- Email: [email protected]
Oxygen toxicity: chemistry and biology of reactive oxygen species
Seminars in Fetal & Neonatal Medicine 15 (2010) 186e190
Contents lists available at ScienceDirect
Seminars in Fetal & Neonatal Medicine journal homepage: www.elsevier.com/locate/siny
Oxygen toxicity: chemistry and biology of reactive oxygen species Giuseppe Buonocore*, Serafina Perrone, Maria Luisa Tataranno Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, Policlinico Santa Maria alle Scotte, V. le Bracci 36, 53100 Siena, Italy
s u m m a r y Keywords: Cell signaling DNA damage Lipid peroxidation Oxidative stress Reactive oxygen species
Oxygen has a central role in the evolution of complex life on Earth mainly because of the biochemical symmetry of oxygenic photosynthesis and aerobic respiration that can maintain homeostasis within our planet biosphere. Oxygen can also produce toxic molecules, reactive oxygen species (ROS). ROS is a collective term that includes both oxygen radicals and certain oxidizing agents that are easily converted into radicals. They can be produced from both endogenous and exogenous substances. ROS play a dual role in biological systems, since they can be either harmful or beneficial to living systems. They can be considered a double-edged sword because on the one hand oxygen-dependent reactions and aerobic respiration have significant advantages but, on the other, overproduction of ROS has the potential to cause damage. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction All aerobic organisms use molecular oxygen to generate ATP that is the chemical energy useful for life. Oxygen has a central role in the evolution of complex life on Earth mainly because of the biochemical symmetry of oxygenic photosynthesis and aerobic respiration (H2O / O2 / H2O) that can maintain homeostasis within our planet biosphere.1 The evolution of multicellular species has involved the uptake, transport and tissue distribution of oxygen,2 due to some of the properties of oxygen: it is completely available, it can easily diffuse across biological membranes, and it can bind heme in proteins (hemoglobin and cytochromes).1 Nevertheless oxygen may also be also toxic and mutagenic through the production of reactive oxygen species (ROS). The single oxygen atom is unstable, which is why each one tends to bind a twin atom, forming molecular oxygen. The stability of this bond is compromised because only one pair of electrons is shared and two unpaired electrons remain; oxygen therefore has a diamagnetic nature and is actually a free biradical. If the oxygen atom does not find a twin atom, it can accept hydrogen to form water (H2O), but there may be one electron less or more, resulting in no stability. This complex represents a free radical. Free radicals are highly reactive substances capable of giving rise to chain reactions, i.e. reactions that involve a number of steps, each of which forms a free radical that triggers the next step. There are three phases: initiation, propagation and termination, and there are different free radical species: oxygen-centred radicals (ROS), nitrogen-centred
* Corresponding author. Tel.: þ39 0577 586542 523; fax: þ39 0577 586182. E-mail address: [email protected] (G. Buonocore). 1744-165X/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.siny.2010.04.003
radicals (RNS), carbon-centred radicals and sulphur-centred radicals.3 Free radical generation can be considered a double-edged sword because on the one hand O2-dependent reactions and aerobic respiration have significant advantages, but on the other the generation of ROS has the potential to cause damage.1 Living organisms have developed different ways of counteracting these harmful substances: preventive mechanisms, repair mechanisms, physical defences and antioxidant defences. The overproduction of ROS and the insufficiency of an antioxidant mechanism results in oxidative stress, a deleterious process that can be an important mediator of damage to cell structures, including lipids and membranes, proteins and DNA. By contrast, beneficial effects of ROS/ RNS occur at low/moderate concentrations and involve physiological roles in cellular responses to noxia, as for example in defence against infectious agents, in the function of a number of cellular signalling pathways and the induction of a mitogenic response.4,5 2. Chemistry of ROS Oxygen radicals represent the most important class of radical species generated in living systems. ROS is a collective term that includes both oxygen radicals and certain non-radicals that are oxidizing agents and/or are easily converted into radicals (HOCl, HOBr, O3, ONOO, 1O2, and H2O2).6 All oxygen toxic species are ROS, but not all ROS are oxygen radicals. ROS can be produced from both endogenous and exogenous substances. Potential endogenous sources include mitochondria, cytochrome P450 metabolism, peroxisomes, microsomes, inflammatory cell activation, monoxygenase system, nitric oxide synthase and several other enzymes involved in the inflammatory process.7,8 Ubisemiquinone has been proposed as the main reductant of oxygen in mitochondrial membranes.7 In addition to ROS generation through cellular respiration, they can also
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be produced by NADPH oxidases (NOX1e3 in smooth muscle and vascular endothelium) and NOX4 (epithelial cells). The burden of ROS can be further amplified by the presence of ‘free’ metals, such as iron, copper and manganese, that are released from metalloprotein complexes.9 Besides mitochondria, there are other cellular sources of free radicals; for example, xanthine oxidase (XO) which is a member of a group of enzymes known as molybdenumeironesulphur flavin hydroxylases and catalyses the hydroxylation of purines. Additional endogenous sources of cellular ROS are neutrophils, eosinophils, macrophages.10 Activated macrophages initiate an increase in oxygen uptake that gives rise to a variety of ROS. Free radicals can also be produced by a host of exogenous processes such as environmental agents and xenobiotics (metal ions, radiations, barbiturates).11 Oxidative stress and a higher ROS production can be also induced by stress factors such as tumour necrosis factor a (TNFa) and the increase in ROS production can be recognized by a redox sensor; this triggers a redox cascade which leads to the activation of both pro-survival and pro-cell-death factors.12 There are many types of ROS in living systems; with an electron addition, superoxide anion (O 2 ) is created from oxygen. Superoxide anion is considered the primary ROS and can generate secondary ROS, interacting with other molecules (directly or through enzyme- or metal-catalysed processes). Superoxide production occurs primarily within the mitochondrial respiratory chain. The respiratory chain is fundamental for the ATP production in mammalian cells. During the respiratory process, oxygen is utilised as an electron acceptor and it is completely reduced to water through the acquisition of four electrons: O2 þ 4e þ 4Hþ / 2H2O Radical formation is possible, when this process is realized through subsequent steps:
