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Targeting oxidative stress in disease: promise and limitations of antioxidant therapy

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• Review Article
• Published: 30 June 2021

Targeting oxidative stress in disease: promise and limitations of antioxidant therapy

• Henry Jay Forman & 
• Hongqiao Zhang 

Nature Reviews Drug Discovery volume 20, pages689–709 (2021)Cite this article

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An Author Correction to this article was published on 13 July 2021

This article has been updated

Abstract

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.

요약

산화 스트레스는 

죽상 동맥 경화증, 만성 폐쇄성 폐 질환, 알츠하이머병, 암을 포함한 

많은 질병의 구성 요소입니다. 

항산화제로 평가되는 

수많은 저분자 물질이 전임상 연구에서 치료 가능성을 보였지만, 

임상시험 결과는 실망스러웠습니다. 

항산화제가 작용하는 메커니즘과 효과가 있는 부위와 시기를 더 잘 이해하면 

약리학적으로 더 큰 성공을 거둘 수 있는 

합리적인 접근 방식을 제공할 수 있습니다. 

여기에서는 

산화 스트레스, 

산화 환원 신호 및 질병 사이의 관계, 

산화 스트레스가 병리에 기여할 수 있는 메커니즘, 

항산화 방어의 작동 방식, 

효과를 제한하는 요소, 

생리적 신호, 

식이 성분 및 잠재적인 약물 개입을 통해 항산화 방어력을 높일 수 있는 방법을 

검토합니다.

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Introduction

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.

소개

'산화스트레스'라는 용어는

헬무트 시에스1가 생체 시스템에 손상을 초래할 수 있는

산화 물질 생산과 항산화 방어 사이의 불균형을 일컫는 말로

처음 사용했습니다.

그 이후로

산화 환원 생물학 분야는

병리학의 산화 스트레스 개념에서 생리학의 산화 환원 신호2,3,4 로 발전해 왔습니다.

산화 스트레스는

죽상 동맥 경화증, 만성 폐쇄성 폐 질환(COPD), 알츠하이머병 및 암을 포함한

광범위한 질병에 관여하는 것으로 밝혀졌으며,

산화제가 세포 손상에 기여하는 여러 메커니즘이 밝혀졌습니다5 .

Free radicals and antioxidants in normal physiological functions and human disease.pdf

1.32MB

그러나

산화 스트레스가 질병의 병리에 관여하는 정도는

매우 다양하기 때문에 일부 질병에서는 항산화 방어력을 높이는 효과가 제한적일 수 있습니다.

산화 스트레스는

산소와 질소에서 파생되는 소위 반응성 종의 화학 반응과 관련이 있습니다(상자 1).

이러한 종 중

어떤 것이 거대 분자에 손상을 일으키는지 이해하면

항산화 방어에 대한 치료적 접근법을 개선하는 데 근거를 제공하는 데 도움이 됩니다.

그러나

지금까지 저분자를 치료적으로 사용하는 것은 실망스러웠는데,

이는 항산화제의 작용 원리에 대한 지나치게 낙관적이고 잘못된 가정 때문이었습니다6.

예를 들어,

하이드록실 라디칼(-OH)을 제거하는 것은 비현실적이지만

과산화수소(H2O2) 생성을 줄임으로써

그 생성을 방지하면 손상을 효과적으로 예방할 수 있습니다.

산화 스트레스 분야의 주요 오해 중 하나는

세포 내부에서도 효과가 없는 소분자에 의한 슈퍼옥사이드(O2--) 또는

H2O2의 청소에 관한 것입니다.

이는 항산화 효소가

저분자보다 수천에서 수백만 배 더 빠르게

이러한 산화 물질과 반응하여 주된 항산화 방어력을 제공하기 때문입니다6,7.

그러나

항산화 효소가 없는 세포 외액에서는

아래에서 설명하는 것처럼 슈퍼옥사이드 디스뮤타아제(SOD)와

카탈라아제의 모방체를 통해 O2-및 H2O2(-OH는 아님)를 제거할 수 있습니다.

