## 4.2.3. Oxidative stress - I.

Reactive oxygen species and antioxidants

Author: Frank van Belleghem

Reviewers: Raymond Niesink, Kees van Gestel, Éva Hideg

Learning objectives:

You should be able to

- explain what oxidative stress is, under which circumstances it arises and why it is important in toxicology.
- describe what reactive oxygen species are and how they are produced.
- describe how levels of reactive oxygen species are kept under control.
- make a distinction between enzymatic and non-enzymatic antioxidants.

Keywords: Reactive oxygen species, Fenton reaction, Enzymatic antioxidants, Non-enzymatic antioxidants, Lipid peroxidation.

Reactive oxygen species

Molecular oxygen (O2) is a byproduct of photosynthesis and essential to all heterotrophic cells because it functions as the terminal electron acceptor during the oxidation of organic substances in aerobic respiration. This process results in the reduction of O2 to water, leading to the formation of chemical energy and reducing power. The reason why O2 can be reduced with relative ease in biological systems can be found in the physicochemical properties of the oxygen molecule (in the triplet ground state, i.e. as it occurs in the atmosphere). Because of its electron configuration, O2 is actually a biradical that can act as an electron acceptor. The outer molecular orbitals of O2 each contain one electron, the spins of these electrons are parallel (Figure 1). As a result, oxygen (in the ground state) is not very reactive because, according to the Pauli exclusion principle, only one electron at a time can react with other electrons in a covalent bond. As a consequence, oxygen can only undergo univalent reductions, and the complete reduction of oxygen to water requires the sequential addition of four electrons leading to the formation of one-, two-, three-electron oxygen intermediates (Figure 1). These oxygen intermediates are, in sequence, the superoxide anion radical (O2●-), hydrogen peroxide (H2O2) and the hydroxyl radical (●OH).

Another reactive oxygen species of importance is singlet oxygen (1O2 or 1Δg). Singlet oxygen is formed by converting ground-state molecular oxygen into an excited energy state, which is much more reactive than the normal ground-state molecular oxygen. Singlet oxygen is typically generated by a process called photosensitization, for example in the lens of the eye. Photosensitization occurs when light (UV) absorption by an endogenous or xenobiotic substance lifts the compound to a higher energy state (a high-energy triplet intermediate) which can transfer its energy to oxygen, forming highly reactive singlet oxygen. Apart from oxygen-dependent photodynamic reactions, singlet oxygen is also produced by neutrophils and this has been suggested to be important for bacterial killing through the formation of ozone (O3) (Onyango, 2016).

Because these oxygen intermediates are potentially deleterious products that can damage cellular components, they are referred to as reactive oxygen species (ROS). ROS are also often termed ‘free radicals’ but this is incorrect because not all ROS are radicals (e.g. H2O2, 1O2 and O3). Moreover, as all radicals are (currently) considered as unattached, the prefix ‘free’ is actually unnecessary (Koppenol & Traynham, 1996).

Figure 1. Consecutive four-step one-electron reduction of oxygen yielding reactive oxygen intermediates and 2 H2O. Step 1 is superoxide anion radical generation by acceptance of one electron. This step is endothermic and hence rate-limiting. The next steps are exothermic and hence spontaneous. In step 2, the superoxide anion radical is reduced by acceptance of one electron and protonated by two H+, resulting in H2O2 formation. In step 3, H2O2 undergoes heterolytic fission in which one oxygen atom receives both electrons from the broken covalent bond. This moiety is protonated yielding one molecule H2O. The other moiety receives one electron (generated by the Fenton reaction, see text) and is transformed into a hydroxyl free radical (●OH). In step 4, ●OH receives one electron, and after protonation, yields one molecule H2O. Figure adapted from Edreva (2005) by Steven Droge.

ROS are byproducts of aerobic metabolism in the different organelles of cells, for instance respiration or photosynthesis, or as part of defenses against pathogens. Endogenous sources of reactive oxygen species include oxidative phosphorylation, P450 metabolism, peroxisomes and inflammatory cell activation. For example, superoxide anion radicals are endogenously formed from the reduction of oxygen by the semiquinone of ubiquinone (coenzyme Q), a coenzyme widely distributed in plants, animals, and microorganisms. Ubiquinones function in conjunction with enzymes in cellular respiration (i.e., oxidation-reduction processes). The superoxide anion radical is formed when one electron is taken up by one of the antibonding π*-orbitals (formed by two 2p atomic orbitals) of molecular oxygen.

Figure 2. A simplified representation of the reaction of the semiquinone anion radical with molecular oxygen to form the superoxide anion radical. Figure adapted from Bolton & Dunlap (2016) by Steven Droge.

