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 Sies1 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 physiology2,3,4.
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 damage5. 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 work6. For example, scavenging of hydroxyl radical (•OH) is impractical, but preventing its formation by reducing hydrogen peroxide (H2O2) production can provide effective prevention of damage. One of the major misunderstandings in the field of oxidative stress concerns the scavenging of superoxide (O2•−) or H2O2 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 defence6,7. However, in extracellular fluids where antioxidant enzymes are absent, scavenging of O2•− and H2O2 (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 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 O2•−, H2O2 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 O2•− production.
Roles of oxidative stress in disease
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 H2O2 generated by cells upon physiological stimulation, can act as second messengers8. In oxidative stress, non-physiological production of H2O2 can cause redox signalling to go awry4. Both types of oxidative stress mechanism can occur in a single disease, such as in diabetes, where both advanced glycation products accumulate and aberrant activation of stress signalling pathways leads to diabetic complications9. Also, the increase in H2O2 production and iron release from proteins in oxidative stress by O2•− (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 signalling12.
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 as the primary cause of pathology
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.
Radiation-induced lung injury
Early pneumonitis followed by fibrosis frequently occur as side effects of radiotherapy for lung and oesophageal cancers13. When cells are exposed to radiation, homolytic cleavage of H2O directly generates •OH, which then oxidizes macromolecules 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 fibrosis14. 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 exposure15.
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 disease16. Paraquat toxicity is initiated by the continuous redox cycling that generates O2•− (ref.17).
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 aorta18, 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 arteries19. In addition, HNE-modified low-density lipoprotein (LDL) was found to be elevated by 50% in plasma of patients with atherosclerosis compared with healthy volunteers20. 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 lesions21. 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 bioavailability22.
Oxidative stress as a secondary contributor to disease progression
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 O2•− or H2O2 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.
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 neutrophil elastase, a major factor in the pathogenesis of COPD23. 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 pathogenesis14. 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 controls24. 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 controls25; and the urinary level of 8-hydroxydeoxyguanosine (8-OHdG), a marker of DNA oxidation, was significantly elevated in patients with COPD26. The level of oxidative stress was inversely correlated with lung function of the patients25. Together, these results suggest that oxidative stress occurs both in the lung and systemically in patients with COPD and contributes to disease pathogenesis.
Idiopathic pulmonary fibrosis
The pathology of idiopathic pulmonary fibrosis (IPF) is characterized by diffuse and progressive mesenchymal fibrosis and mild inflammation in the lung with unknown aetiology. Many studies have shown the presence of oxidative stress in IPF. Oxidative stress markers such as H2O2, 8-isoprostane, 8-isoprostaglandin-F2α (8-iso-PGF2α) and ethane are significantly increased in the exhaled breath condensate of patients with IPF compared with healthy individuals27. In addition, 8-isoprostane is elevated fivefold28 and oxidized proteins twofold29 in bronchoalveolar lavage fluid (BALF) of patients with IPF. HNE in lung30 and 8-isoprostane in blood31 are also significantly elevated in IPF. The glutathione (GSH) level in epithelial lining fluid and sputum of patients with IPF is fourfold lower than in healthy controls32, indicating a deficiency of this important component of antioxidant defence in IPF. H2O2 production is apparently mainly from NOX4 (ref.33) and dysfunctional mitochondria34, and GSH synthesis is downregulated by TGFβ signalling35. Mounting evidence suggests that oxidative stress plays a significant part in IPF, by promoting fibrogenesis through causing apoptosis of alveolar epithelial cells, activating myofibroblasts and inducing an inflammatory response36. Besides oxidative stress, IPF pathogenesis involves a number of processes including apoptosis, senescence, epithelial–mesenchymal transition, endothelial–mesenchymal transition, epithelial cell migration, increased production of chemokines, cytokines and growth factors, as well as mitochondrial dysfunction, endoplasmic reticulum stress, hypoxia and inflammation37. These mechanisms are interrelated, with oxidative stress representing an important component of the IPF pathogenesis.
Multiple risk factors such as diet, smoking, lifestyle, genetics and comorbidities contribute to hypertension. More than 90% of cases are essential hypertension with unclear cause. At the molecular level, however, oxidative stress is a common feature of this condition. Experimental studies suggest that oxidants are mainly from NOXs in hypertension38. Oxidative markers, including H2O2 (ref.39), glutathione disulfide (GSSG) to GSH ratio, malondialdehyde (a lipid peroxidation product)40 and 8-isoprostanes, are significantly increased in the plasma of patients with hypertension41. H2O2 has a role in the development and progression of hypertension, through influencing angiotensin II signalling, NO signalling and other cellular processes42. However, a causative role of oxidative stress in hypertension has not yet been established.
Type 2 diabetes mellitus
Patients with type 2 diabetes mellitus display substantial evidence of oxidative stress that results in microvascular and macrovascular complications43. Markers of oxidative stress, including OxLDL to LDL ratio44, 8-OHdG45, 8-iso-PGF2α46, protein carbonyls47 and GSH conjugation to haemoglobin48, 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 levels49. The increased oxidants in type 2 diabetes mellitus arise from dysfunctional mitochondria50 and NOX1 (ref.51) activated by the diabetic abnormalities of hyperglycaemia and dyslipidaemia.
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 pathways52. Many studies have demonstrated increased oxidative stress in the brain of patients with Alzheimer disease, including increased levels of F2-isoprostane-α in cerebrospinal fluid53 and frontal and temporal poles54, acrolein in amygdala and hippocampus/parahippocampal gyrus55, and HNE in ventricular fluid56, hippocampus and inferior parietal lobule57, and cortex58. 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 subjects59. In addition, protein oxidation in the hippocampus60 and protein carbonyls in the cerebral cortex58 were significantly elevated in the brains of patients with Alzheimer disease. Claims have been made that Aβ(1–42)61, activated microglia62, iron accumulation63 and dysfunctional mitochondria contribute to increased oxidant production64.
Through aberrantly altering signalling transduction pathways that damage DNA and exacerbate inflammation, oxidants are involved in various phases of tumorigenesis, including transformation of normal cells to tumour cells, tumour cell growth, proliferation, invasion, angiogenesis and metastasis65. Conversely, oxidative stress can also trigger apoptosis and ferroptosis, and reduce the opportunity for transformation and thereby prevent tumorigenesis65. In addition, oxidative stress is the main mechanism of action of radiation (see Radiation-induced lung injury subsection above) and many chemotherapeutic drugs66. 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 mitochondria67, NOX4 (ref.68) and 5-lipoxygenase69. 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 non-small-cell lung cancer have been shown to exhale more H2O2 than control individuals70. In addition, increased levels of 8-OHdG71 were detected in breast cancer tissues compared with matched normal tissues, and 8-OHdG was significantly elevated in prostate cancers72 and lung cancers73.
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 insults74. 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-isoprostanes75, HNE76 and 8-OHdG77 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 condensate78 as is nitrotyrosine in BALF79. Oxidants in sepsis originate from several sources depending on the tissues and/or cells, and include iNOS (also known as NOS2)80, NOXs81, xanthine oxidase82 and dysfunctional mitochondria83. In addition, the levels of antioxidants such as vitamin C84, vitamin E85 and GSH86 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−, O2•− and other oxidants is significantly increased from hypoxanthine/xanthine oxidase87, mitochondria, iNOS (NOS2) and NOXs88 in endothelial cells, infiltrated neutrophils and local tissue cells89. 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 disease90, and in patients with coronary angioplasty following carotid reperfusion91. 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 stroke92. Urinary 8-OHdG was also increased after reperfusion in acute myocardial infarction93. 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.
Antioxidant defences and therapeutic implications
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 (refs98,99) (Box 3).
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 scavenging 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 O2•− and removal of O2•− and H2O2. The removal of O2•− also prevents the formation of ONOO−, and the removal of H2O2 prevents formation of •OH and HOX.
SODs and enzymes that remove H2O2 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 processes100. Although the outer surface of some cells binds to EC-SOD, the additional catalase activity of most SOD mimics also catalyses removal of H2O2, which EC-SOD cannot achieve. Intracellular defences include cytosolic SOD1 and mitochondrial matrix SOD2, which remove O2•−, while catalase in peroxisomes (and cardiac mitochondria), GPXs and peroxiredoxins (PRDXs) remove H2O2. 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 O2•− by small molecules is negligible compared with the rate of removal by endogenous SODs, which have rate constants (~2 × 109 M−1 s−1) that are millions of times higher than those of most other reactions with O2•−. The outer surface of some cells binds to EC-SOD, which also outcompetes any potential O2•− scavenger. Nonetheless, SOD mimics are useful in extracellular environments that lack significant EC-SOD. SOD produces H2O2, which would seem to be not much of a gain in terms of antioxidant defence; however, the removal of O2•− 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 TrxS2. 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 H2O2 would adversely alter cellular function101. Scavenging of H2O2 and other hydroperoxides by small molecules is negligible compared with removal by the 15 enzymes that reduce H2O2 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 of research is not directly relevant to the present Review; however, the enzymatic systems for removal of oxidized proteins102, oxidized fatty acid removal and replacement103, and oxidized DNA removal and repair104 are induced by oxidants.
Antioxidant therapeutic strategies
Multiple antioxidant therapeutic strategies are being explored, some of which are currently undergoing clinical trials. These include removal of O2•− before it can react with •NO to form ONOO− (reaction 11) and removal of H2O2 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.
SOD and SOD–catalase mimics
Several antioxidant enzyme mimics have been and are currently in clinical trials (Table 1). SOD is the only enzyme that can eliminate O2•− 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 summarized106,107.The early studies on SOD mimics primarily focused on metalloporphyrins (that is, MnTM-4-PyP5+ and FeTM-4-PyP5+)108,109,110, and since the establishment of the structure–activity relationship between metal-site redox ability and SOD activity in the late 1990s111, 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 function112.