임상시험의 실패로 이어진 한계와 산화 스트레스가

질병의 어느 부위에서,

언제, 어느 정도 발생하는지 현실적으로 파악해야

항산화 방어가 효과적일 수 있다는 점을 인식하는 것이 중요합니다.

실제로

세포 내 대부분의 항산화 방어는

외인성 또는 내인성 소분자가 청소부 역할을 하는 것이 아니라

특정 기질을 사용하는 항산화 효소가 산화 물질을 감소시키는 방식으로 이루어집니다.

따라서

주요 치료 기회는 거대 분자에 직접적인 손상을 일으키는 산화제의 생성을 막고,

염증이나 세포 사멸을 초래하는 산화제의 하류 신호를 억제하며,

항산화 효소와 그 기질을 모두 증가시키는 데 있습니다.

현재

글루타치온 퍼옥시다제(GPX) 모방체인 에셀렌은

메니에르병에 대한 임상 2상(NCT02603081),

SOD 모방체인 GC4419는 편평세포암에 대한 임상 1상(NCT01921426),

설포라판은 NRF2 전사인자 활성화제(NCT01335971)는 COPD 2상 등의 임상시험이 진행 중이며

그 밖에도 여러 임상시험이 진행 중입니다.

이 글에서는

산화 스트레스,

산화 환원 신호 및 질병 사이의 관계를 검토하고

산화 스트레스가 병리에 기여할 수 있는 메커니즘에 대한 개요를 제시합니다.

항산화 방어를 매개하는 메커니즘과

그 효과를 제한하는 요인에 대한 현재의 이해에 초점을 맞추고,

이를 치료적으로 조절하기 위한 새로운 접근법을 강조합니다.

산화제가 작용하는 메커니즘과

항산화 요법의 한계 및 잠재력에 대한 이해를 높임으로써

치료적 개입을 개선할 수 있는 합리적인 접근법을 개발할 수 있습니다.

본 검토에서는

산화 스트레스를

산화제가 단백질, 핵산 및 세포막을 구성하는 지질을 포함한 거대 분자를

비효소적으로 손상시키는 상황으로 지칭합니다.

이 리뷰에서는

산화 물질의 생성을 막거나 효율적으로 제거할 수 있는 요인에만 초점을 맞춥니다.

주요 표적은

O2-, H2O2 및 지질 하이드로퍼옥사이드입니다.

O2•−, H2O2 and lipid hydroperoxides. 

이러한 표적을 제거하면

반응성이 더 높은 -OH,

과산화아질산염(ONOO-) 및

저할로산(HOX)의 생성을 방지할 수 있습니다.

reactive •OH,

peroxynitrite (ONOO−) and the

hypohalous acids (HOX) 

산화질소(-NO) 생성을 억제하여

ONOO- 생성을 제한할 수 있지만,

-NO는 정상적인 생리를 유지하는 데 너무 중요하기 때문에

과도한 O2-생성을 제한하는 것이 더 나은 접근 방식입니다.

Box 1 Mechanisms of oxidative stress

Box 1 Mechanisms of oxidative stress

Both endogenous and exogenous agents cause oxidative stress276. The term reactive oxygen species (ROS) encompasses molecules derived from O2, including superoxide (O2•−), hydrogen peroxide (H2O2), hydroxyl radical (•OH), ozone and singlet oxygen. The use of ROS, as though it were a chemical entity, leads to many imprecise statements because the chemistries of these species are markedly different.

Production of O2•− by one-electron reduction of O2 is primarily through leakage of electrons from the mitochondrial respiratory chain, particularly from ubisemiquinone (QH•−)277 (reaction 1):

QH∙−+O2↔Q+O2∙−

and the NADPH oxidases that catalyse reaction 2 (refs232,278):

NADPH+2O2→NADP++H++2O2∙−

The NADPH oxidases (NOX4, DuOX1 and DuOX2) and some other flavoprotein enzymes reduce O2 to H2O2, by giving O2•− a second electron before it leaves their active sites279.

