Re: 활성산소 연구의 히스토리..

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Indian J Clin Biochem. 2015 Jan; 30(1): 11–26.

Published online 2014 Jul 15. doi: 10.1007/s12291-014-0446-0

PMCID: PMC4310837

PMID: 25646037

Free Radicals: Properties, Sources, Targets, and Their Implication in Various Diseases

Alugoju Phaniendra, Dinesh Babu Jestadi, and Latha Periyasamy

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Abstract

Free radicals and other oxidants have gained importance in the field of biology due to their central role in various physiological conditions as well as their implication in a diverse range of diseases. The free radicals, both the reactive oxygen species (ROS) and reactive nitrogen species (RNS), are derived from both endogenous sources (mitochondria, peroxisomes, endoplasmic reticulum, phagocytic cells etc.) and exogenous sources (pollution, alcohol, tobacco smoke, heavy metals, transition metals, industrial solvents, pesticides, certain drugs like halothane, paracetamol, and radiation). Free radicals can adversely affect various important classes of biological molecules such as nucleic acids, lipids, and proteins, thereby altering the normal redox status leading to increased oxidative stress. The free radicals induced oxidative stress has been reported to be involved in several diseased conditions such as diabetes mellitus, neurodegenerative disorders (Parkinson’s disease-PD, Alzheimer’s disease-AD and Multiple sclerosis-MS), cardiovascular diseases (atherosclerosis and hypertension), respiratory diseases (asthma), cataract development, rheumatoid arthritis and in various cancers (colorectal, prostate, breast, lung, bladder cancers). This review deals with chemistry, formation and sources, and molecular targets of free radicals and it provides a brief overview on the pathogenesis of various diseased conditions caused by ROS/RNS.

자유 라디칼 및 기타 산화제는 

다양한 생리적 조건에서 중심적인 역할을 할 뿐만 아니라 

다양한 질병에 영향을 미치기 때문에 생물학 분야에서 중요성이 커지고 있습니다. 

활성산소종(ROS)과 활성질소종(RNS)인 

자유 라디칼은 

내인성 소스(미토콘드리아, 퍼옥시좀, 소포체, 식세포 등)와 

외인성 소스(공해, 알코올, 담배 연기, 중금속, 전이 금속, 산업용 용매, 살충제, 

할로탄, 파라세타몰 같은 특정 약물 및 방사선) 모두에서 파생됩니다. 

pollution, alcohol, tobacco smoke, heavy metals, transition metals, industrial solvents, pesticides, certain drugs like halothane, paracetamol, and radiation

활성산소는 

핵산, 지질, 단백질과 같은 

다양한 종류의 중요한 생물학적 분자에 악영향을 미쳐 

정상적인 산화 환원 상태를 변화시켜 

산화 스트레스를 증가시킬 수 있습니다. 

활성산소는 

당뇨병, 

신경 퇴행성 질환(파킨슨병-PD, 알츠하이머병-AD 및 다발성 경화증-MS), 

심혈관 질환(죽상경화증 및 고혈압), 

호흡기 질환(천식), 

백내장 발병, 

류마티스 관절염 및 

다양한 암(대장암, 전립선암, 유방암, 폐암, 방광암) 등 

여러 질병 상태에 관여하는 것으로 보고되고 있습니다. 

이 리뷰에서는 

활성산소의 화학, 형성 및 발생원, 분자 표적에 대해 다루며 

ROS/RNS로 인한 다양한 질병의 발병 기전에 대한 간략한 개요를 제공합니다.

Keywords: Free radicals, Reactive oxygen species (ROS), Reactive nitrogen species (RNS), Oxidative stress

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History

In recent years there is an ever-increasing curiosity in studying the role of free radicals in biology, because of their pivotal role in various physiological conditions as well as their involvement in a diverse range of diseases. For the first time in 1900, Moses Gomberg, Professor of Chemistry at the University of Michigan, speculated the existence of an organic free radical, triphenyl methyl radical (Ph3C•) in the living system [1]. Later in 1954, Gershman proposed “free radical theory of oxygen toxicity”, according to which, the toxicity of oxygen is due to its ability to form free radicals [2]. In the same year, the electron paramagnetic resonance (EPR) studies by Commoner et al. 1954 [3] confirmed the presence of free radicals in biological materials. Soon thereafter Denham Harman, in 1956, proposed the “free radical theory of aging”, which states that free radicals play a central role in the aging process. A second era of free radical research began in 1969 by Mc Cord and Fridovich, who discovered superoxide dismutase, the first enzymatic defense system against superoxide anion [4]. For the first time in 1971, Loschen indicated that the Reactive oxygen species are generated in cellular metabolic respiration [5], which was supported by Nohl and Hegner (1978) [6]. In 1977, Mittal and Murad reported that the hydroxyl radical, OH˙ induces the formation of the second messenger cyclic GMP by activating the enzyme guanylate cyclase [7]. Later in 1989, Hallliwell and Gutteridge reported that reactive oxygen species (ROS) include both free radical and non radical derivatives of oxygen [8]. Since then a massive data has been documented on the role of free radicals in various pathophysiological conditions.

