Endogenous Generation of Singlet Oxygen and Ozone in Human and Animal Tissues: Mechanisms, Biological Significance, and Influence of Dietary Components



Recent studies have shown that exposing antibodies or amino acids to singlet oxygen results in the formation of ozone (or an ozone-like oxidant) and hydrogen peroxide and that human neutrophils produce both singlet oxygen and ozone during bacterial killing. There is also mounting evidence that endogenous singlet oxygen production may be a common occurrence in cells through various mechanisms. Thus, the ozone-producing combination of singlet oxygen and amino acids might be a common cellular occurrence. This paper reviews the potential pathways of formation of singlet oxygen and ozone in vivo and also proposes some new pathways for singlet oxygen formation. Physiological consequences of the endogenous formation of these oxidants in human tissues are discussed, as well as examples of how dietary factors may promote or inhibit their generation and activity.


1. Introduction


Singlet oxygen (1O2) is an electronically excited form of oxygen which is well known to be formed when photosensitizers such as chlorophyll or the aromatic dye rose bengal absorb light energy and transfer some of that energy to molecular oxygen [1, 2]. Various nonphotosensitized mechanisms for its formation have also been reported and suggested to occur in biological systems, but the importance of such endogenous singlet oxygen formation has had a controversial history [1, 3]. Ozone (O3) is best known as occurring in the stratosphere where it shields organisms on earth from ultraviolet C and much of ultraviolet B radiations, which are the most damaging UV components of solar radiations because they are readily absorbed by DNA [4, 5]. It is also known as a respiratory system-damaging pollutant in the troposphere and ironically as a therapeutic agent in alternative medicine [6]. More recently, it was shown that antibodies or amino acids catalyze the conversion of singlet oxygen (1O2) to ozone (O3) and that this reaction occurs during the killing of bacteria by activated neutrophils [7, 8]. Since both singlet oxygen and ozone are highly reactive oxygen species, a full understanding of their mechanisms of formation and action in vivo is necessary. Hence, this paper reviews the various reported mechanisms of the endogenous formation of these reactive oxygen species (ROS), the potential relevance of such pathways in human physiology, and how dietary factors affect the generation and activity of these oxidants.


2. Radiation-Induced Formation of Singlet Oxygen


Human beings are frequently exposed to natural and artificial radiation, and most of this interacts primarily with the skin. The spectrum of solar radiation at the earth*s surface consists of ultraviolet (UV) radiation (UVB: 290每320nm and UVA: 320400nm), visible radiation (VIS: 400760nm), and near infrared radiation (IRA: 7601440nm and IRB: 14403000nm) [9]. UV, VIS, and IR contribute 7%, 39%, and 54% of the solar energy reaching the skin [10]. Direct absorption of UVB by cellular DNA leads to formation of cyclobutane pyrimidine dimers and pyrimidine (6每4) pyrimidone products, while UVA is not readily absorbed by DNA, and its direct damage to DNA is therefore not important [5]. Nevertheless, both UVA and UVB as well as visible light convert various photosensitizing compounds to excited states which transfer energy to triplet oxygen, thereby generating reactive oxygen species, particularly singlet oxygen.


UVA makes up 95% of the UV reaching the human skin, and up to 50% of it can penetrate to the dermis, unlike UVB that only penetrates the epidermis [11]. The human skin is rich in UVA and visible light (particularly the blue region) photosensitizers such as porphyrins, bilirubin, flavins, melanin and melanin precursors, pterins, B6 vitamers, and vitamin K [12, 13]. The formation of singlet oxygen in the skin as a result of the interaction of UVA with these photosensitizers has been demonstrated directly by luminescence [14] and by detection of cholesterol-5-hydroperoxide which is preferentially generated by singlet oxygen but not by free radical mediated cholesterol oxidation [2]. The interaction between UVB and various vitamins and fatty acids also results in the generation of singlet oxygen, and some compounds including vitamin E that are ordinarily not UVA photosensitizers can be converted to UVA photosensitizers if they are preirradiated with UVB [11]. Photosensitized formation of singlet oxygen also occurs in the retina, which contains endogenous photosensitizers and is exposed to light [15]. One of the singlet oxygen-generating photosensitizers is lipofuscin, which forms in the retinal pigment epithelium with age or genetic disorders such as Stargardt*s disease [15, 16]. Ground state oxygen can directly absorb visible light of 765nm, even in mammalian cells, leading to formation of singlet oxygen without the involvement of a photosensitizer [17]. Similarly, IRB of 1268nm can cause direct conversion of ground state oxygen to singlet oxygen [18].