The predominant source of H2O2 is dismutation of O2•−, a fast reaction with a rate constant near 105 M−1 s−1 that is accelerated to 2 × 109 M−1 s−1 by superoxide dismutases (reaction 3):

2O2∙−+2H+→H2O2+O2

The rate of H2O2 production largely determines whether redox signalling, oxidative stress or no significant oxidation occurs. H2O2 is reduced enzymatically by 15 enzymes, including catalase (reaction 4):

2H2O2→2H2O+O2

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

H2O2+2Trx(SH)2→TrxS2+2H2O

H2O2+2GSH→GSSG+2H2O

where TrxS2 is thioredoxin disulfide and GSSG is glutathione disulfide.

H2O2 does not easily oxidize most molecules but it can react rapidly with transition metals such as iron to produce hydroxyl radical (reaction 7, often referred to as the Fenton reaction)280:

H2O2+Fe2+→OH−+Fe3++∙OH

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

One reaction responsible for oxidative stress is the lipid peroxidation chain reaction that can be initiated by •OH (reactions 8–10):

LH+∙OH→L∙+H2O

L∙+O2→LOO∙

LOO∙+LH→LOOH+L∙

where LH is a lipid with allylic hydrogens, which are present in polyunsaturated fatty acids including arachidonic acid.

Superoxide can cause release of iron from iron–sulfur proteins, which can then catalyse reaction 7. The major way that the relatively weak oxidant O2•− contributes to oxidative stress, however, is as a precursor of H2O2 and peroxynitrite (ONOO−), which is formed in reaction 11:

O2∙−+∙NO→ONOO−

where •NO is nitric oxide. The danger of producing oxidative stress is not directly from the free radicals, •NO and O2•−, but from the protonated form of peroxynitrite, peroxynitrous acid (ONOOH), a non-radical. Peroxynitrous acid is a very strong oxidant that has the reactivity of the intermediates formed in its decomposition (reaction 12):

HNOO−↔+∙NO2+∙OH→NO3−+H+

nitrogen dioxide •NO2 and •OH. •NO2 can abstract hydrogen as does •OH or add to some molecules including the tyrosines in proteins producing nitrotyrosine that may alter function. ONOO− can also rapidly cause the release of iron from iron–sulfur proteins11, promoting •OH production from H2O2 (reaction 7).

Both •NO2 and •OH are indiscriminate in what they will oxidize, which creates the havoc called oxidative stress. Again, because of their rapid reactions, the best way this can be addressed is prevention of the formation of •NO2 and •OH.

The final oxidants we consider are the hypohalous acids (HOX) that are formed from H2O2 in reaction 13, which is catalysed by phagocytic cell myeloperoxidases:

H2O2+X−+H+→HOX+H2O

where X− may be Cl−, Br− or even SCN− (ref.281). They play a major role in tissue damage associated with phagocyte-mediated inflammation.

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.

질병에서 산화 스트레스의 역할

산화 스트레스가 질병에 기여하는 두 가지 주요 메커니즘이 있습니다.

첫 번째는

산화 스트레스 동안 반응성 종(특히 -OH, ONOO 및 HOCl)이 생성되어

막 지질, 구조 단백질, 효소 및 핵산을 포함한 거대 분자를 직접 산화시켜

세포 기능 이상 및 사멸을 초래하는 것입니다.

산화 스트레스의 두 번째 메커니즘은

비정상적인 산화 환원 신호입니다(상자 2).

산화 물질,

특히 생리적 자극에 의해 세포에서 생성되는 H2O2는

두 번째 메신저 역할을 할 수 있습니다8.

산화 스트레스에서 비생리적으로 생성되는 H2O2는

산화 환원 신호 전달에 이상을 일으킬 수 있습니다4.

두 가지 유형의 산화 스트레스 메커니즘은

모두 당뇨병과 같은 단일 질환에서 발생할 수 있으며,

당뇨병에서 진행된 당화 산물이 축적되고

스트레스 신호 경로의 비정상적인 활성화가 당뇨병 합병증을 유발합니다9.

또한

산화 스트레스를 받는 단백질에서

O2-(참조10) 및 ONOO-(참조11)에 의한 H2O2 생성 및 철 방출의 증가는

4-하이드록시-2-노네날(HNE)을 포함한 지질 과산화 생성물의 생성을 현저하게 증가시켜

비정상적인 신호를 유발할 수 있습니다12.

