key: cord-0701888-6ewgcv0b
authors: Forman, Henry Jay; Zhang, Hongqiao
title: Targeting oxidative stress in disease: promise and limitations of antioxidant therapy
date: 2021-06-30
journal: Nat Rev Drug Discov
DOI: 10.1038/s41573-021-00233-1
sha: fa518f3132d1024f556ca1c452179f1cd7336b49
doc_id: 701888
cord_uid: 6ewgcv0b

Oxidative stress is a component of many diseases, including atherosclerosis, chronic obstructive pulmonary disease, Alzheimer disease and cancer. Although numerous small molecules evaluated as antioxidants have exhibited therapeutic potential in preclinical studies, clinical trial results have been disappointing. A greater understanding of the mechanisms through which antioxidants act and where and when they are effective may provide a rational approach that leads to greater pharmacological success. Here, we review the relationships between oxidative stress, redox signalling and disease, the mechanisms through which oxidative stress can contribute to pathology, how antioxidant defences work, what limits their effectiveness and how antioxidant defences can be increased through physiological signalling, dietary components and potential pharmaceutical intervention.

The term 'oxidative stress' was first coined by Helmut Sies 1 as an imbalance between production of oxidants and antioxidant defences that may result in damage to biological systems. Since then, the field of redox biology has evolved from concepts of oxidative stress in pathology to redox signalling in physiology 2-4 . Oxidative stress has been shown to participate in a wide range of diseases including atherosclerosis, chronic obstructive pulmonary disease (COPD), Alzheimer disease and cancer, which has revealed the multiple mechanisms by which oxidants contribute to cellular damage 5 . However, the extent to which oxidative stress participates in the pathology of diseases is quite variable, such that the effectiveness of increasing antioxidant defence may be limited in some diseases.

Oxidative stress involves the chemistry of reactions of so-called reactive species derived from oxygen and nitrogen (Box 1). Understanding which of these species cause damage to macromolecules helps to provide a rationale for improving therapeutic approaches to antioxidant defence. However, so far, the use of small molecules therapeutically has been disappointing, largely owing to overly optimistic and incorrect assumptions about how antioxidants work 6 . For example, scavenging of hydroxyl radical (•OH) is impractical, but preventing its formation by reducing hydrogen peroxide (H 2 O 2 ) production can provide effective prevention of damage. One of the major misunderstandings in the field of oxidative stress concerns the scavenging of superoxide (O 2

) or H 2 O 2 by small molecules, which are also ineffective inside cells. This is because the antioxidant enzymes react thousands to millions of times more rapidly with those oxidants than small molecules do and provide the predominant antioxidant defence 6, 7 . However, in extracellular fluids where antioxidant enzymes are absent, scavenging of O 2

•− and H 2 O 2 (but not •OH) is possible with mimics of superoxide dismutase (SOD) and catalase, as discussed below.

It is essential to recognize the limitations that have led to failures in clinical trials and how antioxidant defences can be effective if one is realistic about where, when and to what extent oxidative stress is part of a disease. Indeed, most antioxidant defence within cells is not provided by either exogenous or endogenous small molecules acting as scavengers, but by antioxidant enzymes using their specific substrates to reduce oxidants. Therefore, the major therapeutic opportunities lie in preventing the production of oxidants that cause direct injury to macromolecules, inhibiting downstream signalling by oxidants that results in signalling for inflammation or cell death, and increasing both antioxidant enzymes and their substrates. Currently, there are clinical trials ongoing for ebselen, a glutathione peroxidase (GPX) mimic, for Meniere disease in phase II (NCT02603081); GC4419, a SOD mimic, for squamous cell cancers in phase I (NCT01921426); and sulforaphane, an activator of the NRF2 transcription factor, for COPD in phase II (NCT01335971), among others.

This article reviews the relationships between oxidative stress, redox signalling and disease and presents an overview of the mechanisms through which oxidative stress can contribute to pathology. We focus on current understanding of the mechanisms mediating

Imbalance between generation of oxidants and the ability to prevent oxidative damage favouring the latter process.

antioxidant defences and what limits their effectiveness, and highlight emerging approaches to therapeutically modulate them. Through greater understanding of the mechanisms through which oxidants act and the limitations and potential of antioxidant therapies, a rational approach can be developed that will improve therapeutic intervention.

For the purposes of this Review, we refer to oxidative stress as the situation in which oxidants non-enzymatically damage macromolecules, including proteins, nucleic acids and the lipids that compose cell membranes. This Review focuses only on factors that either prevent production of oxidants or allow their efficient removal. The principal targets are O 2 •− , H 2 O 2 and lipid hydroperoxides. By eliminating these targets, production of the more reactive •OH, peroxynitrite (ONOO − ) and the hypohalous acids (HOX) can be prevented. Although ONOO − production can be limited by inhibiting nitric oxide (•NO) production, because •NO is too important in maintaining normal physiology, the better approach is to limit excessive O 2 •− production.

There are two major mechanisms through which oxidative stress contributes to disease. The first involves the production of reactive species during oxidative stress -particularly •OH, ONOO − and HOCl -that directly oxidize macromolecules, including membrane lipids, structural proteins, enzymes and nucleic acids, leading to aberrant cell function and death. The second mechanism of oxidative stress is aberrant redox signalling (Box 2). Oxidants, particularly H 2 O 2 generated by cells upon physiological stimulation, can act as second messengers 8 . In oxidative stress, non-physiological production of H 2 O 2 can cause redox signalling to go awry 4 . Both types of oxidative stress mechanism can occur in a single disease, such as in diabetes, where both advanced glycation products accumulate and aberrant

Nuclear factor E2-related factor 2, which coordinates both the baseline and stress-inducible activation of a great many antioxidant enzymes.

Both endogenous and exogenous agents cause oxidative stress 276 . The term reactive oxygen species (ROS) encompasses molecules derived from O 2 , including superoxide (O 2 •− ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (•OH), ozone and singlet oxygen. The use of ROS, as though it were a chemical entity, leads to many imprecise statements because the chemistries of these species are markedly different.

Production of O 2 •− by one-electron reduction of O 2 is primarily through leakage of electrons from the mitochondrial respiratory chain, particularly from ubisemiquinone (QH •− ) 277 (reaction 1): 2 2 and the NADPH oxidases that catalyse reaction 2 (REFS 232, 278 ):

The NADPH oxidases (NOX4, DuOX1 and DuOX2) and some other flavoprotein enzymes reduce O 2 to H 2 O 2 , by giving O 2 •− a second electron before it leaves their active sites 279 .

, a fast reaction with a rate constant near 10 5 M −1 s −1 that is accelerated to 2 × 10 9 M −1 s −1 by superoxide dismutases (reaction 3):

The rate of H 2 O 2 production largely determines whether redox signalling, oxidative stress or no significant oxidation occurs. H 2 O 2 is reduced enzymatically by 15 enzymes, including catalase (reaction 4):

the five peroxiredoxins that use thioredoxin (a small protein with two crucial cysteines, Trx(SH) 2 ) or the eight glutathione peroxidases and peroxiredoxin 6 that use the tripeptide, glutathione (γ-glutamyl-cysteinyl-glycine, GSH) (reactions 5 and 6):

where TrxS 2 is thioredoxin disulfide and GSSG is glutathione disulfide. H 2 O 2 does not easily oxidize most molecules but it can react rapidly with transition metals such as iron to produce hydroxyl radical (reaction 7, often referred to as the Fenton reaction) 280 :

The hydroxyl radical is an extraordinarily strong oxidant that will rapidly oxidize whatever molecule it is next to.

One reaction responsible for oxidative stress is the lipid peroxidation chain reaction that can be initiated by •OH (reactions 8-10): , but from the protonated form of peroxynitrite, peroxynitrous acid (ONOOH), a non-radical. Peroxynitrous acid is a very strong oxidant that has the reactivity of the intermediates formed in its decomposition (reaction 12):

nitrogen dioxide •NO 2 and •OH. •NO 2 can abstract hydrogen as does •OH or add to some molecules including the tyrosines in proteins producing nitrotyrosine that may alter function. ONOO − can also rapidly cause the release of iron from iron-sulfur proteins 11 where X − may be Cl − , Br − or even SCN − (REF. 281 ). They play a major role in tissue damage associated with phagocyte-mediated inflammation.

www.nature.com/nrd activation of stress signalling pathways leads to diabetic complications 9 . Also, the increase in H 2 O 2 production and iron release from proteins in oxidative stress by O 2 •− (REF. 10 ) and ONOO − (REF. 11 ) causes a marked elevation in the production of lipid peroxidation products including 4-hydroxy-2-nonenal (HNE), which can also cause aberrant signalling 12 .

Oxidative stress has been associated with a wide range of pathologies. On the basis of the contribution of oxidative stress to the aetiology of these pathologies, they have been grouped into two categories below: first, oxidative stress as the primary cause of pathology (including toxicities caused by radiation and paraquat, and in atherosclerosis); second, oxidative stress as the secondary contributor to disease progression (such as in COPD, hypertension and Alzheimer disease). However, as the role of oxidative stress in many diseases is incompletely understood, this categorization is tentative.

Oxidative stress can be a primary factor in toxicity and disease. However, an important caveat is that once damage begins, antioxidant therapy often fails to inhibit the progression of tissue injury as other factors become dominant in the pathology.

Early pneumonitis followed by fibrosis frequently occur as side effects of radiotherapy for lung and oesophageal cancers 13 . When cells are exposed to radiation, homolytic cleavage of H 2 O directly generates •OH, which then oxidizes macro molecules and triggers an inflammatory response leading to infiltration of inflammatory cells into the lung (pneumonitis) and cell death. Over a longer period, aberrant redox signalling for the continuous production of cytokines causes accumulation of collagen and lung fibrosis 14 . In addition, higher lipid peroxidation and DNA oxidation (8-hydroxy-2′-deoxyguanosine) has been observed in lungs of radiation-induced lung injury in rats, which can persist for months after radiation exposure 15 .

Paraquat poisoning. Oxidative stress is also responsible for the toxicity of the widely used chemical herbicide, paraquat. When ingested, paraquat is actively taken up by alveolar type II cells and leads to pneumonitis and progressive lung fibrosis with poor prognosis. Paraquat also causes injury to other organs including liver and kidney. Long-term exposure to paraquat is associated with Parkinson disease 16 . Paraquat toxicity is initiated by the continuous redox cycling that generates O 2

Atherosclerosis. In atherosclerosis, plaque builds up in the intimal layer of arteries and over time the arteries narrow, leading to infarction and stroke. Substantial evidence indicates that oxidative stress has a crucial role in the pathogenesis of atherosclerosis. Since the first identification of lipid hydroperoxides in human atherosclerotic aorta 18 , many studies have shown an increase in oxidized lipids and other oxidative stress markers in the atherosclerotic lesions. For example, 20% of cholesteryl linoleate (Ch18:2) in freshly isolated human plaque was reported to be oxidized, whereas it was undetectable in normal arteries 19 . In addition, HNE-modified lowdensity lipoprotein (LDL) was found to be elevated by 50% in plasma of patients with atherosclerosis compared with healthy volunteers 20 . Furthermore, isoprostanes, peroxidation products of arachidonic acid, have been reported to be increased at least fivefold in human atherosclerotic lesions compared with human umbilical veins, and oxidized linoleic acid was detected only in human lesions 21 . Oxidative stress is responsible for the conversion of LDL cholesterol into the atherogenic form of oxidized-LDL (OxLDL), which has a crucial role in initiating and promoting the inflammatory response and recruitment of leukocytes in the lesion site, and contributes to the development of atherosclerosis through activation of smooth muscle cells and reduced •NO bioavailability 22 .