Although being developed to remove O2•− specifically, most SOD mimics are not specific and can also reduce other reactive oxygen or nitrogen species such as ONOO−, peroxyl radical, H2O2 and CO3•− (refs113,114). In addition, some SOD mimics, such as Mn porphyrins, Mn(ii) cyclic polyamines and M40403, can act as pro-oxidants and react with thiols112, ascorbate115 and tetrahydrobiopterin116, thereby affecting redox-sensitive signalling pathways and cellular transcription117,118. 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 use119. 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 rectitis120,121. 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 patients122. 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 O2•− dismutation activity114. Some of them, such as MnTM-2-pYp5+ and MnTE-2-pYp5+, 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-pYp5+ and MnTDE-2-ImP5+ have been demonstrated in non-human animal models of diseases, including stroke123, radiation injuries124, cancers125,126, diabetes127 and cardiovascular system damage128. 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-ImP5+ in patients with amyotrophic lateral sclerosis showed no toxicity at therapeutic doses129.
Another promising SOD mimetic is GC4419, a novel, highly stable Mn(ii)-containing penta-azamacrocyclic. GC4419 selectively removes superoxide anions without reacting with other oxidants130. In vitro, GC4419 significantly enhanced the toxicity of AscH− to kill cancer cells131. In addition, GC4419 has exhibited therapeutic effects in several non-human animal models of inflammation132, joint disease133 and myocardial IRI134. 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 acceptable135.
Salens, aromatic, substituted ethylenediamine metal complexes, represent an emerging class of SOD mimics. The Mn(iii)-containing salen complexes have both O2•− and H2O2 dismutation activity136. 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 sepsis137, heart ischaemia–reperfusion138, cardiomyopathy139, haemorrhage140 and amyotrophic lateral sclerosis141 (EUK-8); IRI142 and stroke143 (EUK-134); radiation lung fibrosis144, cognitive impairment145, diaphragm muscle weakness in monocrotalin-induced pulmonary hypertension146 and hyperthermia147 (EUK-189). However, no human clinical trial for salens has yet been reported.
Glutathione peroxidase mimics
A variety of mimics of GPXs have been developed148. 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 H2O2 and smaller organic hydroperoxides to membrane-bound phospholipid and cholesterol hydroperoxides149. Ebselen may also induce phase II detoxification enzymes150. In non-human animal studies, ebselen has been shown to reduce oxidative damage150, prevent the acute loss of outer hair cells and reduce hearing loss151, and decrease inflammation152. Accordingly, several clinical trials have been conducted in diseases including Meniere disease (phase III, NCT04677972), bipolar disorder153, complete occlusion of the middle cerebral artery154, delayed neurological deficits after aneurysmal subarachnoid haemorrhage155 and acute ischaemic stroke156. 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 cells157, reduced oxidative damage and prevented neuronal death158, and in a mouse model of heart ischaemia–reperfusion it reduced infarct size159. 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.
Chelation of iron
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 it160. 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 transfusions161.
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.
N-acetylcysteine (NAC) is one of the most studied antioxidant agents for therapeutic treatment (Table 1). It is water soluble and quickly absorbed primarily via the anion exchange protein on the cell membrane162. NAC in cells is deacetylated to produce cysteine. Evidence indicates that the antioxidant function of NAC is primarily mediated via replenishing GSH163. NAC can also reduce cysteine conjugates in plasma162. NAC has been used therapeutically for the treatment of many pathologies, including liver paracetamol (also known as acetaminophen) toxicity164, cystic fibrosis, where it is delivered through the airways165 and nephropathy166. 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.
GSH itself is not effectively transported into most cells, and exogenously administered GSH is rapidly degraded in plasma167. 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 plasma168. 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 monoester169, 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 models170,171,172,173,174,175. Subcutaneous or intraperitoneal injection of GSH esters into animals was able to increase GSH levels in various tissues including liver170, kidney170, spleen, pancreas and heart176, but not brain177. Brain GSH levels can be increased via intracerebroventricular174 delivery of GSH-MEE177. Although oral administration could also increase tissue GSH levels, this is less effective176.
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 levels169. 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 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-MEE169. 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 diseases178, neurodegenerative disorders179 and pulmonary diseases180. 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 polyphenols are effective, they act primarily through antioxidant enzyme induction mediated by NRF2 signalling6. NRF2 activators comprise five categories, according to their mechanisms of action (Fig. 3): modification 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 translation182 and promote BACH1 degradation183.
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 enzymes184,185, and some of these are in clinical trials for disease treatment and/or prevention. For example, 11 clinical trials for turmeric extract and 55 clinical trials for broccoli or broccoli sprout supplement have been completed or are in an active phase for various conditions including COPD, osteoarthritis, joint stiffness and diabetic nephropathy (www.clinicaltrials.gov). Yagishita et al.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 cysteines6.
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 treatment187. Selected electrophilic NRF2 activators and the related clinical trials have previously been summarized187. It is noted that these NRF2 activators may have multiple functions such as anti-inflammatory effects188,189,190, 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.
Challenges facing therapeutic NRF2 activation
There are several concerns and challenges associated with the therapeutic use of NRF2 activators191,192. 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 reversible193 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 blood194 and markedly increased NRF2 activity in vivo at nanomolar concentrations195. 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 expression196 and no effect197 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-κB188 and inflammasome activation198, and cause cell cycle arrest by inhibiting the PI3K–AKT and MAPK–ERK pathways199. 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 development200,201,202. Cell studies showed that higher NRF2 activity and antioxidant capacity can also contribute to the resistance to chemotherapeutic drugs203,204,205,206, as reviewed by others207,208,209. 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 stress192.
NADPH oxidase inhibition
NOXs are important in redox signalling as the source of O2•− and H2O2 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 transport210. 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 inhibition211. Nonetheless, the potential value of inhibition of NOX1, NOX2 and NOX4 has been demonstrated in non-human animal models using genetic deletion212, and a search for low-molecular-weight NOX inhibitors continues.
Small peptides that inhibit the assembly of the NOX complexes have therapeutic potential213. 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.
Mitochondrial antioxidant defence
Leaks of electrons from the respiratory chain results in the production of O2•−. Although inhibiting O2•− 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 diabetes214. One drug, OP2113, which can be used in humans, has been proposed as a specific inhibitor of complex I O2•− production that does not interfere with ATP production215. However, this agent has not yet been investigated in clinical trials.
As discussed above, increasing SOD2 increases the production of H2O2 in mitochondria by pulling reaction 1 (QH•− + O2 ↔ Q + O2•−) (Box 1) forward by dismutation of O2•−. Thus, SOD mimics that enter mitochondria would be expected to increase the rate of production of H2O2. However, as these agents also possess catalase activity, they appear to add protection216, likely by preventing formation of OONO− and protecting iron–sulfur proteins. Ebselen can also enter mitochondria but may produce unexpected toxicity217.
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 them218. 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 molecules219. Vitamin C is also an important antioxidant220, 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 studies221,222,223, especially under conditions of oxidative stress or deficiency of other antioxidants223,224. Vitamin E 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 diseases225,226, 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 cataracts227. 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 models228,229,230. 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.
Inhibition of aberrant redox signalling
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 H2O2 or other oxidants was able to stimulate signalling pathways, it was not until the mid-1990s that NF-κB activation by endogenous generation of H2O2 was first observed231. By the late 1990s, Lambeth and coworkers232 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 area4,233. Nonetheless, as described earlier, H2O2 is the major second messenger in redox signalling and like other second messengers, dysregulation of its production can result in aberrant signalling233. 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 H2O2 or lipid hydroperoxides234.
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 approach235. 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 endotoxin236, 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.
Challenges and limitations in targeting oxidative stress
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.
Pathological role of oxidative stress
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.
Scavenging by small molecules
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 meaningless, 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 H2O2 elimination. For O2•−, scavenging inside the cell is in competition with the already ubiquitous and high activity of SOD, which catalyses reaction 3 (2O2•− + 2H+ → H2O2 + O2) (Box 1), with a rate constant that is at least 105 times higher than most of the reactions of O2•− except that with •NO237. Similarly, the presence of the 15 enzymes that remove H2O2 in reactions 4–6 (2H2O2 → 2H2O + O2; H2O2 + 2Trx(SH)2 → TrxS2 + 2H2O; H2O2 + 2GSH → GSSG + 2H2O) (Box 1) would outcompete most agents that are used intracellularly. Thus, kinetic considerations essentially rule out scavenging as an effective antioxidant defence within cells6. However, outside cells, SOD and catalase mimics that have relatively high kinetic rate constants compared with non-enzymatic reactions of O2•− and H2O2 may be effective. Although not as efficient as the endogenous SOD and catalase, the rate constants for the mimics are approximately 105 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 microenvironment106, where it has been demonstrated that the overexpression of endogenous SOD2 increases H2O2 production238. 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 vivo6. In non-human animal models, lab chow is deficient in vitamin E and selenium239, 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 domain240. However, the long-term effects of the non-physiological increase in ubiquinone is not yet understood.
Achieving effective in vivo concentrations
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 cyanoenones194. 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 NRF2-dependent expression of antioxidant enzymes declines241. Silencing BACH1 reverses this effect in human primary bronchial epithelial cells for some NRF2-regulated genes242, 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 chemoprevention243, a downside of NRF2 activation is the protection of cancer cells against oxidative damage, which helps cancer progression200,201,202. However, in mice, silencing of BACH1 does not appear to increase p53-driven tumorigenesis244. 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 H2O2 and O2•−, H2O2 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 O2•− and H2O2 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 cells99. 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.
Sies, H. Oxidative Stress (ed. Sies, H.) 1–8 (Academic Press, 1985). This article introduces the concept of oxidative stress.
Flohé, L. Looking back at the early stages of redox biology. Antioxidants 9, 1254 (2020). This article provides a history of oxidative stress from a current perspective.
Sies, H. Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: oxidative eustress. Redox Biol. 11, 613–619 (2017). This article explains the central role of hydrogen peroxide in redox signalling.
Sies, H., Berndt, C. & Jones, D. P. Oxidative stress. Annu. Rev. Biochem. 86, 715–748 (2017).
Valko, M. et al. Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell Biol. 39, 44–84 (2007). This article provides a review of redox signalling in disease.
Forman, H. J., Davies, K. J. & Ursini, F. How do nutritional antioxidants really work: nucleophilic tone and para-hormesis versus free radical scavenging in vivo. Free Radic. Biol. Med. 66, 24–35 (2014). This article provides a review of the mechanisms of nutritional antioxidants.