산화 스트레스는 다양한 병리와 관련이 있습니다.

이러한 병리의 원인에 대한 산화 스트레스의 기여도에 따라

첫째, 병리의 주요 원인으로서의 산화 스트레스(방사선 및 파라콰트로 인한 독성, 죽상경화증 등),

둘째, 질병 진행의 이차적 기여자로서 산화 스트레스(COPD, 고혈압 및 알츠하이머병 등)로 분류할 수 있습니다.

그러나

많은 질병에서 산화 스트레스의 역할이 불완전하게 이해되고 있기 때문에

이러한 분류는 잠정적인 것입니다.

Box 2 Redox signalling, homeostasis and antioxidant defences

Redox signalling is dependent on specific interactions of signalling proteins with hydrogen peroxide (H2O2) or other electrophiles that act as second messengers. As with oxidative stress, both endogenous and exogenous sources of H2O2 or other electrophiles may be involved; however, for redox signalling to be physiological rather than pathological, regulation is essential and requires the involvement of specificity that is not part of oxidative stress. An oxidative challenge, as opposed to oxidative stress, involves the stimulation of redox signalling without any damage, a phenomenon that we have called ‘para-hormesis’101. A related term is ‘oxidative eustress’3.

Maintaining redox homeostasis is important for cell function. Despite its name, homeostasis does not imply that nothing is changing. Indeed, a balance between oxidants and reductants, including glutathione, thioredoxin and NADPH, which are the substrates for antioxidant enzymes, is essential for maintaining normal physiology101. Thus, diseases that involve oxidative stress can be due to disruption of redox homeostasis, with type 2 diabetes mellitus as one example9. Adaptive homeostasis, as defined by Kelvin Davies282, involves elevated antioxidant defences brought about by transient alteration of redox homeostasis and redox signalling. However, redox signalling may also occur under pathological conditions, as oxidative stress can stimulate the same pathways as redox signalling under physiological conditions. The difference in this context is that the signalling will be unregulated and accompanied by nonspecific damage.

The effectiveness of this antioxidant system in maintaining the homeostasis relies upon keeping the generation and removal of superoxide (O2•−), H2O2 and nitric oxide (•NO) within a range that does not allow significant production of peroxynitrite (ONOO−) and hydroxyl radical (•OH)101. It is not a perfect system as evidenced by a low rate of oxidized proteins that accumulate with age. Regardless, the ability to induce the enzymes that remove O2•− and H2O2 and damaged proteins in what Davies calls ‘adaptive homeostasis’ provides a major means of enhancing antioxidant defences that will be described elsewhere in this Review282.

박스 2 산화 환원 신호, 항상성 및 항산화 방어력

산화 환원 신호는

신호 단백질과 과산화수소(H2O2) 또는 2차 전달자 역할을 하는 다른 친전해질의 특정 상호 작용에 의존합니다.

산화 스트레스와 마찬가지로

내인성 및 외인성 H2O2 또는 기타 전기성 물질이 모두 관여할 수 있지만,

산화 환원 신호가 병리적이기보다는 생리적이기 위해서는 조절이 필수적이며

산화 스트레스에 포함되지 않는 특이성의 개입이 필요합니다.

산화적 스트레스와는 반대로

산화적 도전은 아무런 손상 없이 산화 환원 신호가 자극되는 것을 말하며,

이를 '파라호메시스'101 라고 부릅니다.

이와 관련된 용어로는 '산화적 유스트레스'3 가 있습니다.