In many diseases, oxidative stress occurs secondary to the initiation of pathology by other factors. Examples of this are the oxidative stress caused by increased production of O 2

•− or H 2 O 2 from NADPH oxidases (NOXs) in the inflammatory response that follows initial tissue injury, and by xanthine oxidase in ischaemia-reperfusion. Oxidative stress can disturb various signalling pathways and affect multiple biological processes through modifying proteins, promoting inflammation, inducing apoptosis, deregulating autophagy, impairing mitochondrial function and many other mechanisms. These effects frequently accelerate pathological progression and exacerbate the symptoms of diseases, as discussed in representative examples below.

Treatment with agents that enhance antioxidant defence.

Inflammation of the lungs caused by irritation of lung tissue, disease, infection, radiation therapy or allergy.

Cessation followed by restoration of blood flow.

A mechanism through which unnecessary or damaged cellular components are degraded.

Redox signalling is dependent on specific interactions of signalling proteins with hydrogen peroxide (H 2 O 2 ) or other electrophiles that act as second messengers. As with oxidative stress, both endogenous and exogenous sources of H 2 O 2 or other electrophiles may be involved; however, for redox signalling to be physiological rather than pathological, regulation is essential and requires the involvement of specificity that is not part of oxidative stress. An oxidative challenge, as opposed to oxidative stress, involves the stimulation of redox signalling without any damage, a phenomenon that we have called 'para-hormesis' 101 . A related term is 'oxidative eustress' 3 . Maintaining redox homeostasis is important for cell function. Despite its name, homeostasis does not imply that nothing is changing. Indeed, a balance between oxidants and reductants, including glutathione, thioredoxin and NADPH, which are the substrates for antioxidant enzymes, is essential for maintaining normal physiology 101 . Thus, diseases that involve oxidative stress can be due to disruption of redox homeostasis, with type 2 diabetes mellitus as one example 9 . Adaptive homeostasis, as defined by Kelvin Davies 282 , involves elevated antioxidant defences brought about by transient alteration of redox homeostasis and redox signalling. However, redox signalling may also occur under pathological conditions, as oxidative stress can stimulate the same pathways as redox signalling under physiological conditions. The difference in this context is that the signalling will be unregulated and accompanied by nonspecific damage.

The effectiveness of this antioxidant system in maintaining the homeostasis relies upon keeping the generation and removal of superoxide (O 101 . It is not a perfect system as evidenced by a low rate of oxidized proteins that accumulate with age. Regardless, the ability to induce the enzymes that remove O 2

and H 2 O 2 and damaged proteins in what Davies calls 'adaptive homeostasis' provides a major means of enhancing antioxidant defences that will be described elsewhere in this Review 282 .

Chronic obstructive pulmonary disease. COPD comprises progressive and irreversible chronic bronchitis and/or emphysema. Cigarette smoking, the main cause of COPD, is an abundant source of oxidants. Oxidative stress can lead to oxidation and inhibition of α1-antitrypsin, thus reducing its ability to inhibit neutro phil elastase, a major factor in the pathogenesis of COPD 23 . In addition, chronic exposure to oxidants in cigarette smoke causes and promotes the inflammatory response and other pathological cascades such as cell death and fibrosis in COPD pathogenesis 14 . The sources of oxidants in COPD are both exogenous (for example, cigarette smoking and air pollution) and endogenous (for example, NOX, mitochondria, inducible nitric oxide synthase (iNOS) and myeloperoxidase) 14 . Increased levels of oxidants and lipid peroxidation products, including 8-isoprostane, have been consistently detected in exhaled breath condensate of patients with COPD compared with healthy controls 24 . In addition, HNE (HNE adducts) levels were found to be significantly elevated by at least 50% in airway and alveolar epithelial cells, endothelial cells and neutrophils in patients with COPD compared with healthy controls 25 ; and the urinary level of 8-hydroxydeoxyguanosine (8-OHdG), a marker of DNA oxidation, was significantly elevated in patients with COPD 26 . The level of oxidative stress was inversely correlated with lung function of the patients 25 . Together, these results suggest that oxidative stress occurs both in the lung and systemically in patients with COPD and contributes to disease pathogenesis. 47 and GSH conjugation to haemoglobin 48 , have been reported to be significantly elevated in the plasma of patients with type 2 diabetes mellitus, as have urine 8-OHdG and 8-iso-PGF2α levels 49 . The increased oxidants in type 2 diabetes mellitus arise from dysfunctional mitochondria 50 and NOX1 (REF. 51 ) activated by the diabetic abnormalities of hyperglycaemia and dyslipidaemia.

Alzheimer disease. Alzheimer disease is characterized by the progressive accumulation of extracellular amyloid-β plaques and neurofibrillary tangles inside neurons. Several risk factors (age, genetics, sex, trauma and air pollution) for Alzheimer disease have been identified, but the exact cause remains unclear. However, accumulating evidence suggests that oxidative stress may have a crucial role through multiple pathways 52 . Many studies have demonstrated increased oxidative stress in the brain of patients with Alzheimer disease, including increased levels of F2-isoprostane-α in cerebrospinal fluid 53 and frontal and temporal poles 54 , acrolein in amygdala and hippocampus/parahippocampal gyrus 55 , and HNE in ventricular fluid 56 , hippocampus and inferior parietal lobule 57 , and cortex 58 . Increased levels of nuclear and mitochondrial DNA oxidation were also found in frontal, parietal and temporal lobes of the brain of patients with Alzheimer disease compared with age-matched control subjects 59 . In addition, protein oxidation in the hippocampus 60 and protein carbonyls in the cerebral cortex 58 were significantly elevated in the brains of patients with Alzheimer disease. Claims have been made that Aβ(1-42) 61 , activated microglia 62 , iron accumulation 63 and dysfunctional mitochondria contribute to increased oxidant production 64 .

Cancer. Through aberrantly altering signalling transduction pathways that damage DNA and exacerbate www.nature.com/nrd inflammation, oxidants are involved in various phases of tumorigenesis, including transformation of normal cells to tumour cells, tumour cell growth, proliferation, invasion, angiogenesis and metastasis 65 . Conversely, oxidative stress can also trigger apoptosis and ferroptosis, and reduce the opportunity for transformation and thereby prevent tumorigenesis 65 . In addition, oxidative stress is the main mechanism of action of radiation (see Radiation-induced lung injury subsection above) and many chemotherapeutic drugs 66 . Therefore, oxidative stress is implicated in almost all phases of cancer. Cancer cells produce more oxidants than normal cells, and therefore cancer cells are exposed to increased oxidative stress in the loci. The increased oxidants in cancer cells are mainly from mitochondria 67 , NOX4 (REF. 68 ) and 5-lipoxygenase 69 . Oxidants in the loci may also come from normal cells in or surrounding the tumour mass, such as endothelial cells and inflammatory immune cells.

The increase in oxidative markers has been observed in various types of cancer. For example, patients with nonsmall-cell lung cancer have been shown to exhale more H 2 O 2 than control individuals 70 . In addition, increased levels of 8-OHdG 71 were detected in breast cancer tissues compared with matched normal tissues, and 8-OHdG was significantly elevated in prostate cancers 72 and lung cancers 73 .

Systemic inflammatory response syndrome. Systemic inflammatory response syndrome (SIRS) is a disorder caused by an exaggerated inflammatory response in the whole body to infectious pathogens or non-infectious insults 74 . SIRS involves the release of oxidants and inflammatory cytokines leading to reversible or irreversible end organ dysfunction and even death. Sepsis is a SIRS caused by infection, which shares common features of inflammation and oxidative stress with SIRS caused by non-infectious insults, and is more frequently studied. Plasma F2-isoprostanes 75 , HNE 76 and 8-OHdG 77 have been reported to be significantly increased in patients with severe sepsis. In patients with acute respiratory distress syndrome from SIRS, the level of 8-iso-PGF2α is increased in exhaled breath condensate 78 as is nitrotyrosine in BALF 79 . Oxidants in sepsis originate from several sources depending on the tissues and/or cells, and include iNOS (also known as NOS2) 80 , NOXs 81 , xanthine oxidase 82 and dysfunctional mitochondria 83 . In addition, the levels of antioxidants such as vitamin C 84 , vitamin E 85 and GSH 86 are decreased in sepsis.

Although timely reperfusion is essential to avoid irreversible injury from ischaemia (interrupted blood flow), extensive damage to both the local and distant organs can occur through initiation of a systemic inflammatory response. Ischaemia-reperfusion injury (IRI) has a major role in the pathophysiological changes of several critical clinical conditions including heart attack, stroke and organ transplantation. The molecular mechanisms underlying IRI are multifactorial and involve the inflammatory response and oxidative stress. In the ischaemic phase, lack of oxygen and nutrients results in accumulation of hypoxanthine, release of calcium, activation of xanthine oxidase and induction of pro-inflammatory cytokines; and in the reperfusion phase, production of NO, ONOO − , O 2 •− and other oxidants is significantly increased from hypoxanthine/xanthine oxidase 87 , mitochondria, iNOS (NOS2) and NOXs 88 in endothelial cells, infiltrated neutrophils and local tissue cells 89 . Markers of oxidative stress including urinary 8-iso-PGF2α are elevated in patients with acute myocardial infarction given thrombolytic therapy, when compared with both age-matched, healthy control subjects and patients with stable coronary heart disease 90 , and in patients with coronary angioplasty following carotid reperfusion 91 . A study involving 66 individuals with stroke and 132 control subjects showed that plasma and urinary F2-isoprostanes were elevated immediately and up to day 7 after onset of ischaemic stroke 92 . Urinary 8-OHdG was also increased after reperfusion in acute myocardial infarction 93 . It should be noted that most oxidative markers measured in IRI studies were systemic and few studies determined the presence of these markers in the lesion site.

To defend against oxidative injury, organisms have evolved defences primarily dependent upon antioxidant enzymes, supply of their substrates and repair of injury. In response to oxidants and other electrophiles, these defences increase and thereby boost the capacity to detoxify oxidants and/or electrophiles and repair oxidative damage. Agents that enhance these defences are the principal strategies underlying antioxidant therapy.

Extensive studies on the induction of antioxidant enzymes have focused on the regulatory mechanisms, the implications in diseases and potential inducers with therapeutic purpose. Although several transcription factors are redox sensitive and are involved in the induction of antioxidant genes (for example, the induction of haem oxygenase 1 (HO1, encoded by HMOX1) through activator protein 1 (AP-1) 94 and peroxisome proliferator-activated receptor-γ (PPARγ) 95 , and the induction of glutamate-cysteine ligase (GCL) 96 and SOD1 (REF. 97 ) through nuclear factor-κB (NF-κB)), the finding with the broadest effect in this area is the induction of antioxidant genes GCLC, GCLM, HMOX1, NQO1, GSTM1, GPX4, TXN and PRDX1 through NRF2

Oxidant species that present immediate danger to the structural integrity and function of cells are •OH, ONOO − and HOX. However, these oxidants react too rapidly with membrane lipids, proteins and nucleic acids to be effectively scavenged by exogenous small molecules. Unfortunately, many erroneous claims have been made for •OH scavengers. Although oxidative stress involves the generation of •OH, the proposed scaven ging of these radicals in biological systems by exogenous molecules is unsound. All organic compounds react with •OH with similar rate constants approaching diffusion limitation. Thus, no compound has more •OH scavenging activity than the thousands of molecules already present in any biological system. To be 50% effective, any compound would have to be present at equal or greater concentration than all of those endogenous molecules.

