metal ions gives rise to the formation of hydroxyl radical (OH˙), an extremely potent oxidant. The Fenton reaction (Fe2+ + H2O2 → Fe3+ + OH˙ + OHˉ) is an important mechanism for H2O2-mediated oxidative damage. Other metal ions such as cuprous ion (Cu1+) can also catalyze the formation of hydroxyl radical from H2O2 via a similar reaction called Fenton-type reaction: Cu1+ + H2O2  → Cu2+ + OH˙ + OHˉ).

3.4. Reaction with Chloride Ion Forming Hypochlorous Acid

Reaction of H2O2 with chloride (Clˉ) generates hypochlorous acid (HOCl), a potent oxidant (H2O2 + Clˉ → HOCl + OHˉ). This reaction is catalyzed by myeloperoxidase (MPO) found in phagocytic cells. The HOCl formed is involved in the killing of invading microorganisms by phagocytic cells. On the other hand, abnormal formation of HOCl also contributes to tissue injury, such as atherosclerosis [27].

3.5. Reaction with Other Molecules

H2O2 oxidizes pyruvate to form acetate and CO2 with a reaction rate constant of 2.2 M‒1s‒1 and as such, pyruvate may act as an efficient biological scavenger of H2O2 [28]. Indeed, pyruvate present in cell culture media or inside the cells has been shown to inhibit the biological activity of H2O2 [29‒31].

H2O2 reacts with CO2 to form peroxymonocarbonate (H2O2 + CO2 → HCO4ˉ + H+), which is much more reactive to thiols and methionine [21]. The biological significance of this reaction remains to be elucidated though peroxymonocarbonate may give rise to carbonate radical (CO3˙), a potent oxidizing species. Reaction of H2O2 with Cu,ZnSOD has also been shown to produce secondary oxidants and inactivation of the enzyme [32, 33].

3.6. Half-Life, Diffusion, and Membrane Permeability

In biological systems, H2O2 has a relatively long half-life in the range of minutes depending on the levels of surrounding H2O2-decomposing enzymes (e.g., catalase, glutathione peroxidase, peroxiredoxin). It has been long known that H2O2 readily crosses mammalian cell membranes. Recently, several specific aquaporin (water channel) isoforms (e.g., AQP3, AQP8, AQP9) are found to facilitate the passive diffusion of H2O2 across cell membranes and influence the cellular effects (e.g., cytotoxicity) of this ROS [34‒36]. This is not surprising as water and H2O2 share similar physicochemical properties.

Notably, aquaporin-facilitated H2O2 transport may also regulate H2O2 signaling [35, 37]. For example, a recent study shows that aquaporin-3-mediated H2O2 transport is required for nuclear factor kappa B (NF-κB) signaling in keratinocytes and development of psoriasis in an animal model [37]. Additionally, aquaporin-3 also controls breast cancer cell migration and metastasis by regulating hydrogen peroxide transport and its downstream cell signaling (e.g., the Akt pathway) [38].

4. CELL AND TISSUE DEFENSES

H2O2 is decomposed to water by several enzymes in mammals including humans. These include catalase, glutathione peroxidase, and peroxiredoxin. As noted earlier in Section 3.5, pyruvate (or pyruvic acid) present in biological systems can spontaneously detoxify H2O2 via a nonenzymatic decarboxylation reaction. In addition to pyruvate, other α-keto acids, such as α-ketoglutarate, oxaloacetate, glyoxylate may also scavenge H2O2 via a similar mechanism [39, 40].

5. BIOLOGY AND MEDICINE

As mentioned above, H2O2 is among the most extensively investigated ROS in biology and medicine. Substantial evidence points to the important roles played by this ROS, ranging from both innate and adaptive immunity to cell signaling involved in stem cell proliferation and wound healing. On the other hand, abnormal production of H2O2 causes oxidative stress and tissue injury, thereby contributing to disease pathophysiology.

5.1. Innate Immunity

As a major product of phagocytic respiratory burst, H2O2 is involved in the killing of the invading pathogens via the formation of hypochlorous acid, a much more potent oxidant (see Section 3.4). H2O2 may also kill the microorganisms via the formation of hydroxyl radical through the Fenton reaction (see Section 3.3). In addition to its antiseptic role, a recent study using zebrafish shows that H2O2 formed by dual oxidase (DUOX) at the wound margin and the resulting H2O2 concentration gradient are required for the rapid recruitment of leukocytes to the wound [41]. This finding reveals a novel role for H2O2 to potentially act as a leukocyte chemoattractant in innate immunity.