개방형 대사 시스템에서 산화 환원 관련 신호는 정상 상태 산화 환원 설정점의 지속적인 모니터링과 미세 조정을 필요로 합니다. 지속적인 산화 대사는 '항상성 공간', '골디락스 영역' 또는 '황금 평균'이라고 불리는 생리적 범위 내에서 작동하는 산화적 유스트레스라고 하는 지속적인 도전 과제입니다. 산화 환원 신호의 시공간적 제어는 산화 물질의 구획화된 생성 및 제거를 통해 이루어집니다. 주요 산화 환원 신호 분자인 이산화탄소의 세포 내 환경은 세포 소기관 간에 농도 차이가 매우 크다는 특징이 있습니다. 이러한 농도 패턴은 산화적으로 변형된 단백질의 패턴에 의해 반영되며, 그 예로 S-글루타치온화 단백질이 있습니다. 이 리뷰는 스트레스와 스트레스 반응의 단기적(비전사적) 및 장기적(전사적/번역적) 항상성 메커니즘에 대한 개념적 배경을 제시합니다. 산화 환원 설정점은 일주기 리듬과 외부 영향에 의해 조절되는 가변적인 이동 목표 값으로, 영양 및 생활 습관 인자를 포함하는 엑스포좀으로 요약할 수 있습니다. 배아 발달과 수명, 골격근과 운동, 수면-각성 리듬, 정신 생물학으로 이어지는 신경계 기능에서 산화적 스트레스에 대한 새로운 통찰을 포함하여 생리적 환경에서 세포별 및 조직별 산화 환원 조절의 새로운 분야를 간략하게 소개합니다.

산화 환원 항상성을 유지하는 것은

세포 기능에 중요합니다.

항상성이란 이름과는 달리 아무것도 변하지 않는다는 것을 의미하지는 않습니다.

실제로

항산화 효소의 기질인

글루타치온, 티오레독신, NADPH를 포함한 산화제와 환원제 사이의 균형은

정상적인 생리를 유지하는 데 필수적입니다101.

따라서

산화 스트레스를 수반하는 질병은

산화 환원 항상성의 파괴로 인해 발생할 수 있으며,

제2형 당뇨병이 한 예입니다9.

켈빈 데이비스가 정의한 적응성 항상성282 은

산화 환원 항상성 및 산화 환원 신호의 일시적인 변화로 인해

항산화 방어력이 높아지는 것을 포함합니다.

그러나

산화 스트레스가 생리적 조건에서 산화 환원 신호와 동일한 경로를 자극할 수 있기 때문에

산화 환원 신호는 병리학적인 조건에서도 발생할 수 있습니다.

이러한 맥락에서 차이점은 신호가 조절되지 않고

비특이적 손상을 동반한다는 것입니다.

항상성을 유지하는 항산화 시스템의 효과는

슈퍼옥사이드(O2--), H2O2 및 산화질소(-NO)의 생성 및 제거를

과산화지질(ONOO-)과 하이드록실 라디칼(-OH)101 의 상당한 생성을 허용하지 않는 범위 내에서 유지하는 데 의존합니다.

나이가 들면서 축적되는

산화 단백질의 비율이 낮은 것에서 알 수 있듯이

완벽한 시스템은 아닙니다.

그럼에도 불구하고

데이비스가 '적응 항상성'이라고 부르는

O2-및 H2O2와 손상된 단백질을 제거하는 효소를 유도하는 능력은

이 리뷰의 다른 곳에서 설명할 항산화 방어력을 강화하는 주요 수단을 제공합니다282.

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.

병리의 주요 원인인 산화 스트레스

산화 스트레스는 독

성 및 질병의 주요 요인이 될 수 있습니다.

그러나

중요한 주의 사항은 일단 손상이 시작되면

다른 요인이 병리에서 지배적이기 때문에

항산화 요법이 조직 손상의 진행을 억제하지 못하는 경우가 많다는 것입니다.

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.

Paraquat poisoning

Oxidative stress is also responsible for the toxicity of the widely used chemical herbicide, paraquat. When ingested, paraquat is actively taken up by alveolar type II cells and leads to pneumonitis and progressive lung fibrosis with poor prognosis. Paraquat also causes injury to other organs including liver and kidney. Long-term exposure to paraquat is associated with Parkinson disease16. Paraquat toxicity is initiated by the continuous redox cycling that generates O2•− (ref.17).