The only effective strategy preventing damage by •OH is prevention of its formation. Strategies that have the potential to be successful in that endeavour are prevention of the formation of O SODs and enzymes that remove H 2 O 2 and lipid hydroperoxides form the front line of defence against oxidative stress. However, there are major differences between the extracellular fluids and within cells, which have therapeutic implications. Extracellular SOD (EC-SOD, SOD3) is generally associated with the outer membrane of cells and is not present in all extracellular fluids. SOD mimics are effective in the extracellular fluids where decreased production of the potentially hazardous ONOO − has the additional advantage of sparing •NO, which participates in vasodilation and other important physiological processes 100 . Although the outer surface of some cells binds to EC-SOD, the additional catalase activity of most SOD mimics also cataly ses removal of H 2 O 2 , which EC-SOD cannot achieve. Intracellular defences include cytosolic SOD1 and mitochondrial matrix SOD2, which remove O 2

•− , while catalase in peroxisomes (and cardiac mitochondria), GPXs and peroxiredoxins (PRDXs) remove H 2 O 2 . Some of the GPXs and PRDXs also reduce lipid hydroperoxides, with two of them (PRDX6 and GPX4) being able to reduce phospholipid hydroperoxides. Within cells, scavenging of O 2

•− by small molecules is negligible compared with the rate of removal by endogenous SODs, which have rate constants (~2 × 10 9 M −1 s −1 ) that are millions of times higher than those of most other reactions with O 2 •− . The outer surface of some cells binds to EC-SOD, which also outcompetes any potential O 2

•− scavenger. Nonetheless, SOD mimics are useful in extracellular environments that lack significant EC-SOD. SOD produces H 2 O 2 , which would seem to be not much of a gain in terms of antioxidant defence; however, the removal of O 2

•− prevents formation of the more dangerous ONOO − , while simultaneously sparing physiologically important •NO. Compounds with combined SOD and catalase activities have an advantage over SOD alone.

The second line of antioxidant defence includes the synthesis of thioredoxin (TRX), GCL and glutathione synthetase responsible for the synthesis of GSH, glutathione reductase and thioredoxin reductase, which use NADPH to reduce GSSG and TrxS 2 . It should be noted that both first-line and second-line enzymes also have a role in physiological redox signalling and the maintenance of redox homeostasis, and that total elimination of H 2 O 2 would adversely alter cellular function 101 . Scavenging of H 2 O 2 and other hydroperoxides by small molecules is negligible compared with removal by the 15 enzymes that reduce H 2 O 2 and lipid hydroperoxides and the two enzymes that reduce phospholipid hydroperoxides. Nonetheless, a few mimics of GPX, including ebselen (see below), have rate constants that approach those of the enzymes. In addition, ebselen may also reduce ONOO − . Although GSH is normally in the millimolar range in cells, it can be depleted during oxidative stress. Thus, compounds that increase GSH by either supplying cysteine, which is limiting for GSH synthesis, or are precursors for GSH, increase the effectiveness of endogenous GPXs or GPX mimics. Increasing synthesis of GSH by induction of GCL, the enzyme that kinetically limits GSH synthesis, also offers a therapeutic advantage. Indeed, finding agents that induce GCL through activation of the NRF2 transcription factor has been a major goal for more than two decades.

A third line of antioxidant defence is repair or removal of oxidized macromolecules. This broad area Box 3 | NrF2-epre signalling Nuclear factor E2-related factor (NRF2) is a member of the 'cap'n'collar' family of bZIP transcription factors (CNC-bZIP). It was first identified as a transcription factor regulating the expression of β-globin by Moi et al. in 1994 (REF. 283 ), and soon after was found to be a transcription activator of NQO1 that bound to the antioxidant response element (ARE) in the promoter 284 . Many detailed studies established that NRF2-ARE signalling has a central role in the regulation of antioxidant gene expression 285 . ARE, the cis element of NRF2 binding, is more accurately called the electrophile response element (EpRE) as the 'antioxidant' inducers are electrophiles and include hydrogen peroxide (H 2 O 2 ), some components of intermediary metabolism and products derived from dietary polyphenols 6 . NRF2-EpRE signalling regulates the basal and inducible expression of more than 200 genes that encode proteins involved in antioxidant defence, detoxification, apoptosis, DNA repair, removal of oxidized protein by the proteasome, inflammation and other processes 102, 286, 287 . The role of NRF2 in the induction of antioxidant enzymes and defence against oxidative stress has been verified in cell and non-human animal models with NRF2 knockout and/or induction. Mounting evidence suggests that deficiency of NRF2 signalling suppresses the induction of target antioxidant enzymes in response to oxidative stress, increases susceptibility to oxidative damage 288 and accelerates the inflammatory response 289 , whereas enhancing NRF2 activity increases the expression of antioxidant enzymes and the defence against oxidative stress.

The molecular mechanism and regulation of NRF2 activation in response to oxidative stress has been discussed in many recent articles. Most relevant to therapeutics is the recent review by Cuadrado et al. 99 . Thus, we only briefly describe the regulation of NRF2 (FIg. 3) . Under basal conditions, most NRF2 protein binds to Kelch-like ECH-associated protein 1 (KEAP1) and/or βtransducin repeat-containing protein (βTrCP) and is rapidly degraded by 26S proteasome after ubiquitylation. KEAP1 is an adaptor for Cullin 3-containing ubiquitin ligase E3 complex 290 , and βTrCP is a substrate receptor for Cul1-based ubiquitin ligase 291 . In response to oxidative stimuli, KEAP1 is oxidatively modified and loses the capacity to present NRF2 for degradation. Simultaneously, oxidative inhibition of glycogen synthase kinase 3β (GSK3β)-mediated NRF2 phosphorylation at the Neh6 domain stops the interaction of NRF2 and βTrCP. NRF2 can also be activated through p62-mediated autophagic degradation of KEAP1 (REF. 292 ). With the activation of these pathways, NRF2, both dissociated from KEAP1 and newly synthesized, escapes from degradation and is then translocated into the nucleus where it forms heterodimers with small Maf or Jun family proteins, binds to EpRE in the promoter and increases transcription of target genes. In the nucleus, NRF2 is competitively suppressed by BACH1 (REF. 293 ). ChIP-seq assays identified a considerable overlap of BACH1 (in HEK293 cells) and NRF2 (in mouse MEF cells) target genes 294 . Evidence suggests that the suppressive effect of BACH1 on NRF2 signalling may be gene selective. BACH1 inactivation is required for the induction of HO1 but not for that of thioredoxin reductase 1, even though both genes are regulated by NRF2 (REF. 295 ). In Bach1knockout mice, fewer than 10% of the upregulated genes are NRF2 target genes 244 . It should be noted that NRF2 regulation is far more complicated than the simplified pathways, as nuclear factor-κB (NF-κB), PKC, p21, BRCA1, HRD1, CRIF1 and microRNAs are involved in regulating NRF2 signalling by acting on NRF2 expression, protein stability, activation and translocation 99 . With more regulators and interaction pathways being identified, NRF2 activity is clearly regulated by a network of signalling pathways allowing it to hold important roles in multiple biological processes and response to multiple circumstances. Some puzzles remain for NRF2 regulation, including how NRF2 is transported in and out of the nucleus, and the dysregulation and ceiling effect of NRF2 induction under some pathophysiological conditions. www.nature.com/nrd of research is not directly relevant to the present Review; however, the enzymatic systems for removal of oxidized proteins 102 , oxidized fatty acid removal and replacement 103 , and oxidized DNA removal and repair 104 are induced by oxidants.

Multiple antioxidant therapeutic strategies are being explored, some of which are currently undergoing clinical trials. These include removal of O 2

•− before it can react with •NO to form ONOO − (reaction 11) and removal of H 2 O 2 before it can form •OH (reaction 7) or HOX (reaction 13); increasing GSH using precursors; increasing the synthesis of antioxidant enzymes, particularly through NRF2 activation (Box 3); inhibition of NOXs (reaction 2); mitochondrial antioxidant defence; supplementing dietary antioxidants; and finally, inhibition of aberrant redox signalling (Box 2). See Box 1 for reactions.

Several antioxidant enzyme mimics have been and are currently in clinical trials (TABlE 1) . SOD is the only enzyme that can eliminate O 2

•− in mammalian cells and is a key component in defence against oxidative stress and in preserving •NO. The therapeutic potential of SOD has therefore generated interest since its discovery in 1969 (REF. 105 ), and many SOD mimetics have since been developed. These mimetics include the metalloporphyrins, Mn cyclic polyamines, nitroxides, Mn-salen complexes and fullerenes, and their chemical properties have previously been well summarized 106, 107 .The early studies on SOD mimics primarily focused on metalloporphyrins (that is, MnTM-4-PyP 5+ and FeTM-4-PyP 5+ ) [108] [109] [110] , and since the establishment of the structure-activity relationship between metal-site redox ability and SOD activity in the late 1990s 111 , more porphyrins or porphyrin-related mimics with higher SOD activity have been developed. The protective effects of many of these compounds have been demonstrated in non-human animal studies or even clinical trials. Mimics of SOD and catalase have rate constants several orders of magnitude lower than the enzymes. Thus, when they enter cells, their contribution to cytosolic antioxidant defence is relatively minor. However, SOD and catalase mimics appear to be effective in extracellular spaces where the concentrations of antioxidant enzymes and substrates are very low or absent (FIg. 1) . Some of the mimics may also be effective in the mitochondrial matrix, but they can act as pro-oxidants instead of as protectors of mitochondrial function 112 .

Although being developed to remove O 2 •− specifically, most SOD mimics are not specific and can also reduce other reactive oxygen or nitrogen species such as ONOO − , peroxyl radical, H 2 O 2 and CO 3

•− (REFS 113, 114 ).

In addition, some SOD mimics, such as Mn porphyrins, Mn(ii) cyclic polyamines and M40403, can act as pro-oxidants and react with thiols 112 , ascorbate 115 and tetrahydrobiopterin 116 , thereby affecting redox-sensitive signalling pathways and cellular transcription 117, 118 . Therefore, some protective effects of SOD mimics might be attributable to activities other than mimicking SOD.

SOD itself was first developed as a drug called orgotein in the late 1970s, but it has not been approved for human use 119 . However, several clinical trials based on the anti-inflammatory property of orgotein have been conducted. A double-blind, placebo-controlled study has demonstrated that orgotein can be used safely and effectively to ameliorate or prevent the side effects of radiation therapy in patients with bladder cancer, such as the incidence of radio-induced acute cystitis and rectitis 120, 121 . However, in another clinical trial, orgotein showed no beneficial effect on radiation response or the acute radiation reactions, and caused side effects such as marked subcutaneous infiltration and redness at local injection site in some patients 122 . Currently orgotein is used as an anti-inflammatory agent in non-human animals. The best-studied class of SOD mimics is probably the Mn porphyrins. Various Mn porphyrin compounds have been synthesized and evaluated for their O 2

•− dismutation activity 114 . Some of them, such as MnTM-2-pYp 5+ and MnTE-2-pYp 5+ , showed very high SOD activity. Although whether the underlying mechanism is via SOD-like activity or another action (for example, pro-oxidant activity) remains elusive in some cases, the protective and therapeutic effects of many Mn porphyrins such as MnTE-2-pYp 5+ and MnTDE-2-ImP 5+ have been demonstrated in non-human animal models of diseases, including stroke 123 , radiation injuries 124 , cancers 125, 126 , diabetes 127 and cardiovascular system damage 128 . These preclinical results suggest the potential of Mn porphyrins in the clinical therapy of diseases in which oxidative stress plays a significant part. Currently, a phase I clinical trial of MnTDE-2-ImP 5+ in patients with amyotrophic lateral sclerosis showed no toxicity at therapeutic doses 129 .