O2 þ 1e þ Hþ 4 HO2 4 Hþ þ Oe 2
HO2 þ 1e þ Hþ 4 H2O2
H2O2 þ 1e þ Hþ 4 [H3O2] 4 H2O þ OH
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oxidation. Mitochondria take up O2 and reduce 95% of it to water by adding four electrons step by step, a process achieved by cytochrome oxidase. The cytochrome oxidase is a large and complex multiprotein assembly, both because it catalyses several reduction steps and also because it must hold on to toxic, partially reduced oxygen species until they can be fully reduced to water. Another important route through which it is possible to form superoxide is represented by heme oxidation. The iron of heme is reduced in a ferrous state (Fe II) in the deoxyhemoglobin, but, when it attaches oxygen, an intermediate structure in which an electron is delocalized between ferrous ion and oxygen is created: Heme Fe2þeO2 4 Heme Fe3þeO 2 The result is an intermediate between Fe II bound to oxygen and Fe III bound to superoxide. Sometimes the oxyhemoglobin molecule goes into decomposition and release superoxide anion:
3þ Heme Fe3þeO 2 / O2 þ Heme Fe
The methemoglobin is the product with Fe III in heme and is unable to bind oxygen. About 3% of hemoglobin in erythrocytes undergoes oxidation every day. There are also several oxidative enzymes that can produce superoxide anion: dihydrorotate dehydrogenase, aldehyde oxidase, xanthine oxidase. In particular, xanthine oxidase causes oxygen reduction both bivalent and monovalent by the transformation of hypoxanthine and xanthine to uric acid, this leads to the production of hydrogen peroxide and superoxide anion. 2.2. Hydroxyl radical The hydroxyl radical has a high oxidant power that makes it a very dangerous radical, probably the most reactive one. It has a very short in-vivo half-life of about 109s.14 Thus when produced in vivo OH reacts close to its site of formation. Hydroxyl radicals are virtually eliminated at the same site of formation because they could interact with the first molecule they meet. This is due to their extreme reactivity. The hydroxyl radical is mainly produced when there is an excess production of superoxide anion and hydrogen peroxide in the HabereWeiss reaction:
OH þ 1e þ Hþ 4 H2O
Three intermediate products are generated from oxygen reduction: superoxide anion (O 2 ), hydrogen peroxide (H2O2) and hydroxyl radical (OH). The second one is the most stable and it can be accumulated in a large quantity. 2.1. Superoxide anion Superoxide anion is considered the ‘primary’ ROS. It can interact with other molecules to generate ‘secondary’ ROS and can be both a reducing and oxidizing factor; by reducing reaction molecular oxygen is produced whereas by oxidizing reaction hydrogen peroxide is generated: 13
þ X þ O 2 þ H / XH þ O2
þ YH þ O 2 þ H / Y þ H2O2
O 2 þ H2O / OH þ OH þ O2
It can be also generated through a variety of mechanisms: ionising radiation causes decomposition of water, resulting in formation of OH and hydrogen atoms, and photolytic decomposition of alkylhydroperoxides. In vivo, a great part of OH comes from the metal-catalysed breakdown of hydrogen peroxide, according to the Fenton reaction: Mnþ (¼ Cuþ, Fe2þ, Ti3þ, Co2þ) þ H2O2 / M(n þ 1) (¼ Cu2þ, Fe3þ, Ti4þ, Co3þ) þ OH þ OHe where Mnþ is a transitional metal ion.15,16 The most realistic in-vivo production of hydroxyl radical according to the Fenton reaction occurs when Mnþ is iron; organisms overloaded by iron (hemochromatosis, b-thalassemia, hemodialysis) contain higher amounts of ‘free available iron’ and this can have deleterious effects. 2.3. Singlet oxygen
In mammalian cells, a bit fraction of total oxygen is utilised for superoxide creation and the most important route of superoxide creation is the mitochondrial respiratory chain and the heme
Singlet oxygen can be generated by an input of energy that rearranges the electrons.17 In both forms of singlet O2 the spin
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restriction is removed and the oxidizing ability greatly increased; singlet O2 can directly oxidize proteins, DNA, and lipids. Because of the differences in their electron shells, singlet and triplet oxygen differ in their chemical properties. The damaging effects of sunlight on many organic materials (polymers, etc.) are often attributed to the effects of singlet oxygen. Singlet oxygen can participate in DielseAlder reactions.17 It can be generated by chemical processes such as spontaneous decomposition of hydrogen trioxide in water or the reaction of hydrogen peroxide with hypochlorite. Singlet oxygen reacts with an alkene (eC¼CeCHe) by abstraction of the allylic proton in an ene-type reaction to the allyl hydroperoxide HOeOeR (R ¼ alkyl), which can then be reduced to the allyl alcohol.
3. Biology of ROS The problem of oxidative stress is rooted in some biological paradoxes (Fig. 1). Oxygen is essential to aerobic organisms. Each cell is characterized by a particular concentration of electrons (redox state) stored in many cellular constituents and the redox state of a cell and its oscillation determines cellular functioning.18 The term ‘redox state’ is not only used to describe the state of a redox pair (GSSH/2GSH) but also to describe more generally the redox environment of a cell. The intracellular redox state is primarily maintained by GSH and thioreoxidin (TRH). The glutathione (2GSH/GSSH couple) represents the major cellular redox buffer and therefore is a representative indicator for the redox environment of the cell.18e20 When a pathological condition occurs the redox state can be altered to lower or higher values. In the cell, reducing and oxidizing molecules modulate the redox state. ROS play an important role in sperm capacitation, acrosome reaction and motility activation.21 During fertilization, ROS levels can influence spermeegg interaction and gamete fusion thanks to testicular sensitivity to the redox status.22 In embryonic and fetal growth, increased oxidative stress may be detrimental, but an oxidized state can also be beneficial.23 This is because redox may also affect key transcription factors that can alter gene expression during development.24,25 In addition, redox may impact on placentation and amniotic membrane integrity during pregnancy.26,27 ROS toxicity comes from the ability to react with all components of the DNA molecule, damaging both the purine and pyrimidine bases and also the deoxyribose backbone. The most extensively studied DNA lesion is the formation of 8-OH-G, which is a good
Fig. 1. Effects of reactive oxygen species (ROS).