최근 몇 년 동안

다양한 생리적 조건에서 중추적인 역할을 할 뿐만 아니라

다양한 질병에 관여하기 때문에

생물학에서 자유 라디칼의 역할에 대한 호기심이 점점 더 커지고 있습니다.

1900년 미시간 대학교 화학과 교수인 모세 곰버그(Moses Gomberg)는

처음으로 생체 내에 유기 자유 라디칼인

트리페닐 메틸 라디칼(Ph3C-)이 존재한다고 추측했습니다[1].

1954년 후반, 거쉬만은

산소의 독성이 자유 라디칼을 형성하는 능력 때문이라는

'산소 독성의 자유 라디칼 이론' free radical theory of oxygen toxicity” 을 제안했습니다[2].

같은 해,

1954년 커먼러 등[3]의 전자기 공명(EPR) 연구를 통해

생물학적 물질에

자유 라디칼이 존재한다는 사실이 확인되었습니다.

얼마 지나지 않아 1956년 덴햄 하먼은

자유 라디칼이 노화 과정에서 중심적인 역할을 한다는

'자유 라디칼 노화 이론 free radical theory of aging '을 제안했습니다.

자유 라디칼 연구의 두 번째 시대는

1969년 맥코드와 프리도비치가

슈퍼옥사이드 음이온에 대한

최초의 효소 방어 시스템인 슈퍼옥사이드 디스뮤타아제를 발견하면서 시작되었습니다[4].

1971년 Loschen은 처음으로

활성산소가 세포 대사 호흡에서 생성된다는 사실을 밝혔으며[5],

이는 Nohl과 Hegner(1978)에 의해 뒷받침되었습니다[6].

1977년 미탈과 무라드는

하이드 록실 라디칼인 OH˙가

효소 구아닐레이트 시클라제를 활성화하여

두 번째 메신저 사이클릭 GMP의 형성을 유도한다고 보고했습니다 [7].

부분적으로 정제된 가용성 쥐 간 구아닐레이트 시클라제[GTP 파이로인산-리아제(순환), EC 4.6.1.2]는 슈퍼옥사이드 디스뮤타제(슈퍼옥사이드: 슈퍼옥사이드 산화환원효소, EC 1.15.1.1)에 의해 활성화되었습니다. 이러한 활성화는 슈퍼옥사이드 디스뮤타제의 억제제인 KCN 또는 글루타치온으로 방지되었습니다. 구아닐레이트 시클라제 제제는 슈퍼옥사이드 이온을 형성했습니다. 슈퍼옥사이드 디스뮤타제에 의한 활성화는 질산염 환원효소를 첨가함으로써 더욱 강화되었습니다. 구아닐레이트 시클라제 활성은 단독 양이온 보조 인자인 Mg2+보다 Mn2+에서 훨씬 더 컸지만, 배양에 Mn2+가 포함되었을 때 슈퍼옥사이드 디스뮤타제에 의한 활성화는 관찰되지 않았습니다. 카탈라아제는 또한 슈퍼옥사이드 디스뮤타제에 의해 유도된 활성화를 감소시켰습니다. 따라서 활성화는 배양에서 슈퍼옥사이드 이온과 H2O2의 형성이 모두 필요했습니다. 구아닐레이트 시클라제의 활성화는 H2O2의 첨가만으로는 달성할 수 없었습니다. 하이드록실 라디칼의 제거제가 활성화를 방해했습니다. 과산화 이온과 과산화수소는 구아닐레이트 시클라제를 활성화하는 하이드 록실 라디칼의 형성으로 이어질 수 있다고 제안됩니다. 이 활성화 메커니즘은 산화 및 환원제를 사용한 조직에서 변화된 구아닐레이트 시클라제 활성과 주기적 GMP 축적에 대한 수많은 관찰을 설명할 수 있습니다. 이 메커니즘은 또한 호르몬, 다른 약제 및 과정에 반응하여 조직에서 산화 환원 또는 자유 라디칼 형성에 변화가 있을 때 구아닐레이트 시클라제 및 순환 GMP 형성의 생리적 조절을 가능하게 합니다.