Another promising SOD mimetic is GC4419, a novel, highly stable Mn(ii)-containing penta-azamacrocyclic. GC4419 selectively removes superoxide anions without reacting with other oxidants 130 . In vitro, GC4419 significantly enhanced the toxicity of AscH − to kill cancer cells 131 . In addition, GC4419 has exhibited therapeutic effects in several non-human animal models of inflammation 132 , joint disease 133 and myocardial IRI 134 . A recent phase I clinical trial in severe oral mucositis of oropharyngeal cancer with radiation and chemotherapy indicates that the safety of GC4419 in patients is acceptable 135 .

Salens, aromatic, substituted ethylenediamine metal complexes, represent an emerging class of SOD mimics. 

•− to hydrogen peroxide (H 2 O 2 ) is accelerated by superoxide dismutase (SOD) mimics, preventing the formation of peroxynitrite (ONOO − ), which spares nitric oxide (•NO). Reduction of H 2 O 2 is accelerated by catalase mimics, preventing the formation of hypohalous acids (HOX) by myeloperoxidase (MPO) and hydroxyl radical (•OH) production via the Fenton reaction. Most SOD mimics appear to have catalase activity. Although NOX4, which is primarily in intracellular organelle membranes, has also been found in the plasma membrane, this has only been reported for one cell type 275 and so its extracellular location remains debatable (indicated by the question mark). NOS, nitric oxide synthase. 136 . Salen compounds are not selective and can also react with other peroxides and ONOO − . The typical representative salens are EUK-8, EUK-134 and EUK-189, which have been shown to be protective in many non-human animal models of human diseases, including sepsis 137 , heart ischaemia-reperfusion 138 , cardiomyopathy 139 , haemorrhage 140 and amyotrophic lateral sclerosis 141 (EUK-8); IRI 142 and stroke 143 (EUK-134); radiation lung fibrosis 144 , cognitive impairment 145 , diaphragm muscle weakness in monocrotalin-induced pulmonary hypertension 146 and hyperthermia 147 (EUK-189). However, no human clinical trial for salens has yet been reported.

A variety of mimics of GPXs have been developed 148 . Among these mimetics, the selenoorganic compound ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is best known, with its broad specificity for substrates from H 2 O 2 and smaller organic hydroperoxides to membrane-bound phospholipid and cholesterol hydroperoxides 149 . Ebselen may also induce phase II detoxification enzymes 150 . In non-human animal studies, ebselen has been shown to reduce oxidative damage 150 , prevent the acute loss of outer hair cells and reduce hearing loss 151 , and decrease inflammation 152 . Accordingly, several clinical trials have been conducted in diseases including Meniere disease (phase III, NCT04677972), bipolar disorder 153 , complete occlusion of the middle cerebral artery 154 , delayed neurological deficits after aneurysmal subarachnoid haemorrhage 155 and acute ischaemic stroke 156 . In these studies, oral administration of ebselen was well tolerated, exerted therapeutic effects and displayed favourable bioavailability.

ALT-2074 (BXT-51072) is a newer analogue of ebselen, displaying increased GPX activity and potency. In vitro, ALT-2074 inhibited the inflammatory response in endothelial cells 157 , reduced oxidative damage and prevented neuronal death 158 , and in a mouse model of heart ischaemia-reperfusion it reduced infarct size 159 . A phase II clinical trial of ALT-2074 (NCT00491543) in diabetes and coronary artery disease has been completed but data are not yet available. Another clinical trial on psoriasis (NCT00782613) was terminated but the reasons for this remain unknown.

It has long been recognized that when iron and copper are released from proteins, they can participate in •OH production, and that some chelators enhance that activity while others inhibit it 160 . In principle, using the inhibitory chelators would be an excellent strategy to prevent •OH production; however, as iron is essential for many biological activities, chelation therapy is generally restricted to the prevention of iron overload in patients with sickle cell disease and thalassaemia, who require frequent transfusions 161 .

Although most cells have a concentration of GSH in the millimolar range, GSH is often significantly decreased by oxidative stress. Thus, approaches to maintaining or replenishing GSH using GSH esters or agents that provide its precursor, cysteine, the limiting amino acid in GSH synthesis, have shown effectiveness in various diseases. (TABlE 1) . It is water soluble and quickly absorbed primarily via the anion exchange protein on the cell membrane 162 . NAC in cells is deacetylated to produce cysteine. Evidence indicates that the antioxidant function of NAC is primarily mediated via replenishing GSH 163 . NAC can also reduce cysteine conjugates in plasma 162 . NAC has been used therapeutically for the treatment of many pathologies, including liver paracetamol (also known as acetaminophen) toxicity 164 , cystic fibrosis, where it is delivered through the airways 165 and nephropathy 166 . In non-human animal studies and clinical trials, NAC is being investigated for prevention or treatment of many other diseases and conditions. The results from these studies are conflicting and a consensus has yet to be reached. Failure of NAC to exert a therapeutic effect may be due to oxidative stress being a secondary contributor to the disease being studied. 167 . Thus, using derivatives of GSH is a strategy for more successful delivery. Ester derivatives of GSH, including monomethyl (GSH-OMe), monoethyl (GSH-MEE), diethyl (GSH-DEE) and isopropyl esters have been synthesized and evaluated for the efficiency of GSH supplementation. In GSH-MEE, the carboxyl group of the glycine residue is esterified (Glu-Cys-Gly-OEt); whereas in GSH-DEE both glutamate and glycine residues are esterified (tEO-Glu-Cys-Gly-OEt). GSH esters are lipophilic, more efficiently transported across the cellular membrane and resistant to degradation by γ-glutamyl transpeptidase in plasma 168 . Once inside cells, GSH esters are rapidly hydrolysed by nonspecific esterases and form GSH. The transport of GSH-DEE into cells seems more efficient than that of the monoester 169 , and human cells can rapidly convert the diethyl ester into the monoester, which is hydrolysed into GSH.

The high efficiency of GSH esters to increase cell and/or tissue GSH has been evidenced in many studies in cell and non-human animal models [170] [171] [172] [173] [174] [175] . Subcutaneous or intraperitoneal injection of GSH esters into animals was able to increase GSH levels in various tissues including liver 170 , kidney 170 , spleen, pancreas and heart 176 , but not brain 177 . Brain GSH levels can be increased via intracerebroventricular 174 delivery of GSH-MEE 177 . Although oral administration could also increase tissue GSH levels, this is less effective 176 .

The relative efficacy of various GSH esters to increase tissue GSH remains unclear owing to limited evidence. Some cell culture-based studies suggest that GSH-DEE is more effective than GSH-MEE in increasing GSH levels 169 . GSH-DEE is metabolized differently in the plasma of non-human animals and humans. In mouse and rat, plasma GSH-DEE is rapidly converted into NATURE REvIEWS | Drug Discovery GSH-MEE by plasma α-esterase, whereas human (and many other species including hamster, guinea pig, rabbit and sheep) plasma has no α-esterase activity, meaning that GSH-DEE can be transported into tissues more efficiently than GSH-MEE 169 . However, no direct comparison study has been conducted on the relative efficacy of the different GSH esters in clinical settings. Although the reports above suggest that humans have apparently been treated with GSH without adverse effects, and the efficacy of GSH esters to increase GSH levels and alleviate oxidative damage in cells and non-human animals has been demonstrated, no clinical trials have been reported with any GSH ester. FIguRE 2 summarizes the strategies for maintaining GSH in cells.

Dysregulation of NRF2 signalling (Box 3; FIg. 3) is implicated in many oxidative stress-related diseases including cardiovascular diseases 178 , neurodegenerative disorders 179 and pulmonary diseases 180 . Therefore, NRF2 activators are regarded as potential agents to induce antioxidant capacity and alleviate pathology. The induction of antioxidant enzymes, particularly through NRF2, is a major way in which antioxidant therapy is being developed. Indeed, when the small molecules such as poly phenols are effective, they act primarily through antioxidant enzyme induction mediated by NRF2 signalling 6 . NRF2 activators comprise five categories, according to their mechanisms of action (FIg. 3) Glutathione (GSH) is synthesized through reactions catalysed by glutamatecysteine ligase (GCL) and GSH synthetase (GS), with GCL as the rate-limiting enzyme and cysteine as the rate-limiting substrate. Both reduced GSH and glutathione disulfide (GSSG) are exported from cells through multidrug resistance protein (MRP), and extracellular GSH is sequentially metabolized by membrane-bound γ-glutamyl transpeptidase (GGT) into cysteinylglycine and γ-glutamyl products, and dipeptidase hydrolyses cysteinylglycine to cysteine and glycine. The amino acids are transported back into cells and participate in GSH synthesis. N-acetylcysteine (NAC) is deacetylated by esterase action into cysteine, while GSH esters (GSH-E) are directly converted by esterase into GSH. γ-Glutamylcysteine (γ-glu-cys) can bypass GCL, the rate-limiting step for GSH synthesis. Electrophiles cause the activation of NRF2, which regulates the transcription of the two subunits of GCL, and also GS. Some transporters have been identified: ASC, sodiumdependent alanine-serine-cysteine transporter; Xc − , system cystine/ glutamate antiporter. Question marks denote the unidentified transporters/ channels for GSH-E, γ-glu-cys and NAC.

www.nature.com/nrd of Kelch-like ECH-associated protein 1 (KEAP1; regulates proteasomal degradation of NRF2), which is inactivated when its sensor cysteines form adducts with electrophiles or when they are oxidized to disulfides; disruption of the interaction between β-transducin repeat-containing protein (βTrCP; ubiquitylates NRF2 for degradation) and NRF2, via oxidative inhibition of the axis of glycogen synthase kinase 3β (GSK3β)-NRF2 phosphorylation at the Neh6 domain-βTrCP; KEAP1 sequestration by p62; de novo synthesis of NRF2 that escapes degradation by inactivated KEAP1 (REF. 181 ); and BACH1 inhibitors that reduce NRF2 suppression by BACH1, including agents that inhibit BACH1 translation 182 and promote BACH1 degradation 183 .

Extracts from tea, cocoa and many dietary vegetables and fruits including broccoli, broccoli sprouts, grape seeds and turmeric can activate NRF2 signalling and induce antioxidant enzymes 184, 185 , and some of these are in clinical trials for disease treatment and/or prevention. For example, 11 clinical trials for turmeric extract and 55

Other positive regulators (1) Transcription factor NRF2 is constantly synthesized in cells but its transport to the nucleus remains low under basal conditions. This is due to its degradation through association with Kelch-like ECH-associated protein 1 (KEAP1), which facilitates its degradation by the 26S proteasome.

Boosting NRF2 synthesis represents a therapeutic antioxidant approach.

(2) Upon exposure to electrophiles, KEAP1 is alkylated and loses its ability to cause degradation of NRF2. Using non-toxic electrophiles to alkylate KEAP1 represents another major therapeutic approach. For KEAP1, SH is the thiol form and SX denotes the adduct formed with the electrophile (X).

(3) In a parallel pathway glycogen synthase kinase 3β (GSK3β) phosphorylates NRF2, which with β-transducin repeat-containing protein (βTrCP) is degraded by the proteasome, a process that is inhibited by oxidative inactivation of GSK3β. The interaction of NRF2 and βTrCP is disrupted owing to oxidant-mediated inhibition of GSK3β and the phosphorylation of NRF2 at the Deh6 domain. Inhibiting GSK3β is another potential therapeutic approach to modulate NRF2 signalling.