방사선 유발 폐 손상

폐암과 식도암에 대한 방사선 치료의 부작용으로 초기 폐렴과 섬유화가 자주 발생합니다13. 세포가 방사선에 노출되면 H2O의 자가분해로 -OH가 직접 생성되고, 이는 거대 분자를 산화시켜 염증 반응을 일으켜 염증 세포가 폐에 침윤(폐렴)하고 세포가 사멸합니다. 장기간에 걸쳐 사이토카인의 지속적인 생성에 대한 비정상적인 산화 환원 신호는 콜라겐 축적과 폐 섬유화를 유발합니다14. 또한, 방사선 노출 후 수개월 동안 지속될 수 있는 방사선 유발 폐 손상 쥐의 폐에서 더 높은 지질 과산화 및 DNA 산화(8-하이드록시-2′-데옥시구아노신)가 관찰되었습니다15.

파라콰트 중독

산화 스트레스는 널리 사용되는 화학 제초제인 파라콰트의 독성에도 영향을 미칩니다. 파라콰트를 섭취하면 폐포 II형 세포에 활발하게 흡수되어 폐렴과 진행성 폐 섬유증을 유발하며 예후가 좋지 않습니다. 파라콰트는 또한 간과 신장을 포함한 다른 장기에 손상을 입힐 수 있습니다. 파라콰트에 장기간 노출되면 파킨슨병과 관련이 있습니다16. 파라콰트 독성은 O2-를생성하는 지속적인 산화 환원 순환에 의해 시작됩니다(참조.17).

Atherosclerosis

In atherosclerosis, plaque builds up in the intimal layer of arteries and over time the arteries narrow, leading to infarction and stroke. Substantial evidence indicates that oxidative stress has a crucial role in the pathogenesis of atherosclerosis. Since the first identification of lipid hydroperoxides in human atherosclerotic 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.

죽상 동맥 경화증

죽상 동맥 경화증에서는 동맥 내막에 플라크가 쌓이고 시간이 지나면서 동맥이 좁아져 경색과 뇌졸중으로 이어집니다. 산화 스트레스가 죽상동맥경화증의 발병에 중요한 역할을 한다는 상당한 증거가 있습니다. 인간 죽상 경화성 대동맥에서 지질 과산화수소가 처음 발견된 이후18, 많은 연구에서 죽상 경화성 병변에서 산화 지질 및 기타 산화 스트레스 마커가 증가하는 것으로 나타났습니다. 예를 들어, 갓 분리한 인간 플라크에서 콜레스테롤 리놀레이트(Ch18:2)의 20%가 산화된 것으로 보고된 반면, 정상 동맥에서는 검출되지 않았습니다19. 또한, 건강한 지원자에 비해 죽상 동맥 경화증 환자의 혈장에서 HNE 변형 저밀도 지단백질(LDL)이 50% 증가한 것으로 밝혀졌습니다20. 또한 아라키돈산의 과산화 산물인 이소프로스탄은 인간 제대 정맥에 비해 인간 죽상 경화성 병변에서 최소 5배 이상 증가하는 것으로 보고되었으며, 산화된 리놀레산은 인간 병변에서만 검출되었습니다21. 산화 스트레스는 LDL 콜레스테롤을 죽종 형성 형태의 산화 LDL(OxLDL)로 전환시키는 역할을 하며, 이는 병변 부위에서 염증 반응과 백혈구 모집을 시작하고 촉진하는 데 중요한 역할을 하고 평활근 세포의 활성화와 -NO 생체 이용률 감소를 통해 죽상 경화증의 발병에 기여합니다22.

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.

Hypertension

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

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.

Cancer

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.

Ischaemia–reperfusion injury

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.

Box 3 NRF2–EpRE signalling

Nuclear factor E2-related factor (NRF2) is a member of the ‘cap‘n’collar’ family of bZIP transcription factors (CNC-bZIP). It was first identified as a transcription factor regulating the expression‎ of β-globin by Moi et al. in 1994 (ref.283), and soon after was found to be a transcription activator of NQO1 that bound to the antioxidant response element (ARE) in the promoter284. Many detailed studies established that NRF2–ARE signalling has a central role in the regulation of antioxidant gene expression‎285. ARE, the cis element of NRF2 binding, is more accurately called the electrophile response element (EpRE) as the ‘antioxidant’ inducers are electrophiles and include hydrogen peroxide (H2O2), some components of intermediary metabolism and products derived from dietary polyphenols6.