Both IRB and IRC penetrate the skin only shallowly, while IRA (which makes up 30% of the total IR radiation reaching the skin) penetrates deeply, with 65% of it reaching the dermis [19, 20]. Unlike UVA, IRA penetration of the skin does not cause photosensitized formation of singlet oxygen but initiates the formation of reactive oxygen species, mainly from the mitochondrial electron transport chain [9, 20, 21]. While singlet oxygen may be one of these ROS [9], its specific detection under such circumstances has not been studied. However, both UVA and IR induce upregulation of matrix metalloproteinases (MMPs) and thereby promote photoaging [9, 19]. The UV-induced MMP expression is dependent on cholesterol-5 hydroperoxide, a product of oxidation of cholesterol by singlet oxygen [2, 22]. Whether IR-induced metalloproteinase activation also depends to a great extent on singlet oxygen and cholesterol-5 hydroperoxide remains to be demonstrated. In this case, the role of IR in singlet oxygen formation may simply involve initiating the formation of superoxide anions, from which singlet oxygen would be generated by various types of radiation-independent reactions (vide infra). Singlet oxygen formation in organs other than the skin and eye mainly depends on such ※dark§ reactions.


Artificial sources of radiation may also contribute to endogenous singlet oxygen formation in humans. For example, during photodynamic therapy, a photosensitizer is inserted into cancerous tissue and irradiated with UV to produce singlet oxygen which serves the purpose of destroying cancer cells [23]. IR irradiation is commonly used in medicine to warm muscle tissue [24] and might also contribute to singlet oxygen formation.



3. Leukocyte-Mediated Formation of Singlet Oxygen


Neutrophils, including human neutrophils, produce singlet oxygen [7, 35每37] and this has been suggested to be important for bacterial killing through the formation of ozone [7]. It is generally considered that production of singlet oxygen by neutrophils is dependent on myeloperoxidase (MPO) which catalyzes the formation of hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) and chloride ion (see equation (1)), followed by reaction of HOCl with hydrogen peroxide anion () (see equation (2)) [7, 37]. However, the significance of the reaction between  and HOCl under physiological environments such as the intraphagosomal milieu may be limited by the presence of other reactive partners for HOCl [36], and it was suggested that alternative pathways of singlet oxygen generation by neutrophils may exist, including the spontaneous dismutation of superoxide anions (see equation (3)) [36, 37]. However, the yield of singlet oxygen from the latter reaction was also found to be minor [38]. Peritoneal macrophages, which are MPO deficient, produce higher yield of singlet oxygen than neutrophils [37]. In the macrophage phagosome, the reaction between nitric oxide () and superoxide anion () occurs at diffusion-controlled rates to form peroxynitrite () (see equation (4)) [39], which reacts with H2O2 to produce singlet oxygen (see equation (5)) [40]. The reaction of  with H2O2 was also found to generate singlet oxygen in a purely chemical system and in a superoxide generating system (see equation (6)) [41, 42]. The eosinophil peroxidase system generates singlet oxygen by a reaction between HOBr and , analogously to (2) [30]:



4. Singlet Oxygen Formation by the Russel Mechanism


Russell [43] proposed the idea that two peroxyl radicals can react to form an unstable tetroxide whose decomposition affords singlet oxygen, an alcohol, and a carbonyl compound, and this mechanism is now believed to contribute to singlet oxygen formation from various biomolecules including proteins, lipids, and nucleic acids [44]. The oxidation of DNA was found to result in singlet oxygen by this mechanism as illustrated in Figure 1, whereby thymine peroxyl radicals 1 react to generate tetroxide 2 whose decomposition produces alcohol 3, carbonyl 4, and 1O2 [25].


5. Singlet Oxygen Formation via the Dismutation of Alkoxyl Radicals


Two alkoxyl radicals () can undergo dismutation to form a carbonyl and an alcohol (Figure 4), and some of the carbonyls are formed in the excited triplet state, with a yield of up to 8% [28]. The triplet carbonyls can transfer energy to triplet oxygen, thereby generating singlet oxygen [28]. Because alkoxyl radicals are major intermediates during decomposition of biological hydroperoxides [28, 48], the potential contribution of this pathway to singlet oxygen formation cannot be ignored.


6. Singlet Oxygen Formation via the Oxidation of Phenolic Substances


Phenolic substances are important components of the human diet, and one of such compounds is the amino acid tyrosine. In many physiological situations, tyrosine 11 gets converted to the tyrosyl radical 12, which in turn gets converted by superoxide anions to tyrosine hydroperoxide 13, whose decomposition may produce singlet oxygen and regenerate tyrosine (Figure 5) [40]. However, tyrosine hydroperoxide 13 also gets converted to its bicyclic isomer 14, whose decomposition does not produce singlet oxygen, and this greatly reduces the amount of singlet oxygen formed from this system [29]. Nevertheless, this mechanism may be important because tyrosine and tryptophan residues are known to be major contributors to protein-dependent singlet oxygen formation [32].


7. Singlet Oxygen Formation via Dioxetanes


Dioxetanes are high energy 1,2-peroxides whose decomposition affords excited carbonyls in high yields [32]. R芍c et al. [47] recently suggested that their observed formation of singlet oxygen in U937 human leukemic cells treated with H2O2 or the Fenton reagent was mainly due to decomposition of dioxetane intermediates.