A second example of an endogenous source of superoxide anion radicals is the auto-oxidation of reduced heme proteins. It is known, for example, that oxyferrocytochrome P-450 substrate complexes may undergo auto-oxidation and subsequently split into (ferri) cytochrome P-450, a superoxide anion radical and the substrate (S). This process is known as the uncoupling of the cytochrome P-450 (CYP) cycle and also referred to as the oxidase activity of cytochrome P-450. However, it should be mentioned that this is not the normal functioning of CYP. Only when the transfer of an oxygen atom to a substrate is not tightly coupled to NADPH utilization, so that electrons derived from NADPH are transferred to oxygen to produce O2●- (and also H2O2).

Table 1 shows the key oxygen species and their biological half-life, their migration distance, the endogenous source and their reaction with biological compounds.

Table 1. The key oxygen species and their characteristics (table adapted from Das & Roychoudhury, 2014)

ROS species | Half-life (T1/2) | Migration distance | Endogenous source | Mode of action
Superoxide anion radical (O2●-) | 1-4 µs | 30 nm | Mitochondria, cytochrome P450, macrophage/ inflammatory cells, membranes, chloroplasts | Reacts with compounds with double bonds
Hydroxyl radical (●OH) | 1 µs | 1 nm | Mitochondria, membranes, chloroplasts | Reacts vigorously with all biomolecules.
Hydrogen peroxide (H2O2) | 1 ms | 1 µm | Mitochondria, membranes, peroxisomes, chloroplasts | Oxidizes proteins by reacting with the Cys residue.
Singlet Oxygen | 1-4 µs | 30 nm | Mitochondria, membranes, chloroplasts | Oxidizes proteins, polyunsaturated fatty acids and DNA

Because of their reactivity, at elevated levels ROS can indiscriminately damage cellular components such as lipids, proteins and nucleic acids. In particular the superoxide anion radical and hydroxyl radicals that possess an unpaired electron are very reactive. In fact, hydroxyl has the highest 1-electron reduction potential, making it the single most reactive radical known. Hydroxyl radicals (Figure 1) can arise from hydrogen peroxide in the presence of redox-active transition metal, notably Fe2+/3+ or Cu+/2+, via the Fenton reaction. In case of iron, for this reaction to take place, the oxidized form (Fe3+) has to be reduced to Fe2+. This means that Fe2+ is only released in an acidic environment (local hypoxia) or in the presence of superoxide anion radicals. The reduction of Fe3+, followed by the interaction with hydrogen peroxide, leading to the generation of hydroxyl radical, is called the iron catalyzed Haber-Weiss reaction.

Keeping reactive oxygen species under control

In order to keep the ROS concentrations at low physiologic levels, aerobic organisms have evolved complex antioxidant defense systems that include antioxidant components that are enzymatic and non-enzymatic. These are cellular mechanisms that are evolved to inhibit oxidation by quenching ROS. Three classes of enzymes are known to provide protection against reactive oxygen species: the superoxide dismutases that catalyze the dismutation of the superoxide anion radical, and the catalases and peroxidases that react specifically with hydrogen peroxide. These antioxidant enzymes can be seen as a first-line defense as they prevent the conversion of the less reactive oxygen species, superoxide anion radical and hydrogen peroxide, to more reactive species such as the hydroxyl radical. The second line of defense largely consists of non-enzymatic substances that eliminate radicals such as glutathione and vitamins E and C. An overview of the cellular defense system is provided in Figure 3.

Figure 3. An overview of the cellular defense system for the inactivation of reactive oxygen species, showing the role of different antioxidant enzyme systems as explained below. The generation of lipid radical (L●), lipid peroxyl radical (LOO●), lipid peroxide (LOOH) & lipid alcohol (LOH) in the lipid peroxidation process is described in the section Oxidative stress II: induction by chemical exposure and possible effects. Figure adapted from Smart & Hodgson (2018) by Steven Droge.

Enzymatic antioxidants

Superoxide dismutases (SODs) are metal-containing proteins (metalloenzymes) that catalyze the dismutation of the superoxide anion radical to molecular oxygen in the ground state and hydrogen peroxide, as illustrated by following reactions:

Dismutation of superoxide anion radicals acts in the first part of the reaction with the superoxide anion radical as a reducing agent (a), and as an oxidant in the second part (b). Different types of SOD are located in different cellular locations, for instance Cu-Zn-SOD are mainly located in the cytosol of eukaryotes, Mn-SOD in mitochondria and prokaryotes, Fe-SOD in chloroplasts and prokaryotes and Ni-SOD in prokaryotes. Mn, Fe, Cu and Ni are the redox active metals in the enzymes, whereas Zn not being catalytic in the Cu-Zn-SOD.

H2O2 is further degraded by catalase and peroxidase. Catalase (CAT) contains four iron-containing heme groups that allow the enzyme to react with the hydrogen peroxide and is usually located in peroxisomes, which are organelles with a high rate of ROS production. Catalase converts hydrogen peroxide to water and oxygen. In fact, catalase cooperates with superoxide dismutase in the removal of the hydrogen peroxide resulting from the dismutation reaction. Catalase acts only on hydrogen peroxide, not on organic hydroperoxide.