5.2. Adaptive Immunity

Mitochondria-derived ROS have recently been demonstrated to play important roles in adaptive immunity, including regulation of T cell activation and CD8+ memory T cell formation, as well as B cell fate determination upon activation [42‒44]. Although the exact ROS involved in the above processes remain unclear, H2O2 appears to be the most likely ROS that acts as a signaling molecule to regulate adaptive immunity [45]. In this regard, H2O2 is among the best characterized ROS involved in cell signal transduction.

5.3. Redox Signaling

It is well recognized that the regulated formation of H2O2 from various sources (including NOX and mitochondria) serves as an important mechanism of cell signaling. Oxidation of the cysteine thiol by H2O2 in signaling proteins (e.g., proteins kinases /phosphatases, receptors, and transcription factors) appears to be a major molecular basis underlying H2O2-mediated redox signaling [25, 26] (Figure 3).

Notably, a recent study shows that peroxiredoxin-2 (Prx2, a H2O2-decomposation enzyme) and STAT3 form a redox relay for H2O2 signaling. Specifically, H2O2 oxidizes Prx2, and the oxidized Prx2 forms a redox relay with the transcription factor STAT3 in which oxidative equivalents flow from Prx2 to STAT3. The redox relay generates disulfide-linked STAT3 oligomers with attenuated transcriptional activity. Cytokine-induced STAT3 signaling is accompanied by Prx2 and STAT3 oxidation and is modulated by Prx2 expression levels [46]. The redox signaling role of H2O2 explains its involvement in diverse conditions, such as stem cell proliferation and wound healing.

5.4. Stem Cell Biology

H2O2 is involved in stem cell biology. While high levels of H2O2 cause injury and shorten the lifespan of stem cells [47], regulated production of H2O2 may be essential for stem cell proliferation. In this regard, Dickinson et al. show that adult hippocampal stem/progenitor cells generate H2O2 through NOX2 to regulate intracellular growth signaling pathways, which in turn maintains their normal proliferation in vitro and in vivo [48].

5.5. Wound Healing

As noted above, wounded epithelial cells release H2O2 and generate a tissue-scale gradient of H2O2, which guides leukocyte recruitment to the wound site to kill pathogens, minimize infection, and promoting healing [41]. In addition, low levels of H2O2 may also cause proliferation of keratinocytes as well as promote angiogenesis via augmenting epithelial growth factor and endothelial growth factor signaling, respectively [49, 50].

5.6. Circadian Rhythm

Light is the key entraining stimulus for the circadian clock, but several features of the signaling pathways that convert the photic signal to clock entrainment remain to be deciphered. Hirayama et al. show that light induces the production of H2O2 that acts as the second messenger coupling photoreception to the circadian clock in zebrafish [51]. Recent studies suggest that mitochondrial release of H2O2 is also likely a circadian event that conveys temporal information on steroidogenesis in the adrenal gland and on energy metabolism in the heart and brown adipose tissue to cytosolic signaling pathways [52, 53].

5.7. Disease Process

Due to its readily commercial availability, H2O2 is perhaps the most widely used chemical for studying oxidative stress in experimental models. Indeed, much of our current knowledge in oxidative stress results from studies using exogenous H2O2. Studies on the involvement of endogenously generated H2O2 in disease process have been frequently done with animal models of catalase gene knockout or overexpression. In this regard, like SOD for selective metabolizing superoxide, catalase is a highly selective enzyme for the detoxification of H2O2. As such, the impact of manipulating cellular or tissue catalase on disease pathogenesis can be reasonably interpreted as a causal involvement of H2O2 in the disease process. Using primarily catalase gene knockout or overexpression animal models, extensive studies over the past decades suggest an important role for H2O2-induced oxidative stress in a wide variety of disease processes and related conditions. These include various forms of cardiovascular disorders [54‒58], diabetes and metabolic syndrome [59‒61], multistage tumorigenesis [62‒64], neurodegeneration [65, 66], pulmonary injury [67, 68], hepatic injury [69], and osteoporosis [70], as well as aging [71‒74], among many others.

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