8. Singlet Oxygen Formation by the Reaction of Superoxide Anion with Hydrogen Peroxide


The reaction of superoxide anion with hydrogen peroxide to form singlet oxygen, hydroxyl radical, and hydroxide ion (see equation (6)), a modified form of the Haber-Weiss reaction, was proposed by Kellogg and Fridovich [63] and demonstrated upon the reaction of potassium superoxide with hydrogen peroxide in a simple reaction system [64]. However, this reaction is controversial: Koppenol [65] registered strong disapproval for it, mainly based on the fact that various studies found that the rate constant for the Haber-Weiss reaction is in the order of 1M1s1 or less.


9. Singlet Oxygen Formation via Cytochrome c-Mediated Formation of Triplet Carbonyls


Cytochrome c converts carbonyls such as lipid-derived aldehydes to triplet carbonyls, which then transfer energy to oxygen, thus generating singlet oxygen [66]. In fact, singlet oxygen formation from a model membrane having polyunsaturated fatty acid-containing cardiolipin in association with cytochrome C was found to be more dependent on triplet carbonyls than on the decomposition of hydroperoxides via the Russel mechanism [66].


10. Singlet Oxygen Formation by the Reaction of Hydroperoxides with Carbonyls


Under certain conditions such as in the presence of pyrogallol, lysine, tryptophan, or superoxide anions, the interaction of H2O2 with carbonyls such as formaldehyde, acetaldehyde, glyoxal, methyl-glyoxal, and even glucose was demonstrated to produce singlet oxygen and reactive aldehydes [51, 67每72], and such conditions should be common in vivo: considering that carbonyls are major lipid oxidation and glycoxidation products, all cells have formaldehyde generating pathways referred to as the formaldehydome [6, 69, 71], and hydrogen peroxide is also generated through many enzymatic and nonenzymatic reactions. The biological relevance of the reaction of H2O2 with carbonyls has been demonstrated in several studies.


Hydroperoxides are very good nucleophiles because of the alpha effect, whereby interaction of lone electron pairs on two adjacent oxygen atoms increases nucleophilicity [78], and this explains the reactivity of hydrogen peroxide with aldehydes.


Treatment of cultured U937 human leukemic cells or human multiple myeloma cells with H2O2 was found to cause singlet oxygen from both the cells and medium components, and this was not dependent on lipid oxidation [47, 96]. The mechanisms in Figures 10, 11, and 15 may be involved in such systems.



Evidence for Endogenous Ozone Formation and the Potential Mechanisms Involved


Wentworth et al. [7] were the first to suggest the possibility of the formation of ozone (O3) in biological systems. One of their key pieces of evidence was that, in solutions of antibodies exposed to singlet oxygen, there was generation of a large amount of hydrogen peroxide, the occurrence of higher bactericidal activity than what could be attributed exclusively to H2O2, as well as the oxidation of cholesterol to secosterol aldehyde A (49 in Figure 9), a well-known product of the ozonolysis of cholesterol. They referred to the generation of H2O2 and ozone under such circumstances as the antibody-catalyzed water oxidation pathway and proposed the idea that this involves an initial reaction of water with 1O2 to form dihydrogen trioxide (H2O3) and that decomposition of the latter affords H2O2 and O3 (see equation (7)). This reaction was suggested to occur in a hydrophobic site in the antibody molecule, where the H2O3 would be shielded from hydrolysis and facilitated to undergo the conversion to H2O2 and O3 [97]. Although antibodies produce much more H2O2 and O3 than other proteins [7], Yamashita et al. [8] reported that antibody catalysis is not essential for this reaction, but rather the presence of one of four amino acids: histidine, tryptophan, cysteine, or methionine. On the other hand, various authors have expressed reservations concerning the generation of ozone under such systems, for example, based on the fact that the catalytic mechanisms for the antibody- or amino acid-catalyzed water oxidation remain ill-defined [1, 3, 35, 37]. Others have reported that cholesterol 5-hydroperoxide 46, a product of the oxidation of cholesterol by singlet oxygen, can also decompose to generate secosterol aldehydes [98, 99]. On the other hand, it has been shown that reaction of cholesterol with ozone predominantly generates secosterol A while decomposition of cholesterol-5 hydroperoxide predominantly generates secosterol B [61]. The fact that secosterol A is the predominant secosterol detected in human tissues and is formed by neutrophils in vitro thus supports the formation of endogenous ozone [61, 100]. There is also indirect evidence consistent with the formation of ozone in plant leaves or in the cyanobacterium Synechocystis PCC 6803 during light-induced damage to their PS II, because singlet oxygen and tryptophan or histidine residues, respectively, are involved [34]. Unlike other commonly generated ROS that only generate single strand breaks in DNA, ozone generates both single strand and double strand breaks [101, 102]. The addition of L-histidine to cultured mammalian cells exposed to H2O2 results in DNA double strand breaks [103每105], and this might be related to histidine-mediated ozone generation in the presence of singlet oxygen.


Oxidative Medicine and Cellular Longevity

Volume 2016, Article ID 2398573, 22 pages


Review Article

Arnold N. Onyango

Department of Food Science and Technology, Jomo Kenyatta University of Agriculture and Technology, P.O. Box 62000, Nairobi 00200, Kenya


Received 27 December 2015; Accepted 8 February 2016


Academic Editor: Sergio Di Meo


Copyright © 2016 Arnold N. Onyango. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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