NRF2–EpRE signalling regulates the basal and inducible expression‎ of more than 200 genes that encode proteins involved in antioxidant defence, detoxification, apoptosis, DNA repair, removal of oxidized protein by the proteasome, inflammation and other processes102,286,287. The role of NRF2 in the induction of antioxidant enzymes and defence against oxidative stress has been verified in cell and non-human animal models with NRF2 knockout and/or induction. Mounting evidence suggests that deficiency of NRF2 signalling suppresses the induction of target antioxidant enzymes in response to oxidative stress, increases susceptibility to oxidative damage288 and accelerates the inflammatory response289, whereas enhancing NRF2 activity increases the expression‎ of antioxidant enzymes and the defence against oxidative stress.

The molecular mechanism and regulation of NRF2 activation in response to oxidative stress has been discussed in many recent articles. Most relevant to therapeutics is the recent review by Cuadrado et al.99. Thus, we only briefly describe the regulation of NRF2 (Fig. 3). Under basal conditions, most NRF2 protein binds to Kelch-like ECH-associated protein 1 (KEAP1) and/or β-transducin repeat-containing protein (βTrCP) and is rapidly degraded by 26S proteasome after ubiquitylation. KEAP1 is an adaptor for Cullin 3-containing ubiquitin ligase E3 complex290, and βTrCP is a substrate receptor for Cul1-based ubiquitin ligase291. In response to oxidative stimuli, KEAP1 is oxidatively modified and loses the capacity to present NRF2 for degradation. Simultaneously, oxidative inhibition of glycogen synthase kinase 3β (GSK3β)-mediated NRF2 phosphorylation at the Neh6 domain stops the interaction of NRF2 and βTrCP. NRF2 can also be activated through p62-mediated autophagic degradation of KEAP1 (ref.292). With the activation of these pathways, NRF2, both dissociated from KEAP1 and newly synthesized, escapes from degradation and is then translocated into the nucleus where it forms heterodimers with small Maf or Jun family proteins, binds to EpRE in the promoter and increases transcription of target genes. In the nucleus, NRF2 is competitively suppressed by BACH1 (ref.293). ChIP-seq assays identified a considerable overlap of BACH1 (in HEK293 cells) and NRF2 (in mouse MEF cells) target genes294. Evidence suggests that the suppressive effect of BACH1 on NRF2 signalling may be gene selective. BACH1 inactivation is required for the induction of HO1 but not for that of thioredoxin reductase 1, even though both genes are regulated by NRF2 (ref.295). In Bach1-knockout mice, fewer than 10% of the upregulated genes are NRF2 target genes244. It should be noted that NRF2 regulation is far more complicated than the simplified pathways, as nuclear factor-κB (NF-κB), PKC, p21, BRCA1, HRD1, CRIF1 and microRNAs are involved in regulating NRF2 signalling by acting on NRF2 expression‎, protein stability, activation and translocation99.

With more regulators and interaction pathways being identified, NRF2 activity is clearly regulated by a network of signalling pathways allowing it to hold important roles in multiple biological processes and response to multiple circumstances. Some puzzles remain for NRF2 regulation, including how NRF2 is transported in and out of the nucleus, and the dysregulation and ceiling effect of NRF2 induction under some pathophysiological conditions.

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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.

Table 1 Clinical status of antioxidant enzyme mimics

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Fig. 1: Reactive species in the extracellular space and defences by SOD or catalase mimics and NOX inhibitors.

Plasma membrane NADPH oxidase (NOX) production of superoxide (O2•−) outside cells may be prevented by NOX inhibitors. Dismutation of O2•− to hydrogen peroxide (H2O2) is accelerated by superoxide dismutase (SOD) mimics, preventing the formation of peroxynitrite (ONOO−), which spares nitric oxide (•NO). Reduction of H2O2 is accelerated by catalase mimics, preventing the formation of hypohalous acids (HOX) by myeloperoxidase (MPO) and hydroxyl radical (•OH) production via the Fenton reaction. Most SOD mimics appear to have catalase activity. Although NOX4, which is primarily in intracellular organelle membranes, has also been found in the plasma membrane, this has only been reported for one cell type275 and so its extracellular location remains debatable (indicated by the question mark). NOS, nitric oxide synthase.