biomarker of oxidative stress.28 This base modification occurs in approximately one in 105 guanidine residues in a normal human cell. The ROS attack on DNA represents the first step involved in mutagenesis, carcinogenesis and ageing. ROS-induced DNA damage involves single- or double-stranded DNA breaks, purine, pyrimidine or deoxyribose modifications, and DNA cross-links. DNA damage can result also in arrest or induction of transcription, induction of signal pathways, replication errors and genomic instability, all of which are associated with carcinogenesis.29 The ROS attack is not only against DNA but also against other cellular components involving polyunsaturated fatty acid residues of phospholipids.30 When Fe IIeFe III or Fe IIeO2eFe III complexes are formed or when there is ADPeFe II, the maximal rate of lipid peroxidation is promoted.31,32 Once formed, peroxyl radicals (ROO) can be rearranged through a cycle reaction to endoperoxides (precursors of malondialdehyde, MDA).33 4-Hydroxy-2-noneal (HNE) is a product of lipid peroxidation.34 MDA is carcinogenic in rats and mutagenic in bacterial and mammalian cells, whereas HNE is just weakly mutagenic but seems to be the major toxic product of lipid peroxidation. Measurement of lipid peroxidation, although useful, suffers from problems related to specificity and sensitivity especially when utilized in vivo. Many of these limitations were addressed with the discovery of prostaglandin (PG)-like compounds, termed isoprostanes (IsoPs), derived by the free radical peroxidation of arachidonic acid.35 More recently, an oxygen insertion step has been demonstrated that diverts intermediates from the IsoP pathway to form compounds termed isofurans (IsoFs) containing a substituted tetrahydrofuran ring.36 Because of this differential method of formation, it has been suggested that oxygen tension can affect lipid peroxidation profile. Like IsoPs, the IsoFs are chemically and metabolically stable, so are well-suited to act as invivo biomarkers of oxidative damage. The ratio of IsoFs to IsoPs also provides information about the relative oxygen tension where the lipid peroxidation is occurring. The same can happen in neuronal cells where docosahexaenoic acid (DHA), a major component of neuronal membranes, is oxidized both in vitro and in vivo to form IsoP-like compounds termed neuroprostanes (NPs).37,38 NPs are the only quantitative in-vivo biomarkers of oxidative damage selective for neurons. An alternative pathway of oxidation of DHA leads to the formation of IsoF-like compounds termed neurofurans (NFs); this happens when there is a higher oxygen tension. Quantitative assessment of NFs in vivo reveals modulated formation under conditions of elevated and diminished oxidative stress. Given the abundance of DHA in the brain, analysis of NFs may have particular value in the quantitative assessment of lipid peroxidation in brain damage. Not only may lipids and DNA suffer oxidative damage, but also proteins. When proteins react with hydroxyl radical there is an abstraction of a hydrogen atom from protein polypeptide, forming a carbon-centred radical, which readily reacts with dioxygen to form peroxyl radicals under aerobic conditions. In the presence of transitional metals an oxidative scission can be observed, along with loss of histidine residues, bityrosine cross-links, an introduction of carbonyl groups and the formation of alkyl, alkoxyl and alkyleperoxyl radicals. The concentration of carbonyl groups, generated by many different mechanisms, is a good measure of ROS-mediated protein oxidation.39 The side chains of all amino acid residues of proteins are susceptible to oxidation by ROS action, expecially cysteine and methionine residues. Iron (II) can bind proteins in specific sites and the Fe IIeprotein complex reacts with H2O2 via the Fenton reaction to yield active oxygen species. Yet oxidative stress is not always bad: formation of ROS at sites of inflammation may not only destroy invading pathogens but also help to modulate an overexuberant inflammatory response under
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certain circumstances.40 These species can sometimes be antiinflammatory, for example by modulating lymphocyte function. This is one of the many paradoxes in the free radical/antioxidant field. In recent years, the dual roles of ROS have been increasingly evident. ROS perform important roles, so the challenge is to evolve antioxidant defences that allow such roles while minimizing damage. ROS production by phagocytes and by other cells in gastrointestinal and respiratory tracts is a defence against microorganisms.41 Oxygen radicals have also a role in cellular processes controlled by enzymes and transcriptional factor phosphorylation and dephosphorylation. Upon stimulation by growth factor and cytokines, NOX of vascular cells produce superoxide and other ROS, which in turn activate multiple intracellular signaling pathways. Thus ROS play an important role in the regulation of cardiac and vascular cell functioning.4,42 Angiotensin II, thrombin, plateletderived growth factor, platelet-activating factor, interleukin-1 and TNFa increase NOX-mediated formation of superoxide in vascular smooth muscle cells and fibroblasts. The role of ROS in the regulation of vascular tone is a special case. Hydrogen peroxide and NO radical are able to activate the enzyme soluble guanylate cyclase (sGC) which catalyses the formation of the ‘second messenger’ cyclic guanosine monophosphate (cGMP). NO binds to Fe2þ-heme group in sGC, resulting in a conformational change at Fe2þ that activated the enzyme.43 Its product, cGMP, modulates the function of protein kinases, ion channels, and other physiologically important