Sies, H. Strategies of antioxidant defense. Eur. J. Biochem. 215, 213–219 (1993).
Forman, H. J., Maiorino, M. & Ursini, F. Signaling functions of reactive oxygen species. Biochemistry 49, 835–842 (2010). This article explains why hydrogen peroxide is the principal redox second messenger.
Evans, J. L., Goldfine, I. D., Maddux, B. A. & Grodsky, G. M. Oxidative stress and stress-activated signaling pathways: a unifying hypothesis of type 2 diabetes. Endocr. Rev. 23, 599–622 (2002). This article examines the links between oxidative stress and type 2 diabetes mellitus.
Liochev, S. I. & Fridovich, I. The role of O2.- in the production of HO·: in vitro and in vivo. Free Radic. Biol. Med. 16, 29–33 (1994).
Castro, L., Tortora, V., Mansilla, S. & Radi, R. Aconitases: non-redox iron-sulfur proteins sensitive to reactive species. Acc. Chem. Res. 52, 2609–2619 (2019).
Zhang, H. & Forman, H. J. 4-hydroxynonenal-mediated signaling and aging. Free Radic. Biol. Med. 111, 219–225 (2017).
Giuranno, L., Ient, J., De Ruysscher, D. & Vooijs, M. A. Radiation-induced lung injury (RILI). Front. Oncol. 9, 877 (2019).
Barnes, P. J. Oxidative stress-based therapeutics in COPD. Redox Biol. 33, 101544 (2020). This article examines the current state of redox-based therapy in COPD.
Fleckenstein, K. et al. Temporal onset of hypoxia and oxidative stress after pulmonary irradiation. Int. J. Radiat. Oncol. Biol. Phys. 68, 196–204 (2007).
Rappold, P. M. et al. Paraquat neurotoxicity is mediated by the dopamine transporter and organic cation transporter-3. Proc. Natl Acad. Sci. USA 108, 20766–20771 (2011).
Bus, J. S. & Gibson, J. E. Paraquat: model for oxidant-initiated toxicity. Env. Health Perspect. 55, 37–46 (1984).
Glavind, J., Hartmann, S., Clemmesen, J., Jessen, K. E. & Dam, H. Studies on the role of lipoperoxides in human pathology. II. The presence of peroxidized lipids in the atherosclerotic aorta. Acta Pathol. Microbiol. Scand. 30, 1–6 (1952).
Suarna, C., Dean, R. T., May, J. & Stocker, R. Human atherosclerotic plaque contains both oxidized lipids and relatively large amounts of alpha-tocopherol and ascorbate. Arterioscler. Thromb. Vasc. Biol. 15, 1616–1624 (1995).
Salomon, R. G. et al. HNE-derived 2-pentylpyrroles are generated during oxidation of LDL, are more prevalent in blood plasma from patients with renal disease or atherosclerosis, and are present in atherosclerotic plaques. Chem. Res. Toxicol. 13, 557–564 (2000).
Gniwotta, C., Morrow, J. D., Roberts, L. J. 2nd & Kuhn, H. Prostaglandin F2-like compounds, F2-isoprostanes, are present in increased amounts in human atherosclerotic lesions. Arterioscler. Thromb. Vasc. Biol. 17, 3236–3241 (1997).
Yang, X. et al. Oxidative stress-mediated atherosclerosis: mechanisms and therapies. Front. Physiol. 8, 600 (2017). This article examines the current state of redox-based therapy in atherosclerosis.
Pryor, W. A., Dooley, M. M. & Church, D. F. Human alpha-1-proteinase inhibitor is inactivated by exposure to sidestream cigarette smoke. Toxicol. Lett. 28, 65–70 (1985).
Montuschi, P. et al. Exhaled 8-isoprostane as an in vivo biomarker of lung oxidative stress in patients with COPD and healthy smokers. Am. J. Respir. Crit. Care Med. 162, 1175–1177 (2000).
Rahman, I. et al. 4-Hydroxy-2-nonenal, a specific lipid peroxidation product, is elevated in lungs of patients with chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med. 166, 490–495 (2002).
Igishi, T. et al. Elevated urinary 8-hydroxydeoxyguanosine, a biomarker of oxidative stress, and lack of association with antioxidant vitamins in chronic obstructive pulmonary disease. Respirology 8, 455–460 (2003).
Psathakis, K. et al. Exhaled markers of oxidative stress in idiopathic pulmonary fibrosis. Eur. J. Clin. Invest. 36, 362–367 (2006).
Montuschi, P. et al. 8-Isoprostane as a biomarker of oxidative stress in interstitial lung diseases. Am. J. Respir. Crit. Care Med. 158, 1524–1527 (1998).
Lenz, A. G., Costabel, U. & Maier, K. L. Oxidized BAL fluid proteins in patients with interstitial lung diseases. Eur. Respir. J. 9, 307–312 (1996).
Tsubouchi, K. et al. Involvement of GPx4-regulated lipid peroxidation in idiopathic pulmonary fibrosis pathogenesis. J. Immunol. 203, 2076–2087 (2019).
Malli, F. et al. 8-Isoprostane levels in serum and bronchoalveolar lavage in idiopathic pulmonary fibrosis and sarcoidosis. Food Chem. Toxicol. 61, 160–163 (2013).
Cantin, A. M., Hubbard, R. C. & Crystal, R. G. Glutathione deficiency in the epithelial lining fluid of the lower respiratory tract in idiopathic pulmonary fibrosis. Am. Rev. Respir. Dis. 139, 370–372 (1989).
Ble-Castillo, J. L. et al. Effect of alpha-tocopherol on the metabolic control and oxidative stress in female type 2 diabetics. Biomed. Pharmacother. 59, 290–295 (2005).
Schuliga, M. et al. Mitochondrial dysfunction contributes to the senescent phenotype of IPF lung fibroblasts. J. Cell Mol. Med. 22, 5847–5861 (2018).
Liu, R. M. et al. Transforming growth factor beta suppresses glutamate-cysteine ligase gene expression and induces oxidative stress in a lung fibrosis model. Free Radic. Biol. Med. 53, 554–563 (2012).
Kliment, C. R. & Oury, T. D. Oxidative stress, extracellular matrix targets, and idiopathic pulmonary fibrosis. Free Radic. Biol. Med. 49, 707–717 (2010).
Phan, T. H. G. et al. Emerging cellular and molecular determinants of idiopathic pulmonary fibrosis. Cell Mol. Life Sci. 78, 2031–2057 (2020).
Montezano, A. C. & Touyz, R. M. Reactive oxygen species, vascular Noxs, and hypertension: focus on translational and clinical research. Antioxid. Redox Signal. 20, 164–182 (2014). This article examines the role of NOXs in disease.
Lacy, F., Kailasam, M. T., O’Connor, D. T., Schmid-Schonbein, G. W. & Parmer, R. J. Plasma hydrogen peroxide production in human essential hypertension: role of heredity, gender, and ethnicity. Hypertension 36, 878–884 (2000).
Redon, J. et al. Antioxidant activities and oxidative stress byproducts in human hypertension. Hypertension 41, 1096–1101 (2003).
Rodrigo, R., Bachler, J. P., Araya, J., Prat, H. & Passalacqua, W. Relationship between (Na + K)-ATPase activity, lipid peroxidation and fatty acid profile in erythrocytes of hypertensive and normotensive subjects. Mol. Cell Biochem. 303, 73–81 (2007).
Touyz, R. M. et al. Oxidative stress: a unifying paradigm in hypertension. Can. J. Cardiol. 36, 659–670 (2020).
Oguntibeju, O. O. Type 2 diabetes mellitus, oxidative stress and inflammation: examining the links. Int. J. Physiol. Pathophysiol. Pharmacol. 11, 45–63 (2019).
Girona, J. et al. Oxidized to non-oxidized lipoprotein ratios are associated with arteriosclerosis and the metabolic syndrome in diabetic patients. Nutr. Metab. Cardiovasc. Dis. 18, 380–387 (2008).
Al-Aubaidy, H. A. & Jelinek, H. F. Oxidative DNA damage and obesity in type 2 diabetes mellitus. Eur. J. Endocrinol. 164, 899–904 (2011).
Gopaul, N. K. et al. Plasma 8-epi-PGF2 alpha levels are elevated in individuals with non-insulin dependent diabetes mellitus. FEBS Lett. 368, 225–229 (1995).
Pandey, K. B., Mishra, N. & Rizvi, S. I. Protein oxidation biomarkers in plasma of type 2 diabetic patients. Clin. Biochem. 43, 508–511 (2010).
Niwa, T., Naito, C., Mawjood, A. H. & Imai, K. Increased glutathionyl hemoglobin in diabetes mellitus and hyperlipidemia demonstrated by liquid chromatography/electrospray ionization-mass spectrometry. Clin. Chem. 46, 82–88 (2000).
Davi, G. et al. In vivo formation of 8-iso-prostaglandin f2alpha and platelet activation in diabetes mellitus: effects of improved metabolic control and vitamin E supplementation. Circulation 99, 224–229 (1999).
Nishikawa, T. & Araki, E. Impact of mitochondrial ROS production in the pathogenesis of diabetes mellitus and its complications. Antioxid. Redox Signal. 9, 343–353 (2007).
Gray, S. P. et al. NADPH oxidase 1 plays a key role in diabetes mellitus-accelerated atherosclerosis. Circulation 127, 1888–1902 (2013).
Butterfield, D. A. & Halliwell, B. Oxidative stress, dysfunctional glucose metabolism and Alzheimer disease. Nat. Rev. Neurosci. 20, 148–160 (2019). This article examines the role of oxidative stress in Alzheimer disease.
Montine, T. J. et al. Increased CSF F2-isoprostane concentration in probable AD. Neurology 52, 562–565 (1999).
Pratico, D., V, M. Y. L., Trojanowski, J. Q., Rokach, J. & Fitzgerald, G. A. Increased F2-isoprostanes in Alzheimer’s disease: evidence for enhanced lipid peroxidation in vivo. FASEB J. 12, 1777–1783 (1998).
Lovell, M. A., Xie, C. & Markesbery, W. R. Acrolein is increased in Alzheimer’s disease brain and is toxic to primary hippocampal cultures. Neurobiol. Aging 22, 187–194 (2001).