이후

1989년 Hallliwell과 Gutteridge는

활성 산소종(ROS)에

산소의 자유 라디칼 및 비라디칼 유도체가 모두 포함된다고 보고했습니다[8].

그 이후로

다양한 병리 생리학적 조건에서

자유 라디칼의 역할에 대한 방대한 데이터가 문서화되었습니다.

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Introduction

Free radicals are the products of normal cellular metabolism. A free radical can be defined as an atom or molecule containing one or more unpaired electrons in valency shell or outer orbit and is capable of independent existence. The odd number of electron(s) of a free radical makes it unstable, short lived and highly reactive. Because of their high reactivity, they can abstract electrons from other compounds to attain stability. Thus the attacked molecule loses its electron and becomes a free radical itself, beginning a chain reaction cascade which finally damages the living cell [9]. Both ROS and RNS collectively constitute the free radicals and other non radical reactive species [10]. The ROS/RNS play a twofold job as both beneficial and toxic compounds to the living system. At moderate or low levels ROS/RNS have beneficial effects and involve in various physiological functions such as in immune function (i.e. defense against pathogenic microorganisms), in a number of cellular signaling pathways, in mitogenic response and in redox regulation [11, 12]. But at higher concentration, both ROS as well as RNS generate oxidative stress and nitrosative stress, respectively, causing potential damage to the biomolecules. The oxidative stress and nitrosative stress are developed when there is an excess production of ROS/RNS on one side and a deficiency of enzymatic and non enzymatic antioxidants on the other side. Most importantly, the excess ROS can damage the integrity of various biomolecules including lipids [13], proteins [14] and DNA [15] leading to increased oxidative stress in various human diseases such as diabetes mellitus, neurodegenerative diseases, rheumatoid arthritis, cataracts, cardiovascular diseases, respiratory diseases as well as in aging process. This review deals with the chemistry, formation and sources, and molecular targets of free radicals, it highlights the implication of free radicals in various diseased conditions.

소개

자유 라디칼은

정상적인 세포 대사의 산물입니다.

자유 라디칼은

원자가 껍질 또는 외부 궤도에

하나 이상의 짝을 이루지 않은 전자를 포함하는

원자 또는 분자로 정의할 수 있으며

독립적으로 존재할 수 있습니다.

자유 라디칼은

전자의 수가 홀수이기 때문에 불안정하고

수명이 짧으며 반응성이 매우 높습니다.

반응성이 높기 때문에

다른 화합물로부터

전자를 빼앗아 안정성을 확보할 수 있습니다.

따라서

공격받은 분자는

전자를 잃고

그 자체로 자유 라디칼이 되어

연쇄 반응이 시작되고

결국 살아있는 세포를 손상시킵니다[9].

ROS와 RNS는

모두 자유 라디칼과 기타 비라디칼 반응성 종을

총체적으로 구성합니다 [10].

ROS/RNS는

생체 시스템에 유익한 화합물인 동시에

독성 화합물로서 두 가지 역할을 합니다.

중간 또는 낮은 수준에서 ROS/RNS는 유익한 영향을 미치며

면역 기능(예: 병원성 미생물에 대한 방어),

여러 세포 신호 경로,

미토겐 반응 및 산화 환원 조절과 같은

다양한 생리적 기능에 관여합니다 [11, 12].

