Free radicals are unstable atoms, ions, or molecules containing one or more unpaired electrons in the outermost electron shell. An unpaired valence electron is unstable and highly reactive. To attain stability, free radicals attack and acquire electrons from other compounds or molecules within their proximity. The attacked entity loses an electron to become oxidized and becomes a free radical itself, thereby initiating a chain reaction that can result in cellular damage [1]. ROS and reactive nitrogen species (RNS) are unstable molecules containing oxygen and/or nitrogen and include both free radical and non-radical species. The oxygen molecule (O₂••) is a weak free radical itself due to the presence of two unpaired electrons in its valence shell; however, it is less reactive than other oxygen species due to the parallel spin of its electrons [2]. Major ROS and RNS are listed in Table 1.

RNS is a family of nitrogen moieties associated with oxygen. They are produced when nitric oxide (•NO) reacts with oxygen species. For example, nitric oxide can react with superoxide (O₂•⁻) to form peroxynitrite (ONOO⁻):

Peroxynitrite is very reactive and readily attacks lipid molecules, resulting in lipid peroxidation and lipoprotein oxidation [3]. However, like ROS, low levels of RNS have important roles in physiological processes. For example, nitric oxide produced by nitric oxide synthase (NOS) regulates blood vessel dilation and is involved in synaptic transmission in the brain ^[4][5][4,5]. On the other hand, high levels of RNS result in nitrosative stress, macromolecule damage, and activation of transcription factors NF-_ΚB and activator protein 1 (AP-1) involved in inflammation and other pathological pathways ^[6][7][6,7]. RNS and ROS often act together to cause cellular damage [8].

ROS are oxidants (i.e., a molecule that removes electrons from other molecules) predominantly produced as byproducts of cellular metabolism and biochemical processes within the cell. Mitochondria are a primary source of ROS produced by aerobic respiration ^{[9][10][11][12]}[9,10,11,12], where the reduction of molecular oxygen in the electron transport chain results in the leaking of superoxide radicals which are readily detoxified to hydrogen peroxide (H₂O₂) by antioxidant enzymes such as catalase and glutathione peroxidase. Hydrogen peroxide may react with transition metals such as iron (Fe²⁺) to produce hydroxyl radicals via the Fenton reaction to further produce hydroxyl radicals (•OH) which are highly reactive toward all components of DNA molecules as well as lipids [13]. Peroxisomes also generate ROS from aerobic metabolism [14], and phagocytic neutrophils and macrophages produce ROS to eliminate invading pathogens [15]. At low to moderate levels, ROS plays an important role in normal cellular processes, serving as secondary messengers in intracellular signalling cascades that mediate cell growth, autophagy, inflammatory and immune function, and contribute to overall redox regulation ^[16][17][16,17]. However, both radical and non-radical ROS can be powerful oxidants that are detrimental to the cell upon high or chronic exposure. Toxic exogenous sources of ROS include pollution, tobacco smoke, alcohol, ozone, environmental and industrial toxins, and radiation [1]. Due to their reactive nature, ROS production and elimination must be strictly regulated by the cell. Figure 1 summarizes the major sources of exogenous and endogenous ROS and their outcomes in the cell.

Extensive or prolonged exposure to ROS results in oxidative stress, a deleterious process that damages lipids, proteins, and nucleic acids in the cell, thereby inhibiting their normal function [2]. In this scenario, there is an imbalance between the production of ROS and cellular defence mechanisms against oxidative stress, i.e., the antioxidant response. Chronic oxidative stress and the resultant oxidative damage have been implicated in many human diseases including cardiovascular disease, neurodegenerative diseases, diabetes, cancer, and the aging process ^{[18][19][20][21][22]}[18,19,20,21,22].

The consequence of ROS or oxidants and the extent of oxidative stress depends on the strength, duration, and context of exposure. In response to oxidative stress, cells typically undergo cell cycle arrest and enter the G₀ phase (i.e., a quiescent, non-dividing stage) due to activation of the p53-regulated cyclin-dependent kinase inhibitor p21, which halts cell cycle progression and inhibits DNA synthesis ^[23][24][23,24]. ROS can also trigger the p53 and p21-mediated dephosphorylation and activation of the tumour-suppressor retinoblastoma protein (Rb) resulting in further inhibition of cell cycle progression [25]. It is interesting to note that p21 is also involved in the regulation of the antioxidant response through its binding to the antioxidant transcription factor, Nrf2 [26] (to be discussed in Section 2.6). Depending on the nature of the exposure, cells can activate adaptive cell survival pathways; however, chronic exposure or excessively high levels of ROS may result in the induction of maladaptive autophagic or apoptotic pathways ^[27][28][27,28].

To preserve the delicate balance between the beneficial and harmful effects of ROS, living organisms have evolved cellular defence mechanisms against oxidative stress to maintain redox homeostasis. Alterations in redox status can lead to the transcriptional activation of pathways and enzymes involved in the detoxification, transport, and elimination of ROS. For further reading on oxidative stress, the reader is encouraged to refer to the comprehensive review by Sies et al., (2017) [29].