Lovell, M. A., Ehmann, W. D., Mattson, M. P. & Markesbery, W. R. Elevated 4-hydroxynonenal in vertricular fluid in Alzheimer’s disease. Neurobiol. Aging 18, 457–461 (1997).
Perluigi, M. et al. Redox proteomics identification of 4-hydroxynonenal-modified brain proteins in Alzheimer’s disease: role of lipid peroxidation in Alzheimer’s disease pathogenesis. Proteom. Clin. Appl. 3, 682–693 (2009).
Ansari, M. A. & Scheff, S. W. Oxidative stress in the progression of Alzheimer disease in the frontal cortex. J. Neuropathol. Exp. Neurol. 69, 155–167 (2010).
Wang, J., Xiong, S., Xie, C., Markesbery, W. R. & Lovell, M. A. Increased oxidative damage in nuclear and mitochondrial DNA in Alzheimer’s disease. J. Neurochem. 93, 953–962 (2005).
Butterfield, D. A. et al. Redox proteomics identification of oxidatively modified hippocampal proteins in mild cognitive impairment: insights into the development of Alzheimer’s disease. Neurobiol. Dis. 22, 223–232 (2006).
Butterfield, D. A., Hensley, K., Harris, M., Mattson, M. & Carney, J. β-Amyloid peptide free radical fragments initiate synaptosomal lipoperoxidation in a sequence-specific fashion: implications to Alzheimer’s disease. Biochem. Biophys. Res. Commun. 200, 710–715 (1994).
Simpson, D. S. A. & Oliver, P. L. ROS generation in microglia: understanding oxidative stress and inflammation in neurodegenerative disease. Antioxidants 9, 743 (2020).
Smith, M. A., Harris, P. L., Sayre, L. M. & Perry, G. Iron accumulation in Alzheimer disease is a source of redox-generated free radicals. Proc. Natl Acad. Sci. USA 94, 9866–9868 (1997).
Swerdlow, R. H. et al. Cybrids in Alzheimer’s disease: a cellular model of the disease? Neurology 49, 918–925 (1997).
Hayes, J. D., Dinkova-Kostova, A. T. & Tew, K. D. Oxidative stress in cancer. Cancer Cell 38, 167–197 (2020).
Conklin, K. A. Chemotherapy-associated oxidative stress: impact on chemotherapeutic effectiveness. Integr. Cancer Ther. 3, 294–300 (2004).
Raimondi, V., Ciccarese, F. & Ciminale, V. Oncogenic pathways and the electron transport chain: a dangeROS liaison. Br. J. Cancer 122, 168–181 (2020).
Graham, K. A. et al. NADPH oxidase 4 is an oncoprotein localized to mitochondria. Cancer Biol. Ther. 10, 223–231 (2010).
Jiang, W. G., Douglas-Jones, A. G. & Mansel, R. E. Aberrant expression of 5-lipoxygenase-activating protein (5-LOXAP) has prognostic and survival significance in patients with breast cancer. Prostaglandins Leukot. Essent. Fat. Acids 74, 125–134 (2006).
Chan, H. P., Tran, V., Lewis, C. & Thomas, P. S. Elevated levels of oxidative stress markers in exhaled breath condensate. J. Thorac. Oncol. 4, 172–178 (2009).
Matsui, A. et al. Increased formation of oxidative DNA damage, 8-hydroxy-2′-deoxyguanosine, in human breast cancer tissue and its relationship to GSTP1 and COMT genotypes. Cancer Lett. 151, 87–95 (2000).
Ohtake, S. et al. Oxidative stress marker 8-hydroxyguanosine is more highly expressed in prostate cancer than in benign prostatic hyperplasia. Mol. Clin. Oncol. 9, 302–304 (2018).
An, A. R. et al. Association between expression of 8-OHdG and cigarette smoking in non-small cell lung cancer. J. Pathol. Transl. Med. 53, 217–224 (2019).
Chakraborty, R. K. & Burns, B. Systemic Inflammatory Response Syndrome (StatPearls 2020).
Ware, L. B., Fessel, J. P., May, A. K. & Roberts, L. J.2nd Plasma biomarkers of oxidant stress and development of organ failure in severe sepsis. Shock 36, 12–17 (2011).
Alonso de Vega, J. M., Diaz, J., Serrano, E. & Carbonell, L. F. Oxidative stress in critically ill patients with systemic inflammatory response syndrome. Crit. Care Med. 30, 1782–1786 (2002).
Bahar, I., Elay, G., Baskol, G., Sungur, M. & Donmez-Altuntas, H. Increased DNA damage and increased apoptosis and necrosis in patients with severe sepsis and septic shock. J. Crit. Care 43, 271–275 (2018).
Carpenter, C. T., Price, P. V. & Christman, B. W. Exhaled breath condensate isoprostanes are elevated in patients with acute lung injury or ARDS. Chest 114, 1653–1659 (1998).
Sittipunt, C. et al. Nitric oxide and nitrotyrosine in the lungs of patients with acute respiratory distress syndrome. Am. J. Respir. Crit. Care Med. 163, 503–510 (2001).
Webber, R. J., Sweet, R. M. & Webber, D. S. Inducible nitric oxide synthase in circulating microvesicles: discovery, evolution, and evidence as a novel biomarker and the probable causative agent for sepsis. J. Appl. Lab. Med. 3, 698–711 (2019).
Joseph, L. C. et al. Inhibition of NADPH oxidase 2 (NOX2) prevents sepsis-induced cardiomyopathy by improving calcium handling and mitochondrial function. JCI Insight 2, e94248 (2017).
Galley, H. F., Davies, M. J. & Webster, N. R. Xanthine oxidase activity and free radical generation in patients with sepsis syndrome. Crit. Care Med. 24, 1649–1653 (1996).
Crouser, E. D., Julian, M. W., Blaho, D. V. & Pfeiffer, D. R. Endotoxin-induced mitochondrial damage correlates with impaired respiratory activity. Crit. Care Med. 30, 276–284 (2002).
Schorah, C. J. et al. Total vitamin C, ascorbic acid, and dehydroascorbic acid concentrations in plasma of critically ill patients. Am. J. Clin. Nutr. 63, 760–765 (1996).
Takeda, K. et al. Plasma lipid peroxides and alpha-tocopherol in critically ill patients. Crit. Care Med. 12, 957–959 (1984).
Lyons, J. et al. Cysteine metabolism and whole blood glutathione synthesis in septic pediatric patients. Crit. Care Med. 29, 870–877 (2001).
Granger, D. N. Role of xanthine oxidase and granulocytes in ischemia-reperfusion injury. Am. J. Physiol. 255, H1269–H1275 (1988). This article documents the sources of oxidative stress in IRI.
Matsushima, S., Tsutsui, H. & Sadoshima, J. Physiological and pathological functions of NADPH oxidases during myocardial ischemia-reperfusion. Trends Cardiovasc. Med. 24, 202–205 (2014).
Duilio, C. et al. Neutrophils are primary source of O2 radicals during reperfusion after prolonged myocardial ischemia. Am. J. Physiol. Heart Circ. Physiol. 280, H2649–H2657 (2001).
Delanty, N. et al. 8-epi PGF2 alpha generation during coronary reperfusion. A potential quantitative marker of oxidant stress in vivo. Circulation 95, 2492–2499 (1997).
Reilly, M. P. et al. 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. Circulation 96, 3314–3320 (1997).
Seet, R. C. et al. Oxidative damage in ischemic stroke revealed using multiple biomarkers. Stroke 42, 2326–2329 (2011).
Nagayoshi, Y. et al. Urinary 8-hydroxy-2′-deoxyguanosine levels increase after reperfusion in acute myocardial infarction and may predict subsequent cardiac events. Am. J. Cardiol. 95, 514–517 (2005).
Gong, P., Stewart, D., Hu, B., Vinson, C. & Alam, J. Multiple basic-leucine zipper proteins regulate induction of the mouse heme oxygenase-1 gene by arsenite. Arch. Biochem. Biophys. 405, 265–274 (2002).
Kronke, G. et al. Expression of heme oxygenase-1 in human vascular cells is regulated by peroxisome proliferator-activated receptors. Arterioscler. Thromb. Vasc. Biol. 27, 1276–1282 (2007).
Peng, Z. et al. Inhibitor of kappaB kinase beta regulates redox homeostasis by controlling the constitutive levels of glutathione. Mol. Pharmacol. 77, 784–792 (2010).
Rojo, A. I., Salinas, M., Martin, D., Perona, R. & Cuadrado, A. Regulation of Cu/Zn-superoxide dismutase expression via the phosphatidylinositol 3 kinase/Akt pathway and nuclear factor-kappaB. J. Neurosci. 24, 7324–7334 (2004).
Mulcahy, R. T. & Gipp, J. J. Identification of a putative antioxidant response element in the 5′-flanking region of the human g-glutamylcycteine synthetase heavy subunit gene. Biochem. Biophys. Res. Commun. 209, 227–233 (1995). This article describes an essential role for NRF2 in GSH biosynthesis.
Cuadrado, A. et al. Therapeutic targeting of the NRF2 and KEAP1 partnership in chronic diseases. Nat. Rev. Drug Discov. 18, 295–317 (2019). This article provides a comprehensive review of NRF2 as a target for therapy.
Moncada, S., Palmer, R. M. J. & Higgs, E. A. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 43, 109–142 (1991). This article provides a review of the primary role of nitric oxide in physiology and disease.
Ursini, F., Maiorino, M. & Forman, H. J. Redox homeostasis: the Golden Mean of healthy living. Redox Biol. 8, 205–215 (2016). This article examines the relationship of redox homeostasis to disease.
Pickering, A. M., Linder, R. A., Zhang, H., Forman, H. J. & Davies, K. J. Nrf2-dependent induction of proteasome and Pa28alphabeta regulator are required for adaptation to oxidative stress. J. Biol. Chem. 287, 10021–10031 (2012).
Chowdhury, I. et al. Oxidant stress stimulates expression of the human peroxiredoxin 6 gene by a transcriptional mechanism involving an antioxidant response element. Free Radic. Biol. Med. 46, 146–153 (2009).