선천 면역 세포의 미토겐 활성화 단백질 키나제(MAPK)는 다양한 패턴 인식 수용체에 의해 활성화되며, 그 중 가장 잘 연구된 것은 톨 유사 수용체(TLR)입니다. TLR 결찰은 IL-1R 관련 키나아제(IRAK)와 TNFR 관련 인자 6(TRAF6)을 포함하는 신호 복합체의 형성을 유도하며, 이는 K63-연결된 다유비퀴티화에 의해 매개됩니다. 이 복합체는 p38α 및 Jun N-말단 키나아제(JNK)의 상류에 있는 MAPK 키나아제(MAP3K)인 TGF-β 활성화 키나아제 1(TAK1)과 상호 작용하여 활성화합니다. TAK1은 또한 IκB 키나제(IKK) 복합체를 활성화하여 전사인자 핵인자-κB와 세포 외 신호 조절 키나제 1(ERK1) 및 ERK2의 업스트림에 있는 MAP3K 종양 진행 유전자좌 2(TPL2)의 활성화를 유도할 수 있습니다. 그러나 최근의 유전학적 증거에 따르면 일차 대식세포에서 TAK1이 MAPK의 TLR 활성화에 필요하지 않으며, 관련된 MAP3K는 아직 밝혀지지 않은 것으로 나타났습니다.MAPK 신호는 사이토카인 및 케모카인과 같은 전염증 매개체의 유도부터 항염증 피드백 경로의 활성화에 이르기까지 선천성 면역 반응에서 여러 가지 역할을 합니다.예를 들어, p38α는 종양 괴사인자(TNF) 생성을 촉진하는 MAPK 활성화 단백질 키나아제 2(MK2)를 활성화하여 면역에 중요한 역할을 하는 다운스트림 키나아제를 활성화할 수 있습니다. 반대로, p38α 또는 ERK1 및 ERK2에 의한 미토겐 및 스트레스 활성화 키나제(MSK)의 활성화는 항염증 사이토카인 인터루킨-10(IL-10)과 IL-1 수용체 길항제(IL-1RA)의 전사 증가를 초래합니다.MAPK 신호는 이중 특이성 포스파타제(DUSP)의 발현을 유도합니다. 이는 DUSP가 MAPK를 탈인산화하고 비활성화하는 부정적인 피드백 루프를 형성합니다. 유전자 연구에 따르면 선천성 면역 반응을 제어하는 데 있어 DUSP의 중요한 역할이 밝혀졌습니다.박테리아 병원체는 숙주 면역 반응을 하향 조절하기 위해 MAPK를 직접 표적으로 삼는 방법을 진화시켜 왔으며, 예를 들어 특정 박테리아 단백질은 MAPK 키나제(MKK) 효소를 비활성화하고 DUSP를 활성화하여 MAPK 신호를 억제하는 것으로 밝혀졌습니다.MAPK 신호를 표적으로 하는 저분자 억제제는 항염증제로서 기능할 가능성이 있습니다. p38 억제제는 최초로 개발된 MAPK 억제제이지만, 이러한 화합물을 사용한 임상 결과는 실망스러웠습니다. 그 결과 제약 업계에서는 업스트림 MAP3K 또는 MK2와 같은 다운스트림 키나아제를 표적으로 삼는 것으로 초점을 옮겼습니다. Abstract 병원체 감염 또는 조직 손상 후, 선천 면역 세포의 세포 표면과 세포질에 있는 패턴 인식 수용체가 자극되면 주요 미토겐 활성화 단백질 키나제(MAPK) 하위 계열인 세포 외 신호 조절 키나제(ERK), p38 및 준 N-말단 키나제(JNK) 하위 계열의 구성원이 각각 활성화됩니다. 핵 인자-κB 및 인터페론 조절 인자 전사인자의 활성화와 함께 MAPK 활성화는 염증 반응을 함께 조절하는 여러 유전자의 발현을 유도합니다. 이 리뷰에서는 선천성 면역에서 MAPK의 조절과 기능에 대한 최신 지식과 숙주 조직 손상을 방지하기 위해 MAPK 활성을 제한하는 데 있어 부정적인 피드백 루프의 중요성에 대해 논의합니다. 또한 병원체가 어떻게 복잡한 메커니즘을 진화시켜 독성을 높이기 위해 MAPK 활성화를 조작했는지 살펴봅니다. 마지막으로 자가면역 및 염증성 질환을 치료하기 위한 MAPK 경로의 약리학적 표적화의 잠재력을 고려합니다.

그러나

농도가 높아지면

ROS와 RNS 모두 각각 산화 스트레스와 질산화 스트레스를 생성하여

생체 분자에 잠재적인 손상을 일으킵니다.

산화 스트레스와 질산화 스트레스는

한쪽에서는 ROS/RNS가 과도하게 생성되고

다른 한쪽에서는 효소 및 비효소 항산화제가 결핍될 때 발생합니다.

oxidative stress and nitrosative stress

가장 중요한 것은

과잉 ROS가

지질[13], 단백질[14], DNA[15] 등

다양한 생체 분자의 완전성을 손상시켜

당뇨병, 신경 퇴행성 질환, 류마티스 관절염, 백내장, 심혈관 질환, 호흡기 질환 등

다양한 인체 질환과

노화 과정에서 산화 스트레스를 증가시킬 수 있다는 점입니다.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3646577/