targets involved in the regulation of smooth vascular tone and the inhibition of platelet adhesion.4 ROS levels have been widely accepted as pro-cell death and prosurvival factors. Under certain circumstances, ROS are able to counteract the cell death. Most likely, the intensity of the oxidative stimulus strongly influences the final effect. The ROS levels are detected by redox-sensitive molecules. Excessive and/or prolonged oxidative stress gives rise to a sustained activation of mitogenactivated protein kinase that leads to cell death, particularly when combined with extensive cell damage.12 Conversely a transient activation results in cell survival through the activation of NF-kB, an important transcription factor able to suppress ROS formation by upregulating the transcription of many pro-survival genes and by inhibiting JNK (pro-cell-death factor) activation. ROS may be thought of as mediators in the cross-talk between NF-kB and JNK and it is the marginal change in their concentrations which produces a vital or pathological result. In addition, nuclear erythroid-related factor 2 (Nrf2) is a transcription factor that regulates an expansive set of antioxidant genes acting in synergy to remove ROS through sequential enzymatic reactions. It is the principal transcription factor that regulates antioxidant response element (ARE)-mediated expression of phase II detoxifying antioxidant enzymes. Under normal conditions, Nrf2 is sequestered in the cytoplasm by an actin-binding (Kelch-like) protein (Keap1); on exposure of cells to oxidative stress, Nrf2 dissociates from Keap1, translocates into the nucleus, binds to ARE, and transactivates phase II detoxifying and antioxidant genes. Among the spectrum of antioxidant genes controlled by Nrf2 are catalase, superoxide dismutase (SOD), glutathione reductase, and glutathione peroxidase.44 4. Conclusions ROS are produced by normal cellular metabolism; they are essential for life, can protect us from infections, are useful for reproduction and fetal development, and they play a physiological role as secondary messengers. They are also involved in intracellular regulation of calcium concentration, in protein phosphorylation and in activation of some transcriptional factors. On the other hand they
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may also trigger death by apoptosis, necrosis, or they can contribute to neurodegenerative disorders and to protein, lipid and DNA damage. Cells may adapt to the stress by upregulation of defence and repair systems. This may protect against damage but not completely. The human body is able to regulate the ROS/antioxidant balance very carefully but the continual injury by free radicals (expecially but not only age-related) can lead to a condition of irreversibility. This finding is much more true in the early stages of life.
Practice points Oxygen is an essential element of aerobic life, and oxidative metabolism represents a principal source of energy. Oxygen when partially reduced induces the generation of reactive oxygen species (ROS). ROS are a double-edged sword acting both as protectors or destructors. A continuous balance exists in the body between ROS production and antioxidants. When the equilibrium is destroyed, the damaging effects of oxidative stress occur.
Conflict of interest statement None declared. Funding sources None. References 1. Thannickal VJ. Oxygen in the evolution of complex life and the price we pay. Am J Respir Cell Molec Biol 2009;40:507e10. 2. Wenger RH. Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia-inducible transcription factors, and O2-regulated gene expression. FASEB J 2002;16:1151e62. 3. Halliwell B. Biochemistry of oxidative stress. Biochem Soc Trans 2007;35:1147e50. 4. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007;39:44e84. 5. Dröge W. Free radicals in the physiologic control of cell function. Physiol Rev 2002;82:47e95. 6. Miller DM, Buettner GR, Aust SD. Transition metals as catalysts of “autoxidation” reactions. Free Radic Biol Med 1990;8:95e108. 7. Inoue M, Sato EF, Nishikawa M, et al. Mitochondrial generation of reactive oxygen species and its role in aerobic life. Curr Med Chem 2003;10:2495e505. 8. Authen RL, Davis JM. Oxygen toxicity and reactive oxygen species: the devil is in the details. Pediatr Res 2009;66:121e7. 9. van Der Vliet A. NADPH oxidases in lung biology and pathology: host defense enzymes, and more. Free Radic Biol Med 2008;44:938e55. 10. Conner EM, Grisham MB. Inflammation, free radicals, and antioxidants. Nutrition 1996;12:274e7. 11. Klaunig JE, Xu Y, Bachowski S, Jiang J. Free-radical oxygen-induced changes in chemical carcinogenesis. In: Wallace KB, editor. Free radical toxicology. London: Taylor & Francis; 1997. p. 375e400. 12. Pourova J, Kottova M, Voprsalova M, Pour M. reactive oxygen and nitrogen species in normal physiological processes. Acta Physiol 2010;198:15e35. 13. Valko M, Morris H, Cronin MTD. Metals, toxicity and oxidative stress. Curr Med Chem 2005;12:1161e208. 14. Pastor N, Weinstein H, Jamison E, Brenowitz M. A detailed interpretation of OH radical footprints in a TBP DNA complex reveals the role of dynamics in the mechanism of sequence- specific binding. J Molec Biol 2000;304:55e68. 15. Liochev I, Fridovich I. The HabereWeiss cycle e70 years later: an alternative view. Redox Rep 2002;7:55e7. 16. Platenik J, Stopka P, Vejrazka M, Stipek S. Quinolinic acideiron(II) complexes: slow autoxidation, but enhanced hydroxyl radical production in the Fenton reaction. Free Radic Res 2001;34:445e59. 17. Foote N, Peterson J, Gadsby PM, Greenwood C, Thomson AJ. Redox-linked spinstate changes in the di-haem cytochrome c-551 peroxidase from Pseudomonas aeruginosa. Biochem J 1985;230:227e37.