Rusyn, I. et al. 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. Cancer Res. 64, 1050–1057 (2004).
McCord, J. M. & Fridovich, I. Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem. 244, 6049–6055 (1969).
Batinic-Haberle, I., Reboucas, J. S. & Spasojevic, I. Superoxide dismutase mimics: chemistry, pharmacology, and therapeutic potential. Antioxid. Redox Signal. 13, 877–918 (2010).
Bonetta, R. Potential therapeutic applications of MnSODs and SOD-mimetics. Chemistry 24, 5032–5041 (2018).
Faraggi, M., Peretz, P. & Weinraub, D. Chemical properties of water-soluble porphyrins. 4. The reaction of a ‘picket-fence-like’ iron (III) complex with the superoxide oxygen couple. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 49, 951–968 (1986).
Pasternack, R. F., Banth, A., Pasternack, J. M. & Johnson, C. S. Catalysis of the disproportionation of superoxide by metalloporphyrins. J. Inorg. Biochem. 15, 261–267 (1981).
Peretz, P., Solomon, D., Weinraub, D. & Faraggi, M. Chemical properties of water-soluble porphyrins 3. The reaction of superoxide radicals with some metalloporphyrins. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 42, 449–456 (1982).
Batinić-Haberle, I. et al. 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. Inorg. Chem. 38, 12 (1999).
Jaramillo, M. C., Briehl, M. M., Batinic-Haberle, I. & Tome, M. E. 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. Biol. Med. 83, 89–100 (2015).
Ferrer-Sueta, G. et al. Reactions of manganese porphyrins with peroxynitrite and carbonate radical anion. J. Biol. Chem. 278, 27432–27438 (2003).
Batinic-Haberle, I., Tovmasyan, A. & Spasojevic, I. An educational overview of the chemistry, biochemistry and therapeutic aspects of Mn porphyrins—from superoxide dismutation to H2O2-driven pathways. Redox Biol. 5, 43–65 (2015). This article examines the potential use of SOD mimics in therapy.
Rawal, M. et al. Manganoporphyrins increase ascorbate-induced cytotoxicity by enhancing H2O2 generation. Cancer Res. 73, 5232–5241 (2013).
Batinic-Haberle, I., Spasojevic, I. & Fridovich, I. Tetrahydrobiopterin rapidly reduces the SOD mimic Mn(iii) ortho-tetrakis(N-ethylpyridinium-2-yl)porphyrin. Free Radic. Biol. Med. 37, 367–374 (2004).
Batinic-Haberle, I. et al. Design of Mn porphyrins for treating oxidative stress injuries and their redox-based regulation of cellular transcriptional activities. Amino Acids 42, 95–113 (2012).
Dorai, T. et al. Amelioration of renal ischemia-reperfusion injury with a novel protective cocktail. J. Urol. 186, 2448–2454 (2011).
Huber, W. Orgotein–(bovine Cu-Zn superoxide dismutase), an anti-inflammatory protein drug: discovery, toxicology and pharmacology. Eur. J. Rheumatol. Inflamm. 4, 173–182 (1981).
Menander-Huber, K. B., Edsmyr, F. & Huber, W. Orgotein (superoxide dismutase): a drug for the amelioration of radiation-induced side effects. A double-blind, placebo-controlled study in patients with bladder tumours. Urol. Res. 6, 255–257 (1978).
Sanchiz, F. et al. Prevention of radioinduced cystitis by orgotein: a randomized study. Anticancer. Res. 16, 2025–2028 (1996).
Nielsen, O. S. et al. Orgotein in radiation treatment of bladder cancer. A report on allergic reactions and lack of radioprotective effect. Acta Oncol. 26, 101–104 (1987).
Mackensen, G. B. et al. Neuroprotection from delayed postischemic administration of a metalloporphyrin catalytic antioxidant. J. Neurosci. 21, 4582–4592 (2001).
Gauter-Fleckenstein, B. et al. Comparison of two Mn porphyrin-based mimics of superoxide dismutase in pulmonary radioprotection. Free Radic. Biol. Med. 44, 982–989 (2008).
Rabbani, Z. N. et al. Antiangiogenic action of redox-modulating 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. Biol. Med. 47, 992–1004 (2009).
Moeller, B. J., Cao, Y., Li, C. Y. & Dewhirst, M. W. Radiation activates HIF-1 to regulate vascular radiosensitivity in tumors: role of reoxygenation, free radicals, and stress granules. Cancer Cell 5, 429–441 (2004).
Piganelli, J. D. et al. A metalloporphyrin-based superoxide dismutase mimic inhibits adoptive transfer of autoimmune diabetes by a diabetogenic T-cell clone. Diabetes 51, 347–355 (2002).
Ganesh, D. et al. Impact of superoxide dismutase mimetic AEOL 10150 on the endothelin system of Fischer 344 rats. PLoS ONE 11, e0151810 (2016).
Benatar, M. Lost in translation: treatment trials in the SOD1 mouse and in human ALS. Neurobiol. Dis. 26, 1–13 (2007).
Aston, K. et al. Computer-aided design (CAD) of Mn(ii) complexes: superoxide dismutase mimetics with catalytic activity exceeding the native enzyme. Inorg. Chem. 40, 1779–1789 (2001).
Heer, C. D. et al. Superoxide dismutase mimetic GC4419 enhances the oxidation of pharmacological ascorbate and its anticancer effects in an H2O2-dependent manner. Antioxidants 7, 18 (2018).
Salvemini, D. et al. Pharmacological manipulation of the inflammatory cascade by the superoxide dismutase mimetic, M40403. Br. J. Pharmacol. 132, 815–827 (2001).
Salvemini, D. et al. Amelioration of joint disease in a rat model of collagen-induced arthritis by M40403, a superoxide dismutase mimetic. Arthritis Rheum. 44, 2909–2921 (2001).
Masini, E. et al. Protective effects of M40403, a selective superoxide dismutase mimetic, in myocardial ischaemia and reperfusion injury in vivo. Br. J. Pharmacol. 136, 905–917 (2002).
Anderson, C. M. et al. Phase 1b/2a trial of the superoxide dismutase mimetic GC4419 to reduce chemoradiotherapy-induced oral mucositis in patients with oral cavity or oropharyngeal carcinoma. Int. J. Radiat. Oncol. Biol. Phys. 100, 427–435 (2018).
Doctrow, S. R. et al. Salen-manganese complexes: combined superoxide dismutase/catalase mimics with broad pharmacological efficacy. Adv. Pharmacol. 38, 247–269 (1997).
McDonald, M. C. et al. A superoxide dismutase mimetic with catalase activity (EUK-8) reduces the organ injury in endotoxic shock. Eur. J. Pharmacol. 466, 181–189 (2003). This article examines the advantage of having catalase activity in SOD mimics.
Xu, Y., Armstrong, S. J., Arenas, I. A., Pehowich, D. J. & Davidge, S. T. Cardioprotection by chronic estrogen or superoxide dismutase mimetic treatment in the aged female rat. Am. J. Physiol. Heart Circ. Physiol. 287, H165–H171 (2004).
van Empel, V. P. et al. EUK-8, a superoxide dismutase and catalase mimetic, reduces cardiac oxidative stress and ameliorates pressure overload-induced heart failure in the harlequin mouse mutant. J. Am. Coll. Cardiol. 48, 824–832 (2006).
Izumi, M., McDonald, M. C., Sharpe, M. A., Chatterjee, P. K. & Thiemermann, C. Superoxide dismutase mimetics with catalase activity reduce the organ injury in hemorrhagic shock. Shock 18, 230–235 (2002).
Jung, C. et al. Synthetic superoxide dismutase/catalase mimetics reduce oxidative stress and prolong survival in a mouse amyotrophic lateral sclerosis model. Neurosci. Lett. 304, 157–160 (2001).
Chatterjee, P. K. et al. EUK-134 reduces renal dysfunction and injury caused by oxidative and nitrosative stress of the kidney. Am. J. Nephrol. 24, 165–177 (2004).
Baker, K. et al. 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. J. Pharmacol. Exp. Ther. 284, 215–221 (1998).
Langan, A. R., Khan, M. A., Yeung, I. W., Van Dyk, J. & Hill, R. P. Partial volume rat lung irradiation: the protective/mitigating effects of Eukarion-189, a superoxide dismutase-catalase mimetic. Radiother. Oncol. 79, 231–238 (2006).
Liu, Z. et al. Frequency modulation of synchronized Ca2+ spikes in cultured hippocampal networks through G-protein-coupled receptors. J. Neurosci. 23, 4156–4163 (2003).
Himori, K. et al. Superoxide dismutase/catalase mimetic EUK-134 prevents diaphragm muscle weakness in monocrotalin-induced pulmonary hypertension. PLoS ONE 12, e0169146 (2017).
Zhang, H. J., Doctrow, S. R., Oberley, L. W. & Kregel, K. C. Chronic antioxidant enzyme mimetic treatment differentially modulates hyperthermia-induced liver HSP70 expression with aging. J. Appl. Physiol. 100, 1385–1391 (2006).
Day, B. J. Catalase and glutathione peroxidase mimics. Biochem. Pharmacol. 77, 285–296 (2009).
Sies, H. Ebselen, a selenoorganic compound as glutathione peroxidase mimic. Free. Radic. Biol. Med. 14, 313–323 (1993).
Nakamura, Y. et al. Ebselen, a glutathione peroxidase mimetic seleno-organic compound, as a multifunctional antioxidant. Implication for inflammation-associated carcinogenesis. J. Biol. Chem. 277, 2687–2694 (2002).
Kil, J., Pierce, C., Tran, H., Gu, R. & Lynch, E. D. Ebselen treatment reduces noise induced hearing loss via the mimicry and induction of glutathione peroxidase. Hear. Res. 226, 44–51 (2007).
Garland, M. et al. The clinical drug ebselen attenuates inflammation and promotes microbiome recovery in mice after antibiotic treatment for CDI. Cell Rep. Med. 1, 100005 (2020).
Singh, N. et al. Effect of the putative lithium mimetic ebselen on brain myo-inositol, sleep, and emotional processing in humans. Neuropsychopharmacology 41, 1768–1778 (2016).