(4) Oxidation-induced KEAP1 degradation also occurs through p62mediated sequestration of KEAP1 and autophagy, a process initiated by phosphorylation of p62 via TANK-binding kinase 1 (TBK1) and mechanistic target of rapamycin complex 1 (mTORC1). p62 therefore provides another potential therapeutic target. Newly synthesized NRF2 that escapes degradation is translocated into the nucleus where it binds to EpRE sequences in the promoters of antioxidant genes and increases their expression. NRF2 activity is also positively regulated through NRF2 phosphorylation by protein kinase C (PKC) 269 and its interaction with other proteins such as p21 (REF. 270 ) and BRCA1 (REF. 271 ). (5) In the nucleus, BACH1 negatively regulates NRF2 activity by competing to form heterodimers with small Maf (sMaf) or Jun proteins and binding to the electrophile response element (EpRE) [272] [273] [274] . Thus, compounds that inhibit BACH1 offer an alternative therapeutic approach for increasing expression of some NRF2regulated genes. Other negative regulators of NRF2, which represent potential therapeutic targets include HRD1, CRIF1, progerin and microRNA for NRF2 (REF. 99 ). 186 summarized the current progress on broccoli/broccoli sprout including the formulation, bioavailability, efficacy and doses for clinical trials. In general, some beneficial effects, including a boost of antioxidant capacity, were observed in the clinical trials, but more effort is required to develop and validate biomarkers of pharmacodynamic action in humans. As pointed out above, an increase in antioxidant defence may be limited in disease treatment or prevention if oxidative stress has only a secondary role in the pathology. The underlying mechanism of the antioxidant properties of these dietary supplements, often the coumarins and polyphenols present in vegetables and fruits, relies upon their oxidation to electrophilic quinones that form adducts with KEAP1 cysteines 6 . The effectiveness of many of these NRF2 activators in inducing antioxidant enzymes and in alleviating oxidative damage has been confirmed in non-human animal studies, and there have been significant advances in drug development based on the mechanism of NRF2 activation and antioxidant induction. Several dietary NRF2 activators, including curcumin, sulforaphane and resveratrol, have been developed as daily supplements, while some NRF2 activators are in clinical trials for disease treatment 187 . Selected electrophilic NRF2 activators and the related clinical trials have previously been summarized 187 . It is noted that these NRF2 activators may have multiple functions such as anti-inflammatory effects [188] [189] [190] , some of which are not dependent on NRF2 activation. TABlE 2 lists the total number of clinical trials of selected dietary NRF2 activators and indicates those that are based on NRF2 activation and/or antioxidant potential. For clarification, it is still possible that some of the agents for which a study of NRF2 activation is not indicated do in fact activate NRF2 even though that was not examined.

There are several concerns and challenges associated with the therapeutic use of NRF2 activators 191, 192 . The first is related to low effective biological concentration, as most NRF2 activators are electrophilic and are metabolized quickly so that their bioavailability in distal organs may be low. However, some evidence suggests that the Michael adducts of nucleophiles (including the cysteines of KEAP1) with some electrophiles, such as cyanoenones, are reversible 193 and this may significantly increase the bioavailability and concentration of these electrophiles in vivo. This concept was demonstrated by a synthesized cyanoenone compound TBE31 that had a 10-h half-life in the blood 194 and markedly increased NRF2 activity in vivo at nanomolar concentrations 195 . It remains unclear whether this reversibility of the covalent adducts also occurs with other electrophiles, especially natural compounds such as sulforaphane and curcumin. In addition, there is controversy regarding the effectiveness of oral sulforaphane to induce antioxidant expression in clinical trials, with both increased antioxidant 196 and no effect 197 being reported. In general, more clinical trial data on NRF2 and antioxidant induction in target organs are needed to further assess the efficacy of these NRF2 activators.

Another key concern is the risk of nonspecific effects. Besides activating NRF2 and inducing antioxidant enzymes, some NRF2 activators may act on other signalling pathways and disrupt related biological processes. For example, sulforaphane can suppress the inflammatory response through inhibition of NF-κB 188 and inflammasome activation 198 , and cause cell cycle arrest by inhibiting the PI3K-AKT and MAPK-ERK pathways 199 . Most of these nonspecific effects have been investigated in in vitro cell studies with >10 μM sulforaphane, a concentration that is less likely to be reached in vivo. Understanding the NRF2-independent effects is important in elucidating the mechanism of the beneficial and therapeutic effects, although for most NRF2 activators this has not been thoroughly studied, especially with regard to their in vivo dose dependency.

Another aspect of nonspecificity is that the effect on NRF2 activation and antioxidant induction is not restricted to a specific cell or organ, and may therefore result in systemic side effects. For example, some evidence suggests that although NRF2 activation could prevent the initiation of cancer, it can, however, promote cancer development [200] [201] [202] . Cell studies showed that higher NRF2 activity and antioxidant capacity can also contribute to the resistance to chemotherapeutic drugs [203] [204] [205] [206] , as reviewed by others [207] [208] [209] . Current evidence is insufficient to draw a definitive conclusion and more systemic in vivo studies are needed to elucidate the role of NRF2 in promoting carcinogenesis and causing resistance to chemotherapies. If increased NRF2 activity does promote tumour growth and/or increase chemoresistance, the systemic administration of NRF2 activators should be avoided, at least in susceptible subjects including cancer patients under chemotherapy. Other side effects of long-term NRF2 activation are less reported. Several strategies have been proposed to avoid systemic side effects, including the development of non-electrophilic drugs and drugs that only become active in loci that exhibit oxidative stress 192 .

NOXs are important in redox signalling as the source of O 2

•− and H 2 O 2 and in the killing of microorganisms, but excessive activation of NOXs can result in damage to normal tissue. There are two types of agent that inhibit NOXs, those that inhibit the enzymatic activity and those that prevent the assembly of the NOX2 enzyme, which is a multiprotein complex. Of the first type, diphenyleneiodonium (DPI) is commonly used in research studies but is a nonspecific inhibitor of flavoproteins as well as an inhibitor of iodide transport 210 . Several agents claimed to be NOX inhibitors, including ebselen, CYR5099, apocynin and GKT137831, some of which show promise in non-human animal models and clinical trials, exhibited effects that were not due to NOX inhibition 211 . Nonetheless, the potential value of inhibition of NOX1, NOX2 and NOX4 has been demonstrated in non-human animal models using genetic deletion 212 , and a search for low-molecular-weight NOX inhibitors continues.

Small peptides that inhibit the assembly of the NOX complexes have therapeutic potential 213 . Although these small peptides would be more specific to the different NOXs than active site inhibitors, none has advanced to clinical trials. A third potential approach is interference with the synthesis of the components of the NOX complexes; however, this too has not yet reached clinical trials.

Leaks of electrons from the respiratory chain results in the production of O 2

. Although inhibiting O 2

•− production by either elevating uncoupling proteins or inhibiting the flow of electrons into the chain is possible, the consequences for ATP production make these approaches difficult. Yet, this strategy has been proposed for preventing hyperglycaemic damage in diabetes 214 . One drug, OP2113, which can be used in humans, has been proposed as a specific inhibitor of complex I O 2

•− production that does not interfere with ATP production 215 . However, this agent has not yet been investigated in clinical trials.

As discussed above, increasing SOD2 increases the production of H 2 O 2 in mitochondria by pulling reaction

•− . Thus, SOD mimics that enter mitochondria would be expected to increase the rate of production of H 2 O 2 . However, as these agents also possess catalase activity, they appear to add protection 216 , likely by preventing formation of OONO − and protecting iron-sulfur proteins. Ebselen can also enter mitochondria but may produce unexpected toxicity 217 .

The large negative inner mitochondrial membrane potential makes it possible to target antioxidants and antioxidant mimics to these organelles by attaching a lipophilic cation to them 218 . This is an area of research that is still under development but basically uses the same principles of antioxidant defence as described in other sections of this Review.

The most widely used and studied dietary antioxidants are l-ascorbic acid (vitamin C) and α-tocopherol (vitamin E). Other dietary nutrients, including selenium, riboflavin and metals, are essential cofactors for antioxidant enzymes, and their adequate supply is essential for the inducers of these enzymes to reach their most effective levels, but discussion of them here is beyond the scope of this Review. Vitamin C is a water-soluble vitamin that cannot be synthesized by the human body and must be provided as an essential dietary component. Vitamin C is required for the biosynthesis of collagen, protein and several other biological molecules 219 . Vitamin C is also an important antioxidant 220 , by providing an electron to neutralize free radicals. Vitamin E, which is lipid soluble, localizes to the plasma membrane and has roles in many biological processes. Almost 100 years after its discovery, the functions and mechanism of action of vitamin E still remain of great interest. Nonetheless, the importance of the antioxidant function of vitamin E has been demonstrated by many studies [221] [222] [223] , especially under conditions of oxidative stress or deficiency of other antioxidants 223, 224 . Vitamin E www.nature.com/nrd reduces peroxyl radicals and forms tocopheroxyl radical, which is subsequently reduced by vitamin C. Thus, vitamin E helps to maintain the integrity of long-chain polyunsaturated fatty acids in the membranes and thereby regulates the bioactivity and signalling related to membrane lipids.

For healthy individuals, sufficient levels of vitamins C and E are provided by normal dietary intake and deficiency rarely occurs. Under some extreme conditions such as malnutrition or imbalanced nutrition and diseases 225, 226 , however, dietary supplementation of vitamins C and E is necessary. As vitamins C and E function as antioxidants, there has been great interest in investigating their therapeutic potential. Many studies and clinical trials have found that vitamins C and E have beneficial effects in reducing various diseases, many of which likely involve oxidative stress, including cancers, cardiovascular diseases and cataracts 227 . But the evidence is inconsistent, as an almost equal number of studies show no significant effect. It was assumed that both vitamin C and vitamin E have low toxicity and were not believed to cause serious adverse effects at much higher intake than needed for their function as vitamins. However, several non-human animal studies showed that antioxidant supplements, including NAC, vitamin E and the soluble vitamin E analogue Trolox, promoted cancer development and metastasis, for example, lung, melanoma and intestinal tumours in mouse models [228] [229] [230] . The potential effect of antioxidants on cancer promotion, including the aforementioned NRF2 activators, raises significant concerns regarding the use of antioxidant supplements, and novel strategies are needed to resolve the double-edged effect of antioxidants.

In the early years of research in redox biology the emphasis was almost entirely on damage caused by oxidants. Although studies demonstrated that the addition of non-lethal doses of H 2 O 2 or other oxidants was able to stimulate signalling pathways, it was not until the mid-1990s that NF-κB activation by endogenous generation of H 2 O 2 was first observed 231 . By the late 1990s, Lambeth and coworkers 232 had described the seven-member NOX family and began to implicate them in cell signalling pathways. Redox signalling is now the major focus of the field, although extensive coverage of the topic is beyond the scope of this article. Readers are referred to specific reviews in this area 4, 233 . Nonetheless, as described earlier, H 2 O 2 is the major second messenger in redox signalling and like other second messengers, dysregulation of its production can result in aberrant signalling 233 . Prevention of dysregulation is tricky because attempts to inhibit the generation of oxidants by NOX proteins or mitochondria, as described in earlier sections, may interfere with physiologically important signalling including the regulation of leukotriene and prostaglandin production, which require a low level of H 2 O 2 or lipid hydroperoxides 234 .