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18. Schafer FQ, Buettner GR. Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic Biol Med 2001;30:1191e212. 19. Dröge W. The plasma redox state and ageing. Ageing Res Rev 2002;1:257e78. 20. Frosali S, Di Simplicio P, Perrone S, et al. Glutathione recycling and antioxidant enzyme activities in erythrocytes of term and preterm newborns at birth. Biol Neonate 2004;85:188e94. 21. De Lamirande E, O’Flaherty C. Sperm activation: role of reactive oxygen species and kinases. Biochim Biophys Acta 2008;1784:106e15. 22. Cohen DJ, Busso D, Da Ros V, et al. Participation of cystein-rich secretory proteins (CRISP) in mammalian spermeegg interaction. Int J Dev Biol 2008;52:737e42. 23. Wong JL, Creton R, Wessel GM. The oxidative burst at fertilization is dependent upon activation of the dual oxidase Udx1. Dev Cell 2004;7:801e14. 24. Thannickal VJ, Fanburg BL. Reactive oxygen species in cell signaling. Am J Physiol Lung Cell Mol Physiol 2000;279:L1005eL1028. 25. Lambeth JD. NOX enzymes and the biology of reactive oxygen. Nat Rev Immunol 2004;4:181e9. 26. Dennery PA. Role of redox in fetal development and neonatal diseases. Antioxid Redox Signal 2004;6:147e53. 27. Longini M, Perrone S, Vezzosi P, et al. Association between oxidative stress in pregnancy and preterm premature rupture of membranes. Clin Biochem 2007;40:793e7. 28. Halliwell B. The wanderings of a free radical. Free Radic Biol Med 2009;46:531e42. 29. Marnett LJ. Oxyradicals and DNA damage. Carcinogenesis 2000;21:361e70. 30. Siems WG, Grune T, Esterbauer H. 4-Hydroxynonenal formation during ischemia and reperfusion of rat small-intestine. Life Sci 1995;57:785e9. 31. Bucher JR, Tien M, Aust SD. The requirement for ferric in the initiation of lipid peroxidation by chelated ferrous iron. Biochem Biophys Res Commun 1983;111:777e84. 32. Nyska A, Kohen R. Oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification. Toxicol Pathol 2002;30:620e50.
33. Marnett LJ. Lipid peroxidationdDNA damage by malondialdehyde. Mutat Res: Fundam Molec Mech Mutagen 1999;424:83e95. 34. Wang MY, Dhingra K, Hittelman WN, Liehr JG, deAndrade M, Li DH. Lipid peroxidation-induced putative malondialdehydeeDNA adducts in human breast tissues. Cancer Epidemiol Biomark Prev 1996;5:705e10. 35. Milne GL, Yin H, Brooks JD, et al. Quantification of F2-isoprostanes in biological fluids and tissues as a measure of oxidant stress. Methods Enzymol 2007;433:113e26. 36. Fessel JP, Porter NA, Moore KP, Sheller JR, Roberts 2nd LJ. Discovery of lipid peroxidation products formed in vivo with a substituted tetrahydrofuran ring (isofurans) that are favored by increased oxygen tension. Proc Natl Acad Sci USA 2002;99:16713e8. 37. Arneson KO, Roberts 2nd LJ. Measurement of products of docosahexaenoic acid peroxidation, neuroprostanes, and neurofurans. Methods Enzymol 2007;433:127e43. 38. Musiek ES, Brooks JD, Joo M, et al. Electrophilic cyclopentenone neuroprostanes are anti-inflammatory mediators formed from the peroxidation of the omega-3 polyunsaturated fatty acid docosahexaenoic acid. J Biol Chem 2008;283:19927e35. 39. Marzocchi B, Perrone S, Paffetti P, et al. Nonprotein-bound iron and plasma protein oxidative stress at birth. Pediatr Res 2005;58:1295e9. 40. Halliwell B. Phagocyte-derived reactive species: salvation or suicide? Trends Biochem Sci 2006;31:509e15. 41. Nauseef WM. Nox enzymes in immune cells. Semin Immunopathol 2008;30:330e63. 42. Griendling KK, Sorescu D, Lasse’gue B, Uschio-Fukai M. Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology. Arterioscler Thromb Vasc Biol 2000;20:2175e83. 43. Wolin MS, Burke-Wolin TM, Mohazzab-H KM. Roles for NAD(P)H oxidases and reactive oxygen species in vascular oxygen sensing mechanisms. Respir Physiol 1999;115:229e38. 44. Lakhan SE, Kirchgessner A, Hofer M. Inflammatory mechanisms in ischemic stroke: therapeutic approaches. J Transl Med 2009;7:97e104.
Contents lists available at ScienceDirect
Seminars in Fetal & Neonatal Medicine journal homepage: www.elsevier.com/locate/siny
Oxygen toxicity: chemistry and biology of reactive oxygen species Giuseppe Buonocore*, Serafina Perrone, Maria Luisa Tataranno Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, Policlinico Santa Maria alle Scotte, V. le Bracci 36, 53100 Siena, Italy
s u m m a r y Keywords: Cell signaling DNA damage Lipid peroxidation Oxidative stress Reactive oxygen species
Oxygen has a central role in the evolution of complex life on Earth mainly because of the biochemical symmetry of oxygenic photosynthesis and aerobic respiration that can maintain homeostasis within our planet biosphere. Oxygen can also produce toxic molecules, reactive oxygen species (ROS). ROS is a collective term that includes both oxygen radicals and certain oxidizing agents that are easily converted into radicals. They can be produced from both endogenous and exogenous substances. ROS play a dual role in biological systems, since they can be either harmful or beneficial to living systems. They can be considered a double-edged sword because on the one hand oxygen-dependent reactions and aerobic respiration have significant advantages but, on the other, overproduction of ROS has the potential to cause damage. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction All aerobic organisms use molecular oxygen to generate ATP that is the chemical energy useful for life. Oxygen has a central role in the evolution of complex life on Earth mainly because of the biochemical symmetry of oxygenic photosynthesis and aerobic respiration (H2O / O2 / H2O) that can maintain homeostasis within our planet biosphere.1 The evolution of multicellular species has involved the uptake, transport and tissue distribution of oxygen,2 due to some of the properties of oxygen: it is completely available, it can easily diffuse across biological membranes, and it can bind heme in proteins (hemoglobin and cytochromes).1 Nevertheless oxygen may also be also toxic and mutagenic through the production of reactive oxygen species (ROS). The single oxygen atom is unstable, which is why each one tends to bind a twin atom, forming molecular oxygen. The stability of this bond is compromised because only one pair of electrons is shared and two unpaired electrons remain; oxygen therefore has a diamagnetic nature and is actually a free biradical. If the oxygen atom does not find a twin atom, it can accept hydrogen to form water (H2O), but there may be one electron less or more, resulting in no stability. This complex represents a free radical. Free radicals are highly reactive substances capable of giving rise to chain reactions, i.e. reactions that involve a number of steps, each of which forms a free radical that triggers the next step. There are three phases: initiation, propagation and termination, and there are different free radical species: oxygen-centred radicals (ROS), nitrogen-centred