Ogawa, A. et al. Ebselen in acute middle cerebral artery occlusion: a placebo-controlled, double-blind clinical trial. Cerebrovasc. Dis. 9, 112–118 (1999).
Saito, I. et al. Neuroprotective effect of an antioxidant, ebselen, in patients with delayed neurological deficits after aneurysmal subarachnoid hemorrhage. Neurosurgery 42, 269–277; discussion 277–278 (1998).
Yamaguchi, T. et al. Ebselen in acute ischemic stroke: a placebo-controlled, double-blind clinical trial. Stroke 29, 12–17 (1998).
d’Alessio, P., Moutet, M., Coudrier, E., Darquenne, S. & Chaudiere, J. ICAM-1 and VCAM-1 expression induced by TNF-alpha are inhibited by a glutathione peroxidase mimic. Free Radic. Biol. Med. 24, 979–987 (1998).
Castagne, V. & Clarke, P. G. Neuroprotective effects of a new glutathione peroxidase mimetic on neurons of the chick embryo’s retina. J. Neurosci. Res. 59, 497–503 (2000).
Blum, S. et al. Haptoglobin genotype determines myocardial infarct size in diabetic mice. J. Am. Coll. Cardiol. 49, 82–87 (2007).
Puntarulo, S. & Cederbaum, A. I. Comparison of the ability of ferric complexes to catalyze microsomal chemiluminescence, lipid peroxidation, and hydroxyl radical generation. Arch. Biochem. Biophys. 264, 482–491 (1988).
Brittenham, G. M. et al. Efficacy of deferoxamine in preventing complications of iron overload in patients with thalassemia major. N. Engl. J. Med. 331, 567–573 (1994).
Raftos, J. E., Whillier, S., Chapman, B. E. & Kuchel, P. W. Kinetics of uptake and deacetylation of N-acetylcysteine by human erythrocytes. Int. J. Biochem. Cell Biol. 39, 1698–1706 (2007).
Rushworth, G. F. & Megson, I. L. Existing and potential therapeutic uses for N-acetylcysteine: the need for conversion to intracellular glutathione for antioxidant benefits. Pharmacol. Ther. 141, 150–159 (2014).
Smilkstein, M. J., Knapp, G. L., Kulig, K. W. & Rumack, B. H. Efficacy of oral N-acetylcysteine in the treatment of acetaminophen overdose. Analysis of the national multicenter study (1976 to 1985). N. Engl. J. Med. 319, 1557–1562 (1988).
Conrad, C. et al. 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. J. Cyst. Fibros. 14, 219–227 (2015).
Xu, R., Tao, A., Bai, Y., Deng, Y. & Chen, G. Effectiveness of N-acetylcysteine for the prevention of contrast-induced nephropathy: a systematic review and meta-analysis of randomized controlled trials. J. Am. Heart Assoc. 5, e003968 (2016).
Wendel, A. & Cikryt, P. The level and half-life of glutathione in human plasma. FEBS Lett. 120, 209–211 (1980).
Anderson, M. E. & Meister, A. Glutathione monoesters. Anal. Biochem. 183, 16–20 (1989).
Levy, E. J., Anderson, M. E. & Meister, A. Transport of glutathione diethyl ester into human cells. Proc. Natl Acad. Sci. USA 90, 9171–9175 (1993). This article demonstrates that glutathione diethyl ester is highly effective as a delivery agent for GSH in human cells to decrease oxidative stress.
Puri, R. N. & Meister, A. Transport of glutathione, as gamma-glutamylcysteinylglycyl ester, into liver and kidney. Proc. Natl Acad. Sci. USA 80, 5258–5260 (1983).
Wellner, V. P., Anderson, M. E., Puri, R. N., Jensen, G. L. & Meister, A. Radioprotection by glutathione ester: transport of glutathione ester into human lymphoid cells and fibroblasts. Proc. Natl Acad. Sci. USA 81, 4732–4735 (1984).
Tsan, M. F., White, J. E. & Rosano, C. L. Modulation of endothelial GSH concentrations: effect of exogenous GSH and GSH monoethyl ester. J. Appl. Physiol. Respir. Environ. Exerc. Physiol. 66, 1029–1034 (1989).
Grattagliano, I., Vendemiale, G. & Lauterburg, B. H. Reperfusion injury of the liver: role of mitochondria and protection by glutathione ester. J. Surg. Res. 86, 2–8 (1999).
Anderson, M. F., Nilsson, M., Eriksson, P. S. & Sims, N. R. Glutathione monoethyl ester provides neuroprotection in a rat model of stroke. Neurosci. Lett. 354, 163–165 (2004).
Chen, T. S., Richie, J. P., Nagasawa, H. T. & Lang, C. A. Glutathione monoethyl ester protects against glutathione deficiencies due to aging and acetaminophen in mice. Mech. Ageing Dev. 120, 127–139 (2000).
Anderson, M. E., Powrie, F., Puri, R. N. & Meister, A. Glutathione monoethyl ester: preparation, uptake by tissues, and conversion to glutathione. Arch. Biochem. Biophys. 239, 538–548 (1985).
Zeevalk, G. D., Manzino, L., Sonsalla, P. K. & Bernard, L. P. Characterization of intracellular elevation of glutathione (GSH) with glutathione monoethyl ester and GSH in brain and neuronal cultures: relevance to Parkinson’s disease. Exp. Neurol. 203, 512–520 (2007).
Howden, R. Nrf2 and cardiovascular defense. Oxid. Med. Cell Longev. 2013, 104308 (2013).
Gan, L. & Johnson, J. A. Oxidative damage and the Nrf2-ARE pathway in neurodegenerative diseases. Biochim. Biophys. Acta 1842, 1208–1218 (2014).
Boutten, A., Goven, D., Artaud-Macari, E. & Bonay, M. Protective role of Nrf2 in the lungs against oxidative airway diseases. Med. Sci. 27, 966–972 (2011).
McMahon, M., Thomas, N., Itoh, K., Yamamoto, M. & Hayes, J. D. 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. J. Biol. Chem. 281, 24756–24768 (2006).
Seo, H. Y. et al. Kahweol activates the Nrf2/HO-1 pathway by decreasing Keap1 expression independently of p62 and autophagy pathways. PLoS ONE 15, e0240478 (2020).
Lee, D. H. et al. The hypertension drug, verapamil, activates Nrf2 by promoting p62-dependent autophagic Keap1 degradation and prevents acetaminophen-induced cytotoxicity. BMB Rep. 50, 91–96 (2017).
Li, J. et al. 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. Mol. Nutr. Food Res. 60, 858–870 (2016).
Pandurangan, A. K., Saadatdoust, Z., Esa, N. M., Hamzah, H. & Ismail, A. Dietary cocoa protects against colitis-associated cancer by activating the Nrf2/Keap1 pathway. Biofactors 41, 1–14 (2015).
Yagishita, Y., Fahey, J. W., Dinkova-Kostova, A. T. & Kensler, T. W. Broccoli or sulforaphane: is it the source or dose that matters? Molecules 24, 3593 (2019). This article evaluates the current knowledge regarding bioavailability and efficacy of glucoraphanin and sulforaphane in terms of dose and route of administration.
Robledinos-Anton, N., Fernandez-Gines, R., Manda, G. & Cuadrado, A. Activators and inhibitors of NRF2: a review of their potential for clinical development. Oxid. Med. Cell Longev. 2019, 9372182 (2019). This article reviewed electrophilic and non-electrophilic NRF2 activators in clinical trials for various chronic diseases including cancer.
Kwon, J. S. et al. Sulforaphane inhibits restenosis by suppressing inflammation and the proliferation of vascular smooth muscle cells. Atherosclerosis 225, 41–49 (2012).
Shimizu, K. et al. Anti-inflammatory action of curcumin and its use in the treatment of lifestyle-related diseases. Eur. Cardiol. 14, 117–122 (2019).
de Sa Coutinho, D., Pacheco, M. T., Frozza, R. L. & Bernardi, A. Anti-inflammatory effects of resveratrol: mechanistic insights. Int. J. Mol. Sci. 19, 1812 (2018).
Jiang, Z. Y., Lu, M. C. & You, Q. D. 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. J. Med. Chem. 59, 10837–10858 (2016).
Satoh, T. & Lipton, S. 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. F1000Res 6, 2138 (2017). This article evaluates NRF2 activators designed to avoid the systemic side effects caused by electrophilic activators.
Couch, R. D. et al. Studies on the reactivity of CDDO, a promising new chemopreventive and chemotherapeutic agent: implications for a molecular mechanism of action. Bioorg. Med. Chem. Lett. 15, 2215–2219 (2005). This article demonstrates that conjugation of electrophilic cyanoenone compounds and KEAP1 is selective and reversable.
Kostov, R. V. et al. Pharmacokinetics and pharmacodynamics of orally administered acetylenic tricyclic bis(cyanoenone), a highly potent Nrf2 activator with a reversible covalent mode of action. Biochem. Biophys. Res. Commun. 465, 402–407 (2015). This article demonstrates that oral administration of acetylenic tricyclic bis(cyanoenone), a NRF2 activator, has a reversible covalent mode of action in various tissues in C57BL/6 mice.
Dinkova-Kostova, A. T. et al. An exceptionally potent inducer of cytoprotective enzymes: elucidation of the structural features that determine inducer potency and reactivity with Keap1. J. Biol. Chem. 285, 33747–33755 (2010).
Sedlak, T. W. et al. Sulforaphane augments glutathione and influences brain metabolites in human subjects: a clinical pilot study. Mol. Neuropsychiatry 3, 214–222 (2018).
Wise, R. A. et al. Lack of effect of oral sulforaphane administration on Nrf2 expression in COPD: a randomized, double-blind, placebo controlled trial. PLoS ONE 11, e0163716 (2016).
Greaney, A. J., Maier, N. K., Leppla, S. H. & Moayeri, M. Sulforaphane inhibits multiple inflammasomes through an Nrf2-independent mechanism. J. Leukoc. Biol. 99, 189–199 (2016).