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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.

Increasing GSH

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

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 esters

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.

Fig. 2: Glutathione metabolism and strategies to increase glutathione.

Glutathione (GSH) is synthesized through reactions catalysed by glutamate–cysteine ligase (GCL) and GSH synthetase (GS), with GCL as the rate-limiting enzyme and cysteine as the rate-limiting substrate. Both reduced GSH and glutathione disulfide (GSSG) are exported from cells through multidrug resistance protein (MRP), and extracellular GSH is sequentially metabolized by membrane-bound γ-glutamyl transpeptidase (GGT) into cysteinylglycine and γ-glutamyl products, and dipeptidase hydrolyses cysteinylglycine to cysteine and glycine. The amino acids are transported back into cells and participate in GSH synthesis. N-acetylcysteine (NAC) is deacetylated by esterase action into cysteine, while GSH esters (GSH-E) are directly converted by esterase into GSH. γ-Glutamylcysteine (γ-glu-cys) can bypass GCL, the rate-limiting step for GSH synthesis. Electrophiles cause the activation of NRF2, which regulates the transcription of the two subunits of GCL, and also GS. Some transporters have been identified: ASC, sodium-dependent alanine-serine-cysteine transporter; Xc−, system cystine/glutamate antiporter. Question marks denote the unidentified transporters/channels for GSH-E, γ-glu-cys and NAC.

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NRF2 activators

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.

Fig. 3: NRF2 signalling pathway and antioxidant therapeutic approaches.

(1) Transcription factor NRF2 is constantly synthesized in cells but its transport to the nucleus remains low under basal conditions. This is due to its degradation through association with Kelch-like ECH-associated protein 1 (KEAP1), which facilitates its degradation by the 26S proteasome. Boosting NRF2 synthesis represents a therapeutic antioxidant approach. (2) Upon exposure to electrophiles, KEAP1 is alkylated and loses its ability to cause degradation of NRF2. Using non-toxic electrophiles to alkylate KEAP1 represents another major therapeutic approach. For KEAP1, SH is the thiol form and SX denotes the adduct formed with the electrophile (X). (3) In a parallel pathway glycogen synthase kinase 3β (GSK3β) phosphorylates NRF2, which with β-transducin repeat-containing protein (βTrCP) is degraded by the proteasome, a process that is inhibited by oxidative inactivation of GSK3β. The interaction of NRF2 and βTrCP is disrupted owing to oxidant-mediated inhibition of GSK3β and the phosphorylation of NRF2 at the Deh6 domain. Inhibiting GSK3β is another potential therapeutic approach to modulate NRF2 signalling. (4) Oxidation-induced KEAP1 degradation also occurs through p62-mediated sequestration of KEAP1 and autophagy, a process initiated by phosphorylation of p62 via TANK-binding kinase 1 (TBK1) and mechanistic target of rapamycin complex 1 (mTORC1). p62 therefore provides another potential therapeutic target. Newly synthesized NRF2 that escapes degradation is translocated into the nucleus where it binds to EpRE sequences in the promoters of antioxidant genes and increases their expression‎. NRF2 activity is also positively regulated through NRF2 phosphorylation by protein kinase C (PKC)269 and its interaction with other proteins such as p21 (ref.270) and BRCA1 (ref.271). (5) In the nucleus, BACH1 negatively regulates NRF2 activity by competing to form heterodimers with small Maf (sMaf) or Jun proteins and binding to the electrophile response element (EpRE)272,273,274. Thus, compounds that inhibit BACH1 offer an alternative therapeutic approach for increasing expression‎ of some NRF2-regulated genes. Other negative regulators of NRF2, which represent potential therapeutic targets include HRD1, CRIF1, progerin and microRNA for NRF2 (ref.99).

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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.

Table 2 NRF2 activators in clinical trials

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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.

Dietary antioxidants

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.

Ageing

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.

Outlook

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.

Change history

• 13 July 2021

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