A more successful approach may be interference with specific redox signalling that is initiated by toxic stimuli. Here, we provide one example to illustrate this approach 235 . Air pollution contains particles of enormously variable composition and includes silicates with iron on their surface. Activation of NF-κB signalling in macrophages by these particles could be inhibited with a SOD and/or catalase mimic, but also by interfering in the signalling pathway initiated by the iron-mediated lipid peroxidation that caused lipid raft disruption and signalling through phosphocholine-specific phospholipase C (PC-PLC) activation. An inhibitor of that enzyme, tricyclodecan-9-yl xanthate (D609), which was unsuccessfully tried as an anticancer agent, stopped particle-induced NF-κB-dependent cytokine production. D609 is an example of an agent that is not an antioxidant but inhibits oxidant-induced aberrant signalling. Interestingly, D609 interferes with the PC-PLC pathway when initiated by endotoxin 236 , which does not involve redox signalling. There are countless agents that have similar potential to inhibit aberrant signalling although they are not specific to redox-mediated signalling.

Oxidative stress is a component of the underlying pathology of many diseases and toxicities, and the antioxidant defences and strategies that have been presented above offer some important opportunities for preventing or reducing pathology. Nonetheless, there are several limitations that challenge our ability to therapeutically apply antioxidant strategies.

The effectiveness of antioxidant defences is limited by the extent to which oxidative stress plays a role in the pathology. When oxidative stress is a secondary contributor to disease, which is more often the case than it being the primary cause, preventing oxidative stress may not have a major impact on disease progression. Indeed, this is one of the major causes of antioxidants exerting little to no effect on pathology, even when they clearly increase antioxidant defence and decrease markers of oxidative stress. This limitation is perhaps the most significant factor that is often overlooked when considering antioxidant defences in clinical trials. The challenge here is to determine to what extent antioxidant strategies may be developed to ameliorate some symptoms if not the underlying cause of the disease. The commercialization of products containing small molecules that are chemical antioxidants but do not function as such in vivo, will ultimately fail to show significant benefit beyond what the antioxidant enzyme-inducing small molecules present in an adequate diet can achieve. This disappointment will add to the challenge of developing and gaining public acceptance of truly effective therapeutics.

The negligible effect of scavenging by small molecules represents a key limitation in antioxidant defence. The claim that an antioxidant is a •OH scavenger is meaning less, as almost all molecules react with •OH at about the same rate. Thus, the only defence against •OH is to prevent its formation, and the most effective way to achieve that is H 2 would outcompete most agents that are used intracellularly. Thus, kinetic considerations essentially rule out scavenging as an effective antioxidant defence within cells 6 . However, outside cells, SOD and catalase mimics that have relatively high kinetic rate constants compared with non-enzymatic reactions of O 2

•− and H 2 O 2 may be effective. Although not as efficient as the endogenous SOD and catalase, the rate constants for the mimics are approximately 10 5 times higher than those of most protein cysteines. SOD mimics can accumulate at high concentrations in the mitochondrial matrix by attachment of a lipophilic cationic group and can be effective in that microenvironment 106 , where it has been demonstrated that the overexpression of endogenous SOD2 increases H 2 O 2 production 238 . However, the long-term effects of the non-physiological increase in mitochondrial SOD activity is unknown.

Vitamin E is the one exception to the limitation of small molecule scavenging by dietary antioxidants because of its relatively rapid rate of reaction with lipid hydroperoxyl radicals as well as its concentration in membranes. Nonetheless, antioxidant therapies that appeared to work in cell culture or in non-human animal models have often failed to achieve significant effects in human trials. A primary reason for this discrepancy is the enormous difference in the ratio of exogenous agents in vitro versus in vivo 6 . In non-human animal models, lab chow is deficient in vitamin E and selenium 239 , which sets up a system in which antioxidants work by restoring redox homeostasis, thereby acting more like vitamins preventing a deficiency than like a drug. Interestingly, mito-Q, made by the attachment of a lipophilic cationic group to ubiquinone, can accumulate in mitochondria and act in a similar manner to vitamin E in that domain 240 . However, the long-term effects of the non-physiological increase in ubiquinone is not yet understood.

Another concern is that compounds that induce antioxidant defences may not be able to reach effective concentrations in vivo, although this may be overcome with cyanoenones 194 . When adequate levels of NRF2 activators are supplied by good nutrition, supplemental NRF2 activators would not provide an advantage. In addition, if oxidative stress occurs in patients, NRF2 is usually already activated to a certain degree and the potential for further induction is limited. As a good diet would be expected for patients in clinical trials, and oxidative stress is frequently seen in patients, the lack of an increase in protection may be due to the existing effects of dietary NRF2 inducers and a lower potential for NRF2 activation. Perhaps the use of NRF2 activators should therefore be considered as similar to that of vitamins that are inadequate in the diet of a significant number of individuals and in patients who have difficulty consuming food.

As we age, the ability of electrophiles to induce NRF2dependent expression of antioxidant enzymes declines 241 . Silencing BACH1 reverses this effect in human primary bronchial epithelial cells for some NRF2-regulated genes 242 , suggesting that BACH1 inhibition has potential in antioxidant therapy, particularly in older patients. However, as older people exhibit an increased risk of cancer, activating NRF2 in this group may be deleterious. Although NRF2 activation has long been associated with chemoprevention 243 , a downside of NRF2 activation is the protection of cancer cells against oxidative damage, which helps cancer progression [200] [201] [202] . However, in mice, silencing of BACH1 does not appear to increase p53-driven tumorigenesis 244 . It is hoped that more studies will further clarify the issue of cancer promotion associated with NRF2, and that additional means of increasing antioxidant defences will be found to benefit older people without adverse effects.

As oxidative stress is a component of many diseases, the development of effective antioxidant therapies is an important goal. Although using small molecules has been largely disappointing, hope lies in the realization that the rationale underlying their use was based on misconceptions that can be overcome. Increased awareness of the fact that, although the goal of antioxidant defence must be to prevent the formation of •OH and ONOO − by decreasing their precursors H 2 O 2 and O 2

, H 2 O 2 is also essential in physiological signalling, will lead to more nuanced approaches to antioxidant defence. In addition, the limitations highlighted in this Review -including consideration of whether oxidative stress plays a primary or secondary role in the pathology, the negligible effect of scavenging by almost all small molecules, difficulty in achieving effective in vivo concentrations and the declining ability to increase NRF2 activation in ageing -must be considered to both avoid unnecessary disappointment and set obtainable goals.

There is promise in agents that scavenge O 2 •− and H 2 O 2 in intracellular spaces and the mitochondrial matrix. SOD, and SOD-catalase and GPX mimics, appear to be effective, with some agents currently in clinical trials. Maintaining GSH, the substrate for GPXs, can be achieved using precursors including NAC and GSH esters. Indeed, NAC is already in human use for the treatment of some toxicities and diseases, although no clinical trials of GSH esters appear to be currently active. In addition to the mimics of antioxidant enzymes and GSH, another major strategy is increasing the synthesis of the endogenous antioxidant enzymes and de novo synthesis of GSH through NRF2 signalling in cells 99 . We expect that all these approaches will contribute to advancing antioxidant therapeutics and hope that this Review will encourage and inform a rational approach to that worthwhile endeavour.

Published online xx xx xxxx www.nature.com/nrd

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8-Isoprostane levels in serum and bronchoalveolar lavage in idiopathic pulmonary fibrosis and sarcoidosis

Glutathione deficiency in the epithelial lining fluid of the lower respiratory tract in idiopathic pulmonary fibrosis

Effect of alpha-tocopherol on the metabolic control and oxidative stress in female type 2 diabetics

Mitochondrial dysfunction contributes to the senescent phenotype of IPF lung fibroblasts

Transforming growth factor beta suppresses glutamate-cysteine ligase gene expression and induces oxidative stress in a lung fibrosis model. Free Radic

Oxidative stress, extracellular matrix targets, and idiopathic pulmonary fibrosis. Free Radic

Emerging cellular and molecular determinants of idiopathic pulmonary fibrosis

Reactive oxygen species, vascular Noxs, and hypertension: focus on translational and clinical research

Plasma hydrogen peroxide production in human essential hypertension: role of heredity, gender, and ethnicity

Antioxidant activities and oxidative stress byproducts in human hypertension

Relationship between (Na + K)-ATPase activity, lipid peroxidation and fatty acid profile in erythrocytes of hypertensive and normotensive subjects

Oxidative stress: a unifying paradigm in hypertension

Type 2 diabetes mellitus, oxidative stress and inflammation: examining the links

Oxidized to non-oxidized lipoprotein ratios are associated with arteriosclerosis and the metabolic syndrome in diabetic patients

Oxidative DNA damage and obesity in type 2 diabetes mellitus

Plasma 8-epi-PGF2 alpha levels are elevated in individuals with non-insulin dependent diabetes mellitus

Protein oxidation biomarkers in plasma of type 2 diabetic patients

Increased glutathionyl hemoglobin in diabetes mellitus and hyperlipidemia demonstrated by liquid chromatography/electrospray ionization-mass spectrometry

In vivo formation of 8-iso-prostaglandin f2alpha and platelet activation in diabetes mellitus: effects of improved metabolic control and vitamin E supplementation

Impact of mitochondrial ROS production in the pathogenesis of diabetes mellitus and its complications

NADPH oxidase 1 plays a key role in diabetes mellitus-accelerated atherosclerosis

Oxidative stress, dysfunctional glucose metabolism and Alzheimer disease

Increased CSF F2-isoprostane concentration in probable AD

Increased F2-isoprostanes in Alzheimer's disease: evidence for enhanced lipid peroxidation in vivo

Acrolein is increased in Alzheimer's disease brain and is toxic to primary hippocampal cultures

Elevated 4-hydroxynonenal in vertricular fluid in Alzheimer's disease

Redox proteomics identification of 4-hydroxynonenal-modified brain proteins in Alzheimer's disease: role of lipid peroxidation in Alzheimer's disease pathogenesis

Oxidative stress in the progression of Alzheimer disease in the frontal cortex

Increased oxidative damage in nuclear and mitochondrial DNA in Alzheimer's disease

Redox proteomics identification of oxidatively modified hippocampal proteins in mild cognitive impairment: insights into the development of Alzheimer's disease

Amyloid peptide free radical fragments initiate synaptosomal lipoperoxidation in a sequencespecific fashion: implications to Alzheimer's disease

ROS generation in microglia: understanding oxidative stress and inflammation in neurodegenerative disease

Iron accumulation in Alzheimer disease is a source of redox-generated free radicals

Cybrids in Alzheimer's disease: a cellular model of the disease?