Peroxidases (Px) are hemoproteins that utilize H2O2 to oxidize a variety of endogenous and exogenous substrates. An important peroxidase enzyme family is the selenium-cysteine containing Glutathione peroxidase (GPx), present in the cytosol and mitochondria. It catalyzes the conversion of hydrogen H2O2 to H2O via the oxidation of reduced glutathione (GSH) into its disulfide form glutathione disulfide (GSSG). Glutathione peroxidase catalyzes not only the conversion of hydrogen peroxide, but also that of organic peroxides. It can transform various peroxides, e.g. the hydroperoxides of lipids. Glutathione peroxidase is found in both the cytosol and in the mitochondria. In the cytosol, the enzyme is present in special vesicles.
Another group of enzymes, not further described here, are the Peroxiredoxins (Prxs), present in the cytosol, mitochondria, and endoplasmic reticulum, use a pair of cysteine residues to reduce and thereby detoxify hydrogen peroxide and other peroxides. It has to be mentioned that no enzymes react with hydroxyl radical or singlet oxygen.

Non-enzymatic antioxidants

The second line of defense largely consists of non-enzymatic substances that eliminate radicals. The major antioxidant is glutathione (GSH), which acts as a nucleophilic scavenger of toxic compounds, trapping electrophilic metabolites by forming a thioether bond between the cysteine residue of GSH and the electrophile. The result generally is a less reactive and more water-soluble conjugate that can easily be excreted (see also phase II biotransformation reactions). GSH also is a co-substrate for the enzymatic (GS peroxidase-catalyzed) degradation of H2O2 and it keeps cells in a reduced state and is involved in the regeneration of oxidized proteins.

Other important radical scavengers of the cell are the vitamins E and C. Vitamin E (α-tocopherol) is lipophilic and is incorporated in cell membranes and subcellular organelles (endoplasmic reticulum , mitochondria, cell nuclei) and reacts with lipid peroxides. α-Tocopherol can be divided into two parts, a lipophilic phytyl tail (intercalating with fatty acid residues of phospholipids ) and a more hydrophilic chroman head with a phenolic group (facing the cytoplasm). This phenolic group can reduce radicals (e.g. lipid peroxy radicals (LOO●, see Figure 2, for explanation of lipid peroxidation, see section on Oxidative stress II: induction by chemical exposure and possible effects) and is thereby oxidized in turn to the tocopheryl radical which is relatively unreactive because it is stabilized by resonance. The radical is regenerated by vitamin C or by reduced glutathione (Figure 4). Oxidized non-enzymatic antioxidants are regenerated by various enzymes such as glutathione.

Figure 4. α-Tocopherol reduces a lipid peroxide radical and prevents the further chain reaction of lipid peroxidation. The oxidized α-tocopherol is regenerated by reduced glutathione. Figure adapted from Niesink et al. (1996) by Steven Droge.

Vitamin C (ascorbic acid) is a water-soluble antioxidant and is present in the cytoplasm. Ascorbic acid is an electron donor which reacts quite rapidly with the superoxide anion radical and peroxyl radicals, but is generally ineffective in detoxifying hydroxyl radicals because of its extreme reactivity it does not reach the antioxidant (See Klaassen, 2013). Moreover, it regenerates α-Tocopherol in combination with reduced GSH or compounds capable of donating reducing equivalents (Nimse and Pal, 2015): Figure 5.

Figure 5. Detoxication of lipid radicals (L·) by vitamin C and subsequent regeneration by reduced glutathione. Figure adapted from Niesink et al. (1996) by Steven Droge.

References

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Das, K., Roychoudhury, A. (2014). Reactive oxygen species (ROS) and response of antioxidants as ROS-scavengers during environmental stress in plants. Frontiers in Environmental Science 2, 53.

Edreva, A. (2005). Generation and scavenging of reactive oxygen species in chloroplasts: a submolecular approach.Agriculture, Ecosystems & Environment 106, 119-133.

Klaassen, C. D. (2013). Casarett & Doull's Toxicology: The Basic Science of Poisons, Eighth Edition, McGraw-Hill Professional.

Koppenol, W.H., Traynham, J.G. (1996). Say NO to nitric oxide: nomenclature for nitrogen-and oxygen-containing compounds. In: Methods in Enzymology (Vol. 268, pp. 3-7). Academic Press.

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Nimse, S.B., Pal, D. (2015). Free radicals, natural antioxidants, and their reaction mechanisms. Rsc Advances 5, 27986-28006.

Onyango, A.N. (2016). Endogenous generation of singlet oxygen and ozone in human and animal tissues: mechanisms, biological significance, and influence of dietary components. Oxidative medicine and cellular longevity, 2016.

Niesink, R.J.M., De Vries, J., Hollinger, M.A. (1996). Toxicology: Principles and Applications. CRC Press.

Smart, R.C., Hodgson, E. (Eds.). (2018). Molecular and Biochemical Toxicology. John Wiley & Sons.