* Corresponding author. Tel.: þ39 0577 586542 523; fax: þ39 0577 586182. E-mail address: [email protected] (G. Buonocore). 1744-165X/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.siny.2010.04.003
radicals (RNS), carbon-centred radicals and sulphur-centred radicals.3 Free radical generation can be considered a double-edged sword because on the one hand O2-dependent reactions and aerobic respiration have significant advantages, but on the other the generation of ROS has the potential to cause damage.1 Living organisms have developed different ways of counteracting these harmful substances: preventive mechanisms, repair mechanisms, physical defences and antioxidant defences. The overproduction of ROS and the insufficiency of an antioxidant mechanism results in oxidative stress, a deleterious process that can be an important mediator of damage to cell structures, including lipids and membranes, proteins and DNA. By contrast, beneficial effects of ROS/ RNS occur at low/moderate concentrations and involve physiological roles in cellular responses to noxia, as for example in defence against infectious agents, in the function of a number of cellular signalling pathways and the induction of a mitogenic response.4,5 2. Chemistry of ROS Oxygen radicals represent the most important class of radical species generated in living systems. ROS is a collective term that includes both oxygen radicals and certain non-radicals that are oxidizing agents and/or are easily converted into radicals (HOCl, HOBr, O3, ONOO, 1O2, and H2O2).6 All oxygen toxic species are ROS, but not all ROS are oxygen radicals. ROS can be produced from both endogenous and exogenous substances. Potential endogenous sources include mitochondria, cytochrome P450 metabolism, peroxisomes, microsomes, inflammatory cell activation, monoxygenase system, nitric oxide synthase and several other enzymes involved in the inflammatory process.7,8 Ubisemiquinone has been proposed as the main reductant of oxygen in mitochondrial membranes.7 In addition to ROS generation through cellular respiration, they can also
G. Buonocore et al. / Seminars in Fetal & Neonatal Medicine 15 (2010) 186e190
be produced by NADPH oxidases (NOX1e3 in smooth muscle and vascular endothelium) and NOX4 (epithelial cells). The burden of ROS can be further amplified by the presence of ‘free’ metals, such as iron, copper and manganese, that are released from metalloprotein complexes.9 Besides mitochondria, there are other cellular sources of free radicals; for example, xanthine oxidase (XO) which is a member of a group of enzymes known as molybdenumeironesulphur flavin hydroxylases and catalyses the hydroxylation of purines. Additional endogenous sources of cellular ROS are neutrophils, eosinophils, macrophages.10 Activated macrophages initiate an increase in oxygen uptake that gives rise to a variety of ROS. Free radicals can also be produced by a host of exogenous processes such as environmental agents and xenobiotics (metal ions, radiations, barbiturates).11 Oxidative stress and a higher ROS production can be also induced by stress factors such as tumour necrosis factor a (TNFa) and the increase in ROS production can be recognized by a redox sensor; this triggers a redox cascade which leads to the activation of both pro-survival and pro-cell-death factors.12 There are many types of ROS in living systems; with an electron addition, superoxide anion (O 2 ) is created from oxygen. Superoxide anion is considered the primary ROS and can generate secondary ROS, interacting with other molecules (directly or through enzyme- or metal-catalysed processes). Superoxide production occurs primarily within the mitochondrial respiratory chain. The respiratory chain is fundamental for the ATP production in mammalian cells. During the respiratory process, oxygen is utilised as an electron acceptor and it is completely reduced to water through the acquisition of four electrons: O2 þ 4e þ 4Hþ / 2H2O Radical formation is possible, when this process is realized through subsequent steps:
O2 þ 1e þ Hþ 4 HO2 4 Hþ þ Oe 2
HO2 þ 1e þ Hþ 4 H2O2
H2O2 þ 1e þ Hþ 4 [H3O2] 4 H2O þ OH
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oxidation. Mitochondria take up O2 and reduce 95% of it to water by adding four electrons step by step, a process achieved by cytochrome oxidase. The cytochrome oxidase is a large and complex multiprotein assembly, both because it catalyses several reduction steps and also because it must hold on to toxic, partially reduced oxygen species until they can be fully reduced to water. Another important route through which it is possible to form superoxide is represented by heme oxidation. The iron of heme is reduced in a ferrous state (Fe II) in the deoxyhemoglobin, but, when it attaches oxygen, an intermediate structure in which an electron is delocalized between ferrous ion and oxygen is created: Heme Fe2þeO2 4 Heme Fe3þeO 2 The result is an intermediate between Fe II bound to oxygen and Fe III bound to superoxide. Sometimes the oxyhemoglobin molecule goes into decomposition and release superoxide anion:
3þ Heme Fe3þeO 2 / O2 þ Heme Fe
The methemoglobin is the product with Fe III in heme and is unable to bind oxygen. About 3% of hemoglobin in erythrocytes undergoes oxidation every day. There are also several oxidative enzymes that can produce superoxide anion: dihydrorotate dehydrogenase, aldehyde oxidase, xanthine oxidase. In particular, xanthine oxidase causes oxygen reduction both bivalent and monovalent by the transformation of hypoxanthine and xanthine to uric acid, this leads to the production of hydrogen peroxide and superoxide anion. 2.2. Hydroxyl radical The hydroxyl radical has a high oxidant power that makes it a very dangerous radical, probably the most reactive one. It has a very short in-vivo half-life of about 109s.14 Thus when produced in vivo OH reacts close to its site of formation. Hydroxyl radicals are virtually eliminated at the same site of formation because they could interact with the first molecule they meet. This is due to their extreme reactivity. The hydroxyl radical is mainly produced when there is an excess production of superoxide anion and hydrogen peroxide in the HabereWeiss reaction:
OH þ 1e þ Hþ 4 H2O