Oxidative stress in cancer

Chemotherapy-associated oxidative stress: impact on chemotherapeutic effectiveness

Oncogenic pathways and the electron transport chain: a dangeROS liaison

NADPH oxidase 4 is an oncoprotein localized to mitochondria

Aberrant expression of 5-lipoxygenase-activating protein (5-LOXAP) has prognostic and survival significance in patients with breast cancer

Elevated levels of oxidative stress markers in exhaled NATURE REvIEWS | Drug Discovery breath condensate

Increased formation of oxidative DNA damage, 8-hydroxy-2′-deoxyguanosine, in human breast cancer tissue and its relationship to GSTP1 and COMT genotypes

Oxidative stress marker 8-hydroxyguanosine is more highly expressed in prostate cancer than in benign prostatic hyperplasia

Association between expression of 8-OHdG and cigarette smoking in non-small cell lung cancer

Systemic Inflammatory Response Syndrome

2nd Plasma biomarkers of oxidant stress and development of organ failure in severe sepsis

Oxidative stress in critically ill patients with systemic inflammatory response syndrome

Increased DNA damage and increased apoptosis and necrosis in patients with severe sepsis and septic shock

Exhaled breath condensate isoprostanes are elevated in patients with acute lung injury or ARDS

Nitric oxide and nitrotyrosine in the lungs of patients with acute respiratory distress syndrome

Inducible nitric oxide synthase in circulating microvesicles: discovery, evolution, and evidence as a novel biomarker and the probable causative agent for sepsis

Inhibition of NADPH oxidase 2 (NOX2) prevents sepsis-induced cardiomyopathy by improving calcium handling and mitochondrial function

Xanthine oxidase activity and free radical generation in patients with sepsis syndrome

Endotoxin-induced mitochondrial damage correlates with impaired respiratory activity

Total vitamin C, ascorbic acid, and dehydroascorbic acid concentrations in plasma of critically ill patients

Plasma lipid peroxides and alphatocopherol in critically ill patients

Cysteine metabolism and whole blood glutathione synthesis in septic pediatric patients

Role of xanthine oxidase and granulocytes in ischemia-reperfusion injury

Physiological and pathological functions of NADPH oxidases during myocardial ischemia-reperfusion

Neutrophils are primary source of O2 radicals during reperfusion after prolonged myocardial ischemia

8-epi PGF2 alpha generation during coronary reperfusion. A potential quantitative marker of oxidant stress in vivo

Increased formation of the isoprostanes IPF2alpha-I and 8-epi-prostaglandin F2alpha in acute coronary angioplasty: evidence for oxidant stress during coronary reperfusion in humans

Oxidative damage in ischemic stroke revealed using multiple biomarkers

Urinary 8-hydroxy-2′-deoxyguanosine levels increase after reperfusion in acute myocardial infarction and may predict subsequent cardiac events

Multiple basic-leucine zipper proteins regulate induction of the mouse heme oxygenase-1 gene by arsenite

Expression of heme oxygenase-1 in human vascular cells is regulated by peroxisome proliferator-activated receptors

Inhibitor of kappaB kinase beta regulates redox homeostasis by controlling the constitutive levels of glutathione

Regulation of Cu/Zn-superoxide dismutase expression via the phosphatidylinositol 3 kinase/Akt pathway and nuclear factor-kappaB

Identification of a putative antioxidant response element in the 5′-flanking region of the human g-glutamylcycteine synthetase heavy subunit gene

This article describes an essential role for NRF2 in GSH biosynthesis

This article provides a comprehensive review of NRF2 as a target for therapy

This article provides a review of the primary role of nitric oxide in physiology and disease

Redox homeostasis: the Golden Mean of healthy living

Nrf2-dependent induction of proteasome and Pa28alphabeta regulator are required for adaptation to oxidative stress

Oxidant stress stimulates expression of the human peroxiredoxin 6 gene by a transcriptional mechanism involving an antioxidant response element. Free Radic

Expression of base excision DNA repair genes is a sensitive biomarker for in vivo detection of chemical-induced chronic oxidative stress: identification of the molecular source of radicals responsible for DNA damage by peroxisome proliferators

Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein)

Superoxide dismutase mimics: chemistry, pharmacology, and therapeutic potential

Potential therapeutic applications of MnSODs and SOD-mimetics

Chemical properties of water-soluble porphyrins. 4. The reaction of a 'picket-fence-like' iron (III) complex with the superoxide oxygen couple

Catalysis of the disproportionation of superoxide by metalloporphyrins

Chemical properties of water-soluble porphyrins 3. The reaction of superoxide radicals with some metalloporphyrins

Relationship among redox potentials, proton dissociation constants of pyrrolic nitrogens, and in vivo and in vitro superoxide dismutating activities of manganese(iii) and iron(iii) water-soluble porphyrins

Manganese (iii) meso-tetrakis N-ethylpyridinium-2-yl porphyrin acts as a pro-oxidant to inhibit electron transport chain proteins, modulate bioenergetics, and enhance the response to chemotherapy in lymphoma cells. Free Radic

Reactions of manganese porphyrins with peroxynitrite and carbonate radical anion

An educational overview of the chemistry, biochemistry and therapeutic aspects of Mn porphyrins-from superoxide dismutation to H 2 O 2 -driven pathways

Manganoporphyrins increase ascorbate-induced cytotoxicity by enhancing H 2 O 2 generation

Tetrahydrobiopterin rapidly reduces the SOD mimic Mn(iii) ortho-tetrakis(N-ethylpyridinium-2-yl) porphyrin. Free Radic

Design of Mn porphyrins for treating oxidative stress injuries and their redoxbased regulation of cellular transcriptional activities

Amelioration of renal ischemiareperfusion injury with a novel protective cocktail

Orgotein-(bovine Cu-Zn superoxide dismutase), an anti-inflammatory protein drug: discovery, toxicology and pharmacology

Orgotein (superoxide dismutase): a drug for the amelioration of radiation-induced side effects. A double-blind, placebo-controlled study in patients with bladder tumours

Prevention of radioinduced cystitis by orgotein: a randomized study

Orgotein in radiation treatment of bladder cancer. A report on allergic reactions and lack of radioprotective effect

Neuroprotection from delayed postischemic administration of a metalloporphyrin catalytic antioxidant

Comparison of two Mn porphyrin-based mimics of superoxide dismutase in pulmonary radioprotection. Free Radic

Antiangiogenic action of redoxmodulating Mn(iii) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin, MnTE-2-PyP(5+), via suppression of oxidative stress in a mouse model of breast tumor. Free Radic

Radiation activates HIF-1 to regulate vascular radiosensitivity in tumors: role of reoxygenation, free radicals, and stress granules

A metalloporphyrin-based superoxide dismutase mimic inhibits adoptive transfer of autoimmune diabetes by a diabetogenic T-cell clone

Impact of superoxide dismutase mimetic AEOL 10150 on the endothelin system of Fischer 344 rats

Lost in translation: treatment trials in the SOD1 mouse and in human ALS

Computer-aided design (CAD) of Mn(ii) complexes: superoxide dismutase mimetics with catalytic activity exceeding the native enzyme

Superoxide dismutase mimetic GC4419 enhances the oxidation of pharmacological ascorbate and its anticancer effects in an H 2 O 2 -dependent manner

Pharmacological manipulation of the inflammatory cascade by the superoxide dismutase mimetic, M40403

Amelioration of joint disease in a rat model of collagen-induced arthritis by M40403, a superoxide dismutase mimetic

Protective effects of M40403, a selective superoxide dismutase mimetic, in myocardial ischaemia and reperfusion injury in vivo

Phase 1b/2a trial of the superoxide dismutase mimetic GC4419 to reduce chemoradiotherapy-induced oral mucositis in patients with oral cavity or oropharyngeal carcinoma

Salen-manganese complexes: combined superoxide dismutase/catalase mimics with broad pharmacological efficacy

A superoxide dismutase mimetic with catalase activity (EUK-8) reduces the organ injury in endotoxic shock

Cardioprotection by chronic estrogen or superoxide dismutase mimetic treatment in the aged female rat

EUK-8, a superoxide dismutase and catalase mimetic, reduces cardiac oxidative stress and ameliorates pressure overload-induced heart failure in the harlequin mouse mutant

Superoxide dismutase mimetics with catalase activity reduce the organ injury in hemorrhagic shock

Synthetic superoxide dismutase/ catalase mimetics reduce oxidative stress and prolong survival in a mouse amyotrophic lateral sclerosis model

EUK-134 reduces renal dysfunction and injury caused by oxidative and nitrosative stress of the kidney

Synthetic combined superoxide dismutase/catalase mimetics are protective as a delayed treatment in a rat stroke model: a key role for reactive oxygen species in ischemic brain injury

Partial volume rat lung irradiation: the protective/mitigating effects of Eukarion-189, a superoxide dismutase-catalase mimetic

Frequency modulation of synchronized Ca 2+ spikes in cultured hippocampal networks through G-protein-coupled receptors

Superoxide dismutase/catalase mimetic EUK-134 prevents diaphragm muscle weakness in monocrotalin-induced pulmonary hypertension

Chronic antioxidant enzyme mimetic treatment differentially modulates hyperthermiainduced liver HSP70 expression with aging

Catalase and glutathione peroxidase mimics

Ebselen, a selenoorganic compound as glutathione peroxidase mimic

Ebselen, a glutathione peroxidase mimetic seleno-organic compound, as a multifunctional antioxidant. Implication for inflammation-associated carcinogenesis

Ebselen treatment reduces noise induced hearing loss via the mimicry and induction of glutathione peroxidase

The clinical drug ebselen attenuates inflammation and promotes microbiome recovery in mice after antibiotic treatment for CDI

Effect of the putative lithium mimetic ebselen on brain myo-inositol, sleep, and emotional processing in humans

Ebselen in acute middle cerebral artery occlusion: a placebo-controlled, double-blind clinical trial

Neuroprotective effect of an antioxidant, ebselen, in patients with delayed neurological deficits after aneurysmal subarachnoid hemorrhage

Ebselen in acute ischemic stroke: a placebo-controlled, double-blind clinical trial

ICAM-1 and VCAM-1 expression induced by TNF-alpha are inhibited by a glutathione peroxidase mimic. Free Radic

Neuroprotective effects of a new glutathione peroxidase mimetic on neurons of the chick embryo's retina

Haptoglobin genotype determines myocardial infarct size in diabetic mice

Comparison of the ability of ferric complexes to catalyze microsomal chemiluminescence, lipid peroxidation, and hydroxyl radical generation

Efficacy of deferoxamine in preventing complications of iron overload in patients with thalassemia major

Kinetics of uptake and deacetylation of N-acetylcysteine by human erythrocytes

Existing and potential therapeutic uses for N-acetylcysteine: the need for conversion to intracellular glutathione for antioxidant benefits

Efficacy of oral N-acetylcysteine in the treatment of acetaminophen overdose. Analysis of the national multicenter study

Long-term treatment with oral N-acetylcysteine: affects lung function but not sputum inflammation in cystic fibrosis subjects. A phase II randomized placebo-controlled trial

Effectiveness of N-acetylcysteine for the prevention of contrast-induced nephropathy: a systematic review and meta-analysis of randomized controlled trials

The level and half-life of glutathione in human plasma

Glutathione monoesters

This article demonstrates that glutathione diethyl ester is highly effective as a delivery agent for GSH

Transport of glutathione, as gamma-glutamylcysteinylglycyl ester, into liver and kidney

Radioprotection by glutathione ester: transport of glutathione ester into human lymphoid cells and fibroblasts

Modulation of endothelial GSH concentrations: effect of exogenous GSH and GSH monoethyl ester

Reperfusion injury of the liver: role of mitochondria and protection by glutathione ester

Glutathione monoethyl ester provides neuroprotection in a rat model of stroke

Glutathione monoethyl ester protects against glutathione deficiencies due to aging and acetaminophen in mice

Glutathione monoethyl ester: preparation, uptake by tissues, and conversion to glutathione

Characterization of intracellular elevation of glutathione (GSH) with glutathione monoethyl ester and GSH in brain and neuronal cultures: relevance to Parkinson's disease

Nrf2 and cardiovascular defense

Oxidative damage and the Nrf2-ARE pathway in neurodegenerative diseases

Protective role of Nrf2 in the lungs against oxidative airway diseases

Dimerization of substrate adaptors can facilitate Cullin-mediated ubiquitylation of proteins by a "tethering" mechanism: a two-site interaction model for the Nrf2-Keap1 complex