Roy, S. K., Srivastava, R. K. & Shankar, S. Inhibition of PI3K/AKT and MAPK/ERK pathways causes activation of FOXO transcription factor, leading to cell cycle arrest and apoptosis in pancreatic cancer. J. Mol. Signal. 5, 10 (2010).
Satoh, H., Moriguchi, T., Takai, J., Ebina, M. & Yamamoto, M. Nrf2 prevents initiation but accelerates progression through the Kras signaling pathway during lung carcinogenesis. Cancer Res. 73, 4158–4168 (2013). 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.
Wiel, C. et al. BACH1 stabilization by antioxidants stimulates lung cancer metastasis. Cell 178, 330–345 e322 (2019). This article demonstrates that long-term supplementation with N-acetylcysteine and vitamin E promoted KRAS-driven lung cancer metastasis, and NRF2 inhibitor BACH1 stimulated glycolysis-dependent lung cancer metastasis in a mouse model.
Tao, S., Rojo de la Vega, M., Chapman, E., Ooi, A. & Zhang, D. D. The effects of NRF2 modulation on the initiation and progression of chemically and genetically induced lung cancer. Mol. Carcinog. 57, 182–192 (2018). This article demonstrates that sulforaphane prevented the initiation of vinyl carbamate-induced lung cancer in mouse models but promoted the progression of pre-existing tumours.
Shibata, T. et al. Genetic alteration of Keap1 confers constitutive Nrf2 activation and resistance to chemotherapy in gallbladder cancer. Gastroenterology 135, 1358–1368 (2008).
Homma, S. et al. Nrf2 enhances cell proliferation and resistance to anticancer drugs in human lung cancer. Clin. Cancer Res. 15, 3423–3432 (2009).
Jiang, T. et al. High levels of Nrf2 determine chemoresistance in type II endometrial cancer. Cancer Res. 70, 5486–5496 (2010).
Roh, J. L., Kim, E. H., Jang, H. & Shin, D. Nrf2 inhibition reverses the resistance of cisplatin-resistant head and neck cancer cells to artesunate-induced ferroptosis. Redox Biol. 11, 254–262 (2017).
Sporn, M. B. & Liby, K. T. NRF2 and cancer: the good, the bad and the importance of context. Nat. Rev. Cancer 12, 564–571 (2012).
Milkovic, L., Zarkovic, N. & Saso, L. Controversy about pharmacological modulation of Nrf2 for cancer therapy. Redox Biol. 12, 727–732 (2017).
Wu, S., Lu, H. & Bai, Y. Nrf2 in cancers: a double-edged sword. Cancer Med. 8, 2252–2267 (2019).
Massart, C. et al. Diphenyleneiodonium, an inhibitor of NOXes and DUOXes, is also an iodide-specific transporter. FEBS Open Bio. 4, 55–59 (2013).
Augsburger, F. et al. Pharmacological characterization of the seven human NOX isoforms and their inhibitors. Redox Biol. 26, 101272 (2019).
Teixeira, G. et al. Therapeutic potential of NADPH oxidase 1/4 inhibitors. Br. J. Pharmacol. 174, 1647–1669 (2017).
Cifuentes-Pagano, M. E., Meijles, D. N. & Pagano, P. J. Nox inhibitors & therapies: rational design of peptidic and small molecule inhibitors. Curr. Pharm. Des. 21, 6023–6035 (2015).
Nishikawa, T. et al. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 404, 787–790 (2000). This article demonstrates that prevention of mitochondrial superoxide production blocked oxidative stress and signal transduction.
Detaille, D., Pasdois, P., Semont, A., Dos Santos, P. & Diolez, P. An old medicine as a new drug to prevent mitochondrial complex I from producing oxygen radicals. PLoS ONE 14, e0216385 (2019).
Craven, R. SOD mimetics to the rescue. Nat. Rev. Neurosci. 2, 858–858 (2001).
Pavon, N., Correa, F., Buelna-Chontal, M., Hernandez-Esquivel, L. & Chavez, E. Ebselen induces mitochondrial permeability transition because of its interaction with adenine nucleotide translocase. Life Sci. 139, 108–113 (2015).
Murphy, M. P. Targeting lipophilic cations to mitochondria. Biochim. Biophys. Acta 1777, 1028–1031 (2008).
Li, Y. & Schellhorn, H. E. New developments and novel therapeutic perspectives for vitamin C. J. Nutr. 137, 2171–2184 (2007).
Frei, B., England, L. & Ames, B. N. Ascorbate is an outstanding antioxidant in human blood plasma. Proc. Natl Acad. Sci. USA 86, 6377–6381 (1989).
Buettner, G. R. The pecking order of free radicals and antioxidants: lipid peroxidation, α-tocopherol, and ascorbate. Arch. Biochem. Biophys. 300, 535–543 (1993).
Bruno, R. S. et al. Faster plasma vitamin E disappearance in smokers is normalized by vitamin C supplementation. Free Radic. Biol. Med. 40, 689–697 (2006).
Hill, K. E. et al. Combined deficiency of vitamins E and C causes paralysis and death in guinea pigs. Am. J. Clin. Nutr. 77, 1484–1488 (2003).
Traber, M. G. & Atkinson, J. Vitamin E, antioxidant and nothing more. Free Radic. Biol. Med. 43, 4–15 (2007).
Maxfield, L. & Crane, J. S. Vitamin C Deficiency. StatPearls [online] https://www.ncbi.nlm.nih.gov/books/NBK493187/ (updated 2 Jul 2020).
Traber, M. G. Vitamin E inadequacy in humans: causes and consequences. Adv. Nutr. 5, 503–514 (2014).
Fang, Y. Z., Yang, S. & Wu, G. Free radicals, antioxidants, and nutrition. Nutrition 18, 872–879 (2002).
Sayin, V. I. et al. Antioxidants accelerate lung cancer progression in mice. Sci. Transl Med. 6, 221ra215 (2014). This article demonstrates that N-acetylcysteine and vitamin E markedly increased tumour progression and reduced survival in mouse models of lung cancer.
Le Gal, K. et al. Antioxidants can increase melanoma metastasis in mice. Sci. Transl Med. 7, 308re308 (2015).
Zou, Z. V. et al. Antioxidants promote intestinal tumor progression in mice. Antioxidants 10, 241 (2021).
Kaul, N. & Forman, H. J. Activation of NF kappa B by the respiratory burst of macrophages. Free Radic. Biol. Med. 21, 401–405 (1996). This is probably the first article to demonstrate stimulation of cell signalling by endogenous generation of H2O2 through activation of NOX.
Lambeth, J. D. NOX enzymes and the biology of reactive oxygen. Nat. Rev. Immunol. 4, 181–189 (2004). This article reviews the NOX family of enzymes.
Forman, H. J., Ursini, F. & Maiorino, M. An overview of mechanisms of redox signaling. J. Mol. Cell. Cardiol. 73, 2–9 (2014).
Marshall, P. J., Kulmacz, R. J. & Lands, W. E. M. in Hydroperoxides, Free Radicals and Prostaglandin Synthesis (eds Bors, W., Saran, M. & Tait, D) 299–304 (Walter de Gruyter, 1984).
Premasekharan, G. et al. Iron-mediated lipid peroxidation and lipid raft disruption in low-dose silica-induced macrophage cytokine production. Free Radic. Biol. Med. 51, 1184–1194 (2011).
Monick, M. M. et al. A phosphatidylcholine-specific phospholipase C regulates activation of p42/44 mitogen-activated protein kinases in lipopolysaccharide- stimulated human alveolar macrophages. J. Immunol. 162, 3005–3012 (1999).
Huie, R. E. & Padmaja, S. The reaction of NO with superoxide. Free Radic. Res. Commun. 18, 195–199 (1993).
Buettner, G. R., Ng, C. F., Wang, M., Rodgers, V. G. & Schafer, F. Q. A new paradigm: manganese superoxide dismutase influences the production of H2O2 in cells and thereby their biological state. Free Radic. Biol. Med. 41, 1338–1350 (2006).
Farnsworth, C. C., Stone, W. L. & Dratz, E. A. Effects of vitamin E and selenium deficiency on the fatty acid composition of rat retinal tissues. Biochim. Biophys. Acta 552, 281–293 (1979).
Smith, R. A. et al. Mitochondria-targeted antioxidants in the treatment of disease. Ann. NY Acad. Sci. 1147, 105–111 (2008).
Zhang, H., Davies, K. J. & Forman, H. J. Oxidative stress response and Nrf2 signaling in aging. Free Radic. Biol. Med. 88, 314–336 (2015). This article reviews the evidence that NRF2 activation declines with ageing.
Zhang, H., Zhou, L., Davies, K. J. A. & Forman, H. J. Silencing Bach1 alters aging-related changes in the expression of Nrf2-regulated genes in primary human bronchial epithelial cells. Arch. Biochem. Biophys. 672, 108074 (2019). This article demonstrates that inhibition of BACH1 partially reversed the decline of NRF2 activation in ageing.
Surh, Y. J., Kundu, J. K., Na, H. K. & Lee, J. S. Redox-sensitive transcription factors as prime targets for chemoprevention with anti-inflammatory and antioxidative phytochemicals. J. Nutr. 135, 2993S–3001S (2005). This article describes how NRF2 and other transcription factors are targets for protective phytochemicals.
Ota, K., Brydun, A., Itoh-Nakadai, A., Sun, J. & Igarashi, K. Bach1 deficiency and accompanying overexpression of heme oxygenase-1 do not influence aging or tumorigenesis in mice. Oxid. Med. Cell Longev. 2014, 757901 (2014). This article demonstrates the effects of Bach1 knockout on the transcriptome, p53−/− induced tumorigenesis and oxidative damage in a mouse model.
Sharpley, A. L. et al. A phase 2a randomised, double-blind, placebo-controlled, parallel-group, add-on clinical trial of ebselen (SPI-1005) as a novel treatment for mania or hypomania. Psychopharmacology 237, 3773–3782 (2020).
Masaki, C. et al. Effects of the potential lithium-mimetic, ebselen, on impulsivity and emotional processing. Psychopharmacology 233, 2655–2661 (2016).
Bowler, R. P. et al. A catalytic antioxidant (AEOL 10150) attenuates expression of inflammatory genes in stroke. Free Radic. Biol. Med. 33, 1141–1152 (2002).