Three intermediate products are generated from oxygen reduction: superoxide anion (O 2 ), hydrogen peroxide (H2O2) and hydroxyl radical (OH). The second one is the most stable and it can be accumulated in a large quantity. 2.1. Superoxide anion Superoxide anion is considered the ‘primary’ ROS. It can interact with other molecules to generate ‘secondary’ ROS and can be both a reducing and oxidizing factor; by reducing reaction molecular oxygen is produced whereas by oxidizing reaction hydrogen peroxide is generated: 13
þ X þ O 2 þ H / XH þ O2
þ YH þ O 2 þ H / Y þ H2O2
O 2 þ H2O / OH þ OH þ O2
It can be also generated through a variety of mechanisms: ionising radiation causes decomposition of water, resulting in formation of OH and hydrogen atoms, and photolytic decomposition of alkylhydroperoxides. In vivo, a great part of OH comes from the metal-catalysed breakdown of hydrogen peroxide, according to the Fenton reaction: Mnþ (¼ Cuþ, Fe2þ, Ti3þ, Co2þ) þ H2O2 / M(n þ 1) (¼ Cu2þ, Fe3þ, Ti4þ, Co3þ) þ OH þ OHe where Mnþ is a transitional metal ion.15,16 The most realistic in-vivo production of hydroxyl radical according to the Fenton reaction occurs when Mnþ is iron; organisms overloaded by iron (hemochromatosis, b-thalassemia, hemodialysis) contain higher amounts of ‘free available iron’ and this can have deleterious effects. 2.3. Singlet oxygen
In mammalian cells, a bit fraction of total oxygen is utilised for superoxide creation and the most important route of superoxide creation is the mitochondrial respiratory chain and the heme
Singlet oxygen can be generated by an input of energy that rearranges the electrons.17 In both forms of singlet O2 the spin
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restriction is removed and the oxidizing ability greatly increased; singlet O2 can directly oxidize proteins, DNA, and lipids. Because of the differences in their electron shells, singlet and triplet oxygen differ in their chemical properties. The damaging effects of sunlight on many organic materials (polymers, etc.) are often attributed to the effects of singlet oxygen. Singlet oxygen can participate in DielseAlder reactions.17 It can be generated by chemical processes such as spontaneous decomposition of hydrogen trioxide in water or the reaction of hydrogen peroxide with hypochlorite. Singlet oxygen reacts with an alkene (eC¼CeCHe) by abstraction of the allylic proton in an ene-type reaction to the allyl hydroperoxide HOeOeR (R ¼ alkyl), which can then be reduced to the allyl alcohol.
3. Biology of ROS The problem of oxidative stress is rooted in some biological paradoxes (Fig. 1). Oxygen is essential to aerobic organisms. Each cell is characterized by a particular concentration of electrons (redox state) stored in many cellular constituents and the redox state of a cell and its oscillation determines cellular functioning.18 The term ‘redox state’ is not only used to describe the state of a redox pair (GSSH/2GSH) but also to describe more generally the redox environment of a cell. The intracellular redox state is primarily maintained by GSH and thioreoxidin (TRH). The glutathione (2GSH/GSSH couple) represents the major cellular redox buffer and therefore is a representative indicator for the redox environment of the cell.18e20 When a pathological condition occurs the redox state can be altered to lower or higher values. In the cell, reducing and oxidizing molecules modulate the redox state. ROS play an important role in sperm capacitation, acrosome reaction and motility activation.21 During fertilization, ROS levels can influence spermeegg interaction and gamete fusion thanks to testicular sensitivity to the redox status.22 In embryonic and fetal growth, increased oxidative stress may be detrimental, but an oxidized state can also be beneficial.23 This is because redox may also affect key transcription factors that can alter gene expression during development.24,25 In addition, redox may impact on placentation and amniotic membrane integrity during pregnancy.26,27 ROS toxicity comes from the ability to react with all components of the DNA molecule, damaging both the purine and pyrimidine bases and also the deoxyribose backbone. The most extensively studied DNA lesion is the formation of 8-OH-G, which is a good
Fig. 1. Effects of reactive oxygen species (ROS).
biomarker of oxidative stress.28 This base modification occurs in approximately one in 105 guanidine residues in a normal human cell. The ROS attack on DNA represents the first step involved in mutagenesis, carcinogenesis and ageing. ROS-induced DNA damage involves single- or double-stranded DNA breaks, purine, pyrimidine or deoxyribose modifications, and DNA cross-links. DNA damage can result also in arrest or induction of transcription, induction of signal pathways, replication errors and genomic instability, all of which are associated with carcinogenesis.29 The ROS attack is not only against DNA but also against other cellular components involving polyunsaturated fatty acid residues of phospholipids.30 When Fe IIeFe III or Fe IIeO2eFe III complexes are formed or when there is ADPeFe II, the maximal rate of lipid peroxidation is promoted.31,32 Once formed, peroxyl radicals (ROO) can be rearranged through a cycle reaction to endoperoxides (precursors of malondialdehyde, MDA).33 4-Hydroxy-2-noneal (HNE) is a product of lipid peroxidation.34 MDA is carcinogenic in rats and mutagenic in bacterial and mammalian cells, whereas HNE is just weakly mutagenic but seems to be the major toxic product of lipid peroxidation. Measurement of lipid peroxidation, although useful, suffers from problems related to specificity and sensitivity especially when utilized in vivo. Many of these limitations were addressed with the discovery of prostaglandin (PG)-like compounds, termed isoprostanes (IsoPs), derived by the free radical peroxidation of arachidonic acid.35 More recently, an oxygen insertion step has been demonstrated that diverts intermediates from the IsoP pathway to form compounds termed isofurans (IsoFs) containing a substituted tetrahydrofuran ring.36 Because of this differential method of formation, it has been suggested that oxygen tension can affect lipid peroxidation profile. Like IsoPs, the IsoFs are chemically and metabolically stable, so are well-suited to act as invivo biomarkers of oxidative damage. The ratio of IsoFs to IsoPs also provides information about the relative oxygen tension where the lipid peroxidation is occurring. The same can happen in neuronal cells where docosahexaenoic acid (DHA), a major component of neuronal membranes, is oxidized both in vitro and in vivo to form IsoP-like compounds termed neuroprostanes (NPs).37,38 NPs are the only quantitative in-vivo biomarkers of oxidative damage