Kahweol activates the Nrf2/HO-1 pathway by decreasing Keap1 expression independently of p62 and autophagy pathways

The hypertension drug, verapamil, activates Nrf2 by promoting p62-dependent autophagic Keap1 degradation and prevents acetaminopheninduced cytotoxicity

Green tea extract provides extensive Nrf2-independent protection against lipid accumulation and NFkappaB pro-inflammatory responses during nonalcoholic steatohepatitis in mice fed a high-fat diet

Dietary cocoa protects against colitis-associated cancer by activating the Nrf2/Keap1 pathway

This article evaluates the current knowledge regarding bioavailability and efficacy of glucoraphanin and sulforaphane in terms of dose and route of administration

This article reviewed electrophilic and nonelectrophilic NRF2 activators in clinical trials for various chronic diseases including cancer

Sulforaphane inhibits restenosis by suppressing inflammation and the proliferation of vascular smooth muscle cells

Anti-inflammatory action of curcumin and its use in the treatment of lifestyle-related diseases

Anti-inflammatory effects of resveratrol: mechanistic insights

Discovery and development of Kelch-like ECH-associated protein 1. Nuclear factor erythroid 2-related factor 2 (KEAP1:NRF2) protein-protein interaction inhibitors: achievements, challenges, and future directions

Recent advances in understanding NRF2 as a druggable target: development of pro-electrophilic and non-covalent NRF2 activators to overcome systemic side effects of electrophilic drugs like dimethyl fumarate

Studies on the reactivity of CDDO, a promising new chemopreventive and chemotherapeutic agent: implications for a molecular mechanism of action

Pharmacokinetics and pharmacodynamics of orally administered acetylenic tricyclic bis(cyanoenone), a highly potent Nrf2 activator with a reversible covalent mode of action

An exceptionally potent inducer of cytoprotective enzymes: elucidation of the structural features that determine inducer potency and reactivity with Keap1

Sulforaphane augments glutathione and influences brain metabolites in human subjects: a clinical pilot study

Lack of effect of oral sulforaphane administration on Nrf2 expression in COPD: a randomized, double-blind, placebo controlled trial

Sulforaphane inhibits multiple inflammasomes through an Nrf2-independent mechanism

Inhibition of PI3K/AKT and MAPK/ERK pathways causes activation of FOXO transcription factor, leading to cell cycle arrest and apoptosis in pancreatic cancer

Nrf2 prevents initiation but accelerates progression through the Kras signaling pathway during lung carcinogenesis

This article demonstrates that NRF2-deficient mice exhibited an increase in tumour foci after urethane induction but a reduction in tumours with more malignant characteristics

BACH1 stabilization by antioxidants stimulates lung cancer metastasis

This article demonstrates that long-term supplementation with N-acetylcysteine and vitamin E promoted KRAS-driven lung cancer metastasis, and NRF2 inhibitor BACH1 stimulated glycolysisdependent lung cancer metastasis in a mouse model

This article demonstrates that sulforaphane prevented the initiation of vinyl carbamate-induced lung cancer in mouse models but

Genetic alteration of Keap1 confers constitutive Nrf2 activation and resistance to chemotherapy in gallbladder cancer

Nrf2 enhances cell proliferation and resistance to anticancer drugs in human lung cancer

High levels of Nrf2 determine chemoresistance in type II endometrial cancer

Nrf2 inhibition reverses the resistance of cisplatin-resistant head and neck cancer cells to artesunate-induced ferroptosis

NRF2 and cancer: the good, the bad and the importance of context

Controversy about pharmacological modulation of Nrf2 for cancer therapy

Nrf2 in cancers: a doubleedged sword

Diphenyleneiodonium, an inhibitor of NOXes and DUOXes, is also an iodide-specific transporter

Pharmacological characterization of the seven human NOX isoforms and their inhibitors

Therapeutic potential of NADPH oxidase 1/4 inhibitors

Nox inhibitors & therapies: rational design of peptidic and small molecule inhibitors

This article demonstrates that prevention of mitochondrial superoxide production blocked oxidative stress and signal transduction

An old medicine as a new drug to prevent mitochondrial complex I from producing oxygen radicals

SOD mimetics to the rescue

Ebselen induces mitochondrial permeability transition because of its interaction with adenine nucleotide translocase

Targeting lipophilic cations to mitochondria

New developments and novel therapeutic perspectives for vitamin C

Ascorbate is an outstanding antioxidant in human blood plasma

The pecking order of free radicals and antioxidants: lipid peroxidation, α-tocopherol, and ascorbate

Faster plasma vitamin E disappearance in smokers is normalized by vitamin C supplementation. Free Radic

Combined deficiency of vitamins E and C causes paralysis and death in guinea pigs

Vitamin E, antioxidant and nothing more. Free Radic

Vitamin E inadequacy in humans: causes and consequences

Free radicals, antioxidants, and nutrition

This article demonstrates that N-acetylcysteine and vitamin E markedly increased tumour progression and reduced survival in mouse models of lung cancer

Antioxidants can increase melanoma metastasis in mice

Antioxidants promote intestinal tumor progression in mice

This is probably the first article to demonstrate stimulation of cell signalling by endogenous generation of H 2 O 2 through activation of NOX

NOX enzymes and the biology of reactive oxygen

An overview of mechanisms of redox signaling

Iron-mediated lipid peroxidation and lipid raft disruption in low-dose silica-induced macrophage cytokine production. Free Radic

A phosphatidylcholine-specific phospholipase C regulates activation of p42/44 mitogen-activated protein kinases in lipopolysaccharide-stimulated human alveolar macrophages

The reaction of NO with superoxide

A new paradigm: manganese superoxide dismutase influences the production of H 2 O 2 in cells and thereby their biological state. Free Radic

Effects of vitamin E and selenium deficiency on the fatty acid composition of rat retinal tissues

Mitochondria-targeted antioxidants in the treatment of disease

Oxidative stress response and Nrf2 signaling in aging. Free Radic

Silencing Bach1 alters aging-related changes in the expression of Nrf2-regulated genes in primary human bronchial epithelial cells

Redox-sensitive transcription factors as prime targets for chemoprevention with anti-inflammatory and antioxidative phytochemicals

This article describes how NRF2 and other transcription factors are targets for protective phytochemicals

Bach1 deficiency and accompanying overexpression of heme oxygenase-1 do not influence aging or tumorigenesis in mice

A phase 2a randomised, doubleblind, placebo-controlled, parallel-group, add-on clinical trial of ebselen (SPI-1005) as a novel treatment for mania or hypomania

Effects of the potential lithiummimetic, ebselen, on impulsivity and emotional processing

A catalytic antioxidant (AEOL 10150) attenuates expression of inflammatory genes in stroke. Free Radic

Long-term administration of a small molecular weight catalytic metalloporphyrin antioxidant, AEOL 10150, protects lungs from radiation-induced injury

Superoxide dismutase mimetic with catalase activity, EUK-134, attenuates the multiple organ injury and dysfunction caused by endotoxin in the rat

Biomarker exploration in human peripheral blood mononuclear cells for monitoring sulforaphane treatment responses in autism spectrum disorder

Sulforaphane treatment of autism spectrum disorder (ASD)

Resveratrol confers endothelial protection via activation of the antioxidant transcription factor Nrf2

Action of Nrf2 and Keap1 in ARE-mediated NQO1 expression by quercetin

Curcumin activates the haem oxygenase-1 gene via regulation of Nrf2 and the antioxidant-responsive element

Bardoxolone methyl and kidney function in CKD with type 2 diabetes

Structure of the BTB domain of Keap1 and its interaction with the triterpenoid antagonist CDDO

C151 in KEAP1 is the main cysteine sensor for the cyanoenone class of NRF2 activators, irrespective of molecular size or shape

Safety, pharmacodynamics, and potential benefit of omaveloxolone in Friedreich ataxia

Safety and efficacy of omaveloxolone in patients with mitochondrial myopathy: MOTOR trial

RTA 408, a novel synthetic triterpenoid with broad anticancer and antiinflammatory activity

Topical application of the synthetic triterpenoid RTA 408 activates Nrf2 and induces cytoprotective genes in rat skin

Placebo-controlled phase 3 study of oral BG-12 or glatiramer in multiple sclerosis

Placebo-controlled phase 3 study of oral BG-12 for relapsing multiple sclerosis

Dimethyl fumarate and monoethyl fumarate exhibit differential effects on KEAP1, NRF2 activation, and glutathione depletion in vitro

Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice

Nrf2-dependent and -independent responses to nitro-fatty acids in human endothelial cells: identification of heat shock response as the major pathway activated by nitro-oleic acid

Andrographolide activates Keap1/Nrf2/ ARE/HO-1 pathway in HT22 cells and suppresses microglial activation by Abeta42 through Nrf2-related inflammatory response

Ursodeoxycholic acid stimulates Nrf2-mediated hepatocellular transport, detoxification, and antioxidative stress systems in mice

Phosphorylation of Nrf2 at Ser-40 by protein kinase C regulates antioxidant response element-mediated transcription

Direct interaction between Nrf2 and p21(Cip1/WAF1) upregulates the Nrf2-mediated antioxidant response

BRCA1 interacts with Nrf2 to regulate antioxidant signaling and cell survival

Role of Bach1 and Nrf2 in up-regulation of the heme oxygenase-1 gene by cobalt protoporphyrin

Bach1 differentially regulates distinct Nrf2-dependent genes in human venous and coronary artery endothelial cells adapted to physiological oxygen levels. Free Radic

Bach1: function, regulation, and involvement in disease

From form to function: the role of Nox4 in the cardiovascular system

Free Radicals in Biology and Medicine

Role of ubiquinone in the mitochondrial generation of hydrogen peroxide

The production by leukocytes of superoxide, a potential bactericidal agent

Nox4: a hydrogen peroxide-generating oxygen sensor

The centennial of the Fenton reaction. Free Radic

Reactions and reactivity of myeloperoxidase-derived oxidants: differential biological effects of hypochlorous and hypothiocyanous acids

This article describes adaptive homeostasis, the endogenous defence that antioxidant therapies are intended to mimic

Isolation of NF-E2-related factor 2 (Nrf2), a NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP1 repeat of the betaglobin locus control region

Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductase1 gene

Introduction to special issue on 'Nrf2 regulated redox signaling and metabolism in physiology and medicine' . Free Radic

Sulforaphane accelerates acetaldehyde metabolism by inducing aldehyde dehydrogenases: relevance to ethanol intolerance

Nrf2 suppresses macrophage inflammatory response by blocking proinflammatory cytokine transcription

Role of nrf2 in oxidative stress and toxicity

Nrf2 is a critical regulator of the innate immune response and survival during experimental sepsis

Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2

Nrf2 is controlled by two distinct beta-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK-3 activity

This article demonstrates that overproduction of p62 or a deficiency in autophagy competed with NRF2-KEAP1 interaction and activated NRF2

Heme regulates the dynamic exchange of Bach1 and NF-E2-related factors in the Maf transcription factor network

The BTB and CNC homology 1 (BACH1) target genes are involved in the oxidative stress response and in control of the cell cycle

Heme oxygenase-1 induction by NRF2 requires inactivation of the transcriptional repressor BACH1

The authors thank their many colleagues with whom conversations and collaborations concerning antioxidants have occurred over decades. The authors' work in this area was supported by several past NIH grants and currently by P01 AG055367.

Both authors were involved in researching data, discussion of content, writing the article and reviewing/editing the manuscript before submission.

The authors declare no competing interests.

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