Rabbani, Z. N. et al. Long-term administration of a small molecular weight catalytic metalloporphyrin antioxidant, AEOL 10150, protects lungs from radiation-induced injury. Int. J. Radiat. Oncol. Biol. Phys. 67, 573–580 (2007).
Bianca, R. et al. Superoxide dismutase mimetic with catalase activity, EUK-134, attenuates the multiple organ injury and dysfunction caused by endotoxin in the rat. Med. Sci. Monit. 8, BR1–BR7 (2002).
Liu, H. et al. Biomarker exploration in human peripheral blood mononuclear cells for monitoring sulforaphane treatment responses in autism spectrum disorder. Sci. Rep. 10, 5822 (2020).
Singh, K. et al. Sulforaphane treatment of autism spectrum disorder (ASD). Proc. Natl Acad. Sci. USA 111, 15550–15555 (2014).
Ungvari, Z. et al. Resveratrol confers endothelial protection via activation of the antioxidant transcription factor Nrf2. Am. J. Physiol. Heart Circ. Physiol. 299, H18–H24 (2010).
Tanigawa, S., Fujii, M. & Hou, D. X. Action of Nrf2 and Keap1 in ARE-mediated NQO1 expression by quercetin. Free. Radic. Biol. Med. 42, 1690–1703 (2007).
Balogun, E. et al. Curcumin activates the haem oxygenase-1 gene via regulation of Nrf2 and the antioxidant-responsive element. Biochem. J. 371, 887–895 (2003).
Pergola, P. E. et al. Bardoxolone methyl and kidney function in CKD with type 2 diabetes. N. Engl. J. Med. 365, 327–336 (2011).
Cleasby, A. et al. Structure of the BTB domain of Keap1 and its interaction with the triterpenoid antagonist CDDO. PLoS ONE 9, e98896 (2014).
Dayalan Naidu, S. et al. C151 in KEAP1 is the main cysteine sensor for the cyanoenone class of NRF2 activators, irrespective of molecular size or shape. Sci. Rep. 8, 8037 (2018).
Lynch, D. R. et al. Safety, pharmacodynamics, and potential benefit of omaveloxolone in Friedreich ataxia. Ann. Clin. Transl. Neurol. 6, 15–26 (2019).
Madsen, K. L. et al. Safety and efficacy of omaveloxolone in patients with mitochondrial myopathy: MOTOR trial. Neurology 94, e687–e698 (2020).
Probst, B. L. et al. RTA 408, a novel synthetic triterpenoid with broad anticancer and anti-inflammatory activity. PLoS ONE 10, e0122942 (2015).
Reisman, S. A., Lee, C. Y., Meyer, C. J., Proksch, J. W. & Ward, K. W. Topical application of the synthetic triterpenoid RTA 408 activates Nrf2 and induces cytoprotective genes in rat skin. Arch. Dermatol. Res. 306, 447–454 (2014).
Fox, R. J. et al. Placebo-controlled phase 3 study of oral BG-12 or glatiramer in multiple sclerosis. N. Engl. J. Med. 367, 1087–1097 (2012).
Gold, R. et al. Placebo-controlled phase 3 study of oral BG-12 for relapsing multiple sclerosis. N. Engl. J. Med. 367, 1098–1107 (2012).
Brennan, M. S. et al. Dimethyl fumarate and monoethyl fumarate exhibit differential effects on KEAP1, NRF2 activation, and glutathione depletion in vitro. PLoS ONE 10, e0120254 (2015).
Ramos-Gomez, M. et al. Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice. Proc. Natl Acad. Sci. USA 98, 3410–3415 (2001).
Kansanen, E. et al. 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. J. Biol. Chem. 284, 33233–33241 (2009).
Seo, J. Y. et al. Andrographolide activates Keap1/Nrf2/ARE/HO-1 pathway in HT22 cells and suppresses microglial activation by Abeta42 through Nrf2-related inflammatory response. Mediators Inflamm. 2017, 5906189 (2017).
Okada, K. et al. Ursodeoxycholic acid stimulates Nrf2-mediated hepatocellular transport, detoxification, and antioxidative stress systems in mice. Am. J. Physiol. Gastrointest. Liver Physiol. 295, G735–G747 (2008).
Huang, H. C., Nguyen, T. & Pickett, C. B. Phosphorylation of Nrf2 at Ser-40 by protein kinase C regulates antioxidant response element-mediated transcription. J. Biol. Chem. 277, 42769–42774 (2002).
Chen, W. et al. Direct interaction between Nrf2 and p21(Cip1/WAF1) upregulates the Nrf2-mediated antioxidant response. Mol. Cell 34, 663–673 (2009).
Gorrini, C. et al. BRCA1 interacts with Nrf2 to regulate antioxidant signaling and cell survival. J. Exp. Med. 210, 1529–1544 (2013).
Shan, Y., Lambrecht, R. W., Donohue, S. E. & Bonkovsky, H. L. Role of Bach1 and Nrf2 in up-regulation of the heme oxygenase-1 gene by cobalt protoporphyrin. FASEB J. 20, 2651–2653 (2006).
Chapple, S. J. et al. Bach1 differentially regulates distinct Nrf2-dependent genes in human venous and coronary artery endothelial cells adapted to physiological oxygen levels. Free Radic. Biol. Med. 92, 152–162 (2016).
Zhang, X. et al. Bach1: function, regulation, and involvement in disease. Oxid. Med. Cell Longev. 2018, 1347969 (2018).
Chen, F., Haigh, S., Barman, S. & Fulton, D. J. From form to function: the role of Nox4 in the cardiovascular system. Front. Physiol. 3, 412 (2012).
Halliwell, B. & Gutteridge, J. M. C. Free Radicals in Biology and Medicine (Clarendon Press, 1989).
Boveris, A., Cadenas, E. & Stoppani, A. O. M. Role of ubiquinone in the mitochondrial generation of hydrogen peroxide. Biochem. J. 156, 435–435 (1976).
Babior, B. M., Kipnes, R. S. & Curnutte, J. T. The production by leukocytes of superoxide, a potential bactericidal agent. J. Clin. Invest. 52, 741–741 (1973).
Nisimoto, Y., Diebold, B. A., Cosentino-Gomes, D. & Lambeth, J. D. Nox4: a hydrogen peroxide-generating oxygen sensor. Biochemistry 53, 5111–5120 (2014).
Koppenol, W. H. The centennial of the Fenton reaction. Free Radic. Biol. Med. 15, 645–651 (1993).
Pattison, D. I., Davies, M. J. & Hawkins, C. L. Reactions and reactivity of myeloperoxidase-derived oxidants: differential biological effects of hypochlorous and hypothiocyanous acids. Free Radic. Res. 46, 975–995 (2012).
Davies, K. J. A. Adaptive homeostasis. Mol. Asp. Med. 49, 1–7 (2016). This article describes adaptive homeostasis, the endogenous defence that antioxidant therapies are intended to mimic.
Moi, P., Chan, K., Asunis, I., Cao, A. & Kan, Y. W. 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 beta-globin locus control region. Proc. Natl Acad. Sci. USA 91, 9926–9930 (1994).
Venugopal, R. & Jaiswal, A. K. 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. J. Clin. Invest. 93, 14960–14965 (1996).
Mann, G. E. & Forman, H. J. Introduction to special issue on ‘Nrf2 regulated redox signaling and metabolism in physiology and medicine’. Free Radic. Biol. Med. 88, 91–92 (2015). This article introduces an important group of reviews on NRF2 signalling.
Ushida, Y. & Talalay, P. Sulforaphane accelerates acetaldehyde metabolism by inducing aldehyde dehydrogenases: relevance to ethanol intolerance. Alcohol Alcohol. 48, 526–534 (2013).
Kobayashi, E. H. et al. Nrf2 suppresses macrophage inflammatory response by blocking proinflammatory cytokine transcription. Nat. Commun. 7, 11624 (2016).
Ma, Q. Role of nrf2 in oxidative stress and toxicity. Annu. Rev. Pharmacol. Toxicol. 53, 401–426 (2013).
Thimmulappa, R. K. et al. Nrf2 is a critical regulator of the innate immune response and survival during experimental sepsis. J. Clin. Invest. 116, 984–995 (2006).
Kobayashi, A. et al. Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2. Mol. Cell. Biol. 24, 7130–7139 (2004).
Chowdhry, S. et al. Nrf2 is controlled by two distinct beta-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK-3 activity. Oncogene 32, 3765–3781 (2013).
Komatsu, M. et al. The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1. Nat. Cell Biol. 12, 213–223 (2010). This article demonstrates that overproduction of p62 or a deficiency in autophagy competed with NRF2–KEAP1 interaction and activated NRF2.
Sun, J. et al. Heme regulates the dynamic exchange of Bach1 and NF-E2-related factors in the Maf transcription factor network. Proc. Natl Acad. Sci. USA 101, 1461–1466 (2004).
Warnatz, H. J. et al. The BTB and CNC homology 1 (BACH1) target genes are involved in the oxidative stress response and in control of the cell cycle. J. Biol. Chem. 286, 23521–23532 (2011).
Reichard, J. F., Motz, G. T. & Puga, A. Heme oxygenase-1 induction by NRF2 requires inactivation of the transcriptional repressor BACH1. Nucleic Acids Res. 35, 7074–7086 (2007).
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.
The authors declare no competing interests.
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- Oxidative stress
Imbalance between generation of oxidants and the ability to prevent oxidative damage favouring the latter process.
- Redox signalling
Signal transduction in which oxidants act as second messengers.
- Antioxidant defence
Prevention or repair of oxidative damage.
- Antioxidant enzymes
Strictly, enzymes that remove oxidants; broadly, enzymes that contribute to the prevention or repair of oxidative damage. The broader definition is used in this Review.
- NRF2 transcription factor
Nuclear factor E2-related factor 2, which coordinates both the baseline and stress-inducible activation of a great many antioxidant enzymes.
- Antioxidant therapy
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.
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Forman, H.J., Zhang, H. Targeting oxidative stress in disease: promise and limitations of antioxidant therapy. Nat Rev Drug Discov 20, 689–709 (2021). https://doi.org/10.1038/s41573-021-00233-1