Complex antioxidant defense systems have evolved to protect cells and tissue against oxidative stress. Halliwell and Gutteridge have defined antioxidants as “any substance that, when present in low concentrations compared to that of an oxidizable substrate, significantly delays or inhibits the oxidation of that substrate” [30]. Key antioxidant defenses include (1) antioxidants that directly scavenge ROS, such as glutathione, vitamin C, and vitamin E, and (2) antioxidant enzymes including superoxide dismutase, catalase, and glutathione peroxidase.

Superoxide dismutases (SOD) are a class of enzymes found within the cytosol and mitochondria of nearly all aerobic cells and contain metal ion cofactors such as copper, zinc, manganese, or iron. SOD isoenzymes include Cu/Zn-SOD (SOD1), Mn-SOD (SOD2), and extracellular (EC) SOD (SOD3) ^[31][32][31,32]. SODs are responsible for the dismutation (simultaneous oxidation and reduction) and breakdown of superoxide radicals into molecular oxygen and hydrogen peroxide

Molecular oxygen and hydrogen peroxide are weak oxidants that are relatively stable; however, hydrogen peroxide can be converted into extremely reactive hydroxyl radicals and must therefore be targeted for further breakdown. Two enzymes responsible for the decomposition of hydrogen peroxide are catalase and glutathione peroxidase.

Catalase is found in nearly all living eukaryotic organisms and exists primarily within peroxisomes as well as in the mitochondria and nucleus [33]. Catalases catalyze the breakdown of hydrogen peroxide into molecular oxygen and water:

Glutathione peroxidases (GPx) are a class of enzymes that also break down hydrogen peroxide but do so specifically through the oxidation of a glutathione (GSH) cofactor:

Paraoxonase 2 (PON2) is a ubiquitously expressed member of the paraoxonase family of enzymes with dual functions as a lactonase and as an antioxidant enzyme. PON2 has been shown to prevent oxidation and modification of low-density lipoproteins and also counteract lipid peroxidation in the plasma membrane [34]. Additionally, PON2 interacts with the electron transport chain in the inner mitochondrial membrane to significantly reduce the production of superoxide ions [35]. The importance of PON2 as an antioxidant is demonstrated by work showing that the downregulation of PON2 significantly sensitizes cells to oxidative stress [36].

Glutathione (GSH) is a tripeptide comprised of three amino acids (cysteine, glutamic acid, and glycine) and is the most abundant and important low molecular weight antioxidant synthesized in both eukaryotic and prokaryotic cells. GSH plays a critical role in protecting cells from oxidative damage through direct antioxidant activity or coupled to GPx enzymatic activity ^[37][38][37,38]. Enzymes in the GPx family include GPx1 through 8, each with different expression patterns within the body [39]. GPx1 is the most abundant isoform and is ubiquitously expressed in the cytosol and mitochondria. GPx2 is an intestinal extracellular enzyme, while GPx3 is extracellular, and GPx4 prefers lipid peroxides. Four additional isoforms of GPx (GPx5–8) have been identified in humans but are not well studied. GPx enzymes are part of a family of critical proteins known as the phase II enzymes responsible for the conjugation of xenobiotics with peptides and sugars for detoxification.

Xenobiotic metabolism consists of phase I, phase II, and phase III enzymes involved in oxidation, conjugation/detoxification, and elimination, respectively ^[40][41][40,41]. Phase II enzymes are particularly important in cellular responses to oxidative stress and include GPx, glutathione S-transferase (GST), and UDP-glucuronosyltransferase (UGT). Other important antioxidant enzymes include sulfiredoxin (Srx), thioredoxin (Trx), thioredoxin reductase (TrxR), heme oxygenase 1 (HO-1), and NAD(P)H:quinone oxidoreductase 1 (NQO1). Activation of these enzymes leads to robust xenobiotic detoxification and/or antioxidant effects. Early mechanistic studies on the induction of the rat glutathione S-transferase subunit genes, GSTA1 and GSTA2, led to the discovery of a specific enhancer sequence within their promoter region termed the antioxidant response element (ARE) [42]. Since then, AREs have been found in many other antioxidant genes including, among others, NQO1 and HMOX1 ^[42][43][42,43].

The antioxidant response element (ARE) [42], also referred to as the electrophile response element (EpRE), is a cis-acting enhancer sequence found within the promoter region of many cytoprotective antioxidant and phase II enzyme genes. It has a core sequence of 5′-TGACnnnGC-3′ and participates in inducible gene expression in response to oxidative stress [42]. The ARE is also responsible for low-level basal gene expression to mitigate the ROS produced by cellular respiration. Thus, the ARE is important for redox regulation under both stressed and non-stressed conditions. Using in vivo studies in mice, Itoh et al. discovered that the induction of phase II enzymes through the ARE is mediated by a protein transcription factor called Nrf2 [44] (Figure 2). Nrf2-deficient mice showed marked reductions in the expression of the phase II enzyme GST α₁ subunit and the antioxidant enzyme NQO1 [44], and ensuing studies demonstrated increased sensitivity to carcinogens and impaired detoxification of acetaminophen in Nrf2^−/− mice ^[45][46][47][45,46,47]. This illustrates the key role of Nrf2 in the activation of ARE-regulated antioxidant and phase II enzyme genes.