selective for neurons. An alternative pathway of oxidation of DHA leads to the formation of IsoF-like compounds termed neurofurans (NFs); this happens when there is a higher oxygen tension. Quantitative assessment of NFs in vivo reveals modulated formation under conditions of elevated and diminished oxidative stress. Given the abundance of DHA in the brain, analysis of NFs may have particular value in the quantitative assessment of lipid peroxidation in brain damage. Not only may lipids and DNA suffer oxidative damage, but also proteins. When proteins react with hydroxyl radical there is an abstraction of a hydrogen atom from protein polypeptide, forming a carbon-centred radical, which readily reacts with dioxygen to form peroxyl radicals under aerobic conditions. In the presence of transitional metals an oxidative scission can be observed, along with loss of histidine residues, bityrosine cross-links, an introduction of carbonyl groups and the formation of alkyl, alkoxyl and alkyleperoxyl radicals. The concentration of carbonyl groups, generated by many different mechanisms, is a good measure of ROS-mediated protein oxidation.39 The side chains of all amino acid residues of proteins are susceptible to oxidation by ROS action, expecially cysteine and methionine residues. Iron (II) can bind proteins in specific sites and the Fe IIeprotein complex reacts with H2O2 via the Fenton reaction to yield active oxygen species. Yet oxidative stress is not always bad: formation of ROS at sites of inflammation may not only destroy invading pathogens but also help to modulate an overexuberant inflammatory response under
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certain circumstances.40 These species can sometimes be antiinflammatory, for example by modulating lymphocyte function. This is one of the many paradoxes in the free radical/antioxidant field. In recent years, the dual roles of ROS have been increasingly evident. ROS perform important roles, so the challenge is to evolve antioxidant defences that allow such roles while minimizing damage. ROS production by phagocytes and by other cells in gastrointestinal and respiratory tracts is a defence against microorganisms.41 Oxygen radicals have also a role in cellular processes controlled by enzymes and transcriptional factor phosphorylation and dephosphorylation. Upon stimulation by growth factor and cytokines, NOX of vascular cells produce superoxide and other ROS, which in turn activate multiple intracellular signaling pathways. Thus ROS play an important role in the regulation of cardiac and vascular cell functioning.4,42 Angiotensin II, thrombin, plateletderived growth factor, platelet-activating factor, interleukin-1 and TNFa increase NOX-mediated formation of superoxide in vascular smooth muscle cells and fibroblasts. The role of ROS in the regulation of vascular tone is a special case. Hydrogen peroxide and NO radical are able to activate the enzyme soluble guanylate cyclase (sGC) which catalyses the formation of the ‘second messenger’ cyclic guanosine monophosphate (cGMP). NO binds to Fe2þ-heme group in sGC, resulting in a conformational change at Fe2þ that activated the enzyme.43 Its product, cGMP, modulates the function of protein kinases, ion channels, and other physiologically important targets involved in the regulation of smooth vascular tone and the inhibition of platelet adhesion.4 ROS levels have been widely accepted as pro-cell death and prosurvival factors. Under certain circumstances, ROS are able to counteract the cell death. Most likely, the intensity of the oxidative stimulus strongly influences the final effect. The ROS levels are detected by redox-sensitive molecules. Excessive and/or prolonged oxidative stress gives rise to a sustained activation of mitogenactivated protein kinase that leads to cell death, particularly when combined with extensive cell damage.12 Conversely a transient activation results in cell survival through the activation of NF-kB, an important transcription factor able to suppress ROS formation by upregulating the transcription of many pro-survival genes and by inhibiting JNK (pro-cell-death factor) activation. ROS may be thought of as mediators in the cross-talk between NF-kB and JNK and it is the marginal change in their concentrations which produces a vital or pathological result. In addition, nuclear erythroid-related factor 2 (Nrf2) is a transcription factor that regulates an expansive set of antioxidant genes acting in synergy to remove ROS through sequential enzymatic reactions. It is the principal transcription factor that regulates antioxidant response element (ARE)-mediated expression of phase II detoxifying antioxidant enzymes. Under normal conditions, Nrf2 is sequestered in the cytoplasm by an actin-binding (Kelch-like) protein (Keap1); on exposure of cells to oxidative stress, Nrf2 dissociates from Keap1, translocates into the nucleus, binds to ARE, and transactivates phase II detoxifying and antioxidant genes. Among the spectrum of antioxidant genes controlled by Nrf2 are catalase, superoxide dismutase (SOD), glutathione reductase, and glutathione peroxidase.44 4. Conclusions ROS are produced by normal cellular metabolism; they are essential for life, can protect us from infections, are useful for reproduction and fetal development, and they play a physiological role as secondary messengers. They are also involved in intracellular regulation of calcium concentration, in protein phosphorylation and in activation of some transcriptional factors. On the other hand they
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may also trigger death by apoptosis, necrosis, or they can contribute to neurodegenerative disorders and to protein, lipid and DNA damage. Cells may adapt to the stress by upregulation of defence and repair systems. This may protect against damage but not completely. The human body is able to regulate the ROS/antioxidant balance very carefully but the continual injury by free radicals (expecially but not only age-related) can lead to a condition of irreversibility. This finding is much more true in the early stages of life.
Practice points Oxygen is an essential element of aerobic life, and oxidative metabolism represents a principal source of energy. Oxygen when partially reduced induces the generation of reactive oxygen species (ROS). ROS are a double-edged sword acting both as protectors or destructors. A continuous balance exists in the body between ROS production and antioxidants. When the equilibrium is destroyed, the damaging effects of oxidative stress occur.
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