단백질 산화로도 알려진 단백질 산화 변형은 단백질 번역 후 변형의 주요 유형입니다. 단백질 아미노산 잔기와 활성 산소종(ROS) 또는 활성 질소종(RNS) 사이의 반응으로 인해 발생하며 비가역적 변형과 가역적 변형의 두 가지 범주로 분류할 수 있습니다. 단백질 산화는 종종 표적 단백질의 기능 저하와 관련이 있으며, 이는 정상적인 노화 및 노화 관련 발병에 기여하는 것으로 여겨져 왔습니다. 그러나 이제는 단백질 산화 변형이 질병과 건강에도 유익한 역할을 할 수 있다는 사실이 밝혀졌습니다. 이 리뷰에서는 문헌에 기록된 단백질 산화 변형의 긍정적인 역할을 요약하고 강조합니다. 다루는 산화적 변형 단백질 부가체에는 카르보닐화, 3-니트로티로신, s-설펜화, s-니트로실화, s-글루타티오닐화 및 이황화물 형성이 포함됩니다. 이 모든 것들은 산화 환원 스트레스 조건과 관련된 수많은 실험 시스템에서 광범위하게 분석되었습니다. 저자들은 전처치 또는 허혈성 내성과 같은 예방적 접근 방식에서 가역적인 방식으로 선택된 단백질 표적을 수정하면 건강을 유지하고 질병과 싸우는 데 잠재적인 가능성을 제공할 수 있다고 믿습니다.

이 리뷰에서는 비가역적 및 가역적 단백질 산화 생성물에 대해 설명합니다. 비가역적 산화에는 단백질 카르보닐과 3-니트로티로신이 포함되며 가역적 산화에는 설펜산, 니트로소티올, s-글루타티온과 같은 시스테인 변형 산물이 포함됩니다.

Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS)

In general pro-oxidants/oxidants are termed as ROS/RNS. The most important free radicals produced during metabolic reactions are radicals derived from oxygen, ROS. Both the ROS and RNS can be classified into two groups of compounds namely; radicals and non-radicals (see Table 1). Radicals are the species which contain at least one unpaired electron in the shells around the atomic nucleus and are capable of independent existence. The oxygen molecule itself is a radical, and because of the presence of two unpaired electrons it is referred as biradical. The examples for the radicals include Superoxide (O∙−2), Oxygen radical (O∙∙2), Hydroxyl (OH∙), Alkoxyradical (RO∙), Peroxyl radical (ROO∙), Nitric oxide (nitrogen monoxide) (NO∙) and nitrogen dioxide (NO∙2) [17]. The high reactivity of these radicals is due to the presence of one unpaired electron which tends to donate it or to obtain another electron to attain stability. The non radical species include hydrogen peroxide (H2O2), hypochlorous acid (HOCl), hypobromous acid (HOBr), ozone (O3), singlet oxygen (1O2), nitrous acid (HNO2), nitrosyl cation (NO+), nitroxyl anion (NO−), dinitrogen trioxide (N2O3), dinitrogen tetraoxide (N2O4), nitronium (nitryl) cation (NO2+), organic peroxides (ROOH), aldehydes (HCOR) and peroxynitrite (ONOOH) [16, 17]. These non radical species are not free radicals but can easily lead to free radical reactions in living organisms [18].

활성 산소종(ROS) 및 활성 질소종(RNS)

일반적으로

활성산소/산화제는 ROS/RNS로 불립니다.

대사 반응 중에 생성되는

가장 중요한 자유 라디칼은

산소로부터 파생된 라디칼인 ROS입니다.

ROS와 RNS는

라디칼과 비라디칼의 두 가지 화합물 그룹으로 분류할 수 있습니다(표 1 참조).

라디칼은

원자핵 주변의 껍질에 짝을 이루지 않은 전자가 하나 이상 포함되어 있고

독립적으로 존재할 수 있는 종입니다.

산소 분자 자체는

라디칼이며,

두 개의 짝을 이루지 않은 전자가 존재하기 때문에

바이라디칼이라고 합니다.

라디칼의 예로는

슈퍼옥사이드(O∙-2),

산소 라디칼(O∙∙2),

하이드록실(OH∙),

알콕시라디칼(RO∙),

퍼옥실 라디칼(ROO∙),

산화질소(일산화질소)(NO∙) 및

이산화질소(NO∙2) 등이 있습니다[17].

Superoxide (O∙−2),

Oxygen radical (O∙∙2),

Hydroxyl (OH∙),

Alkoxyradical (RO∙),

Peroxyl radical (ROO∙),

Nitric oxide (nitrogen monoxide) (NO∙) and

nitrogen dioxide (NO∙2)

이러한 라디칼의 높은 반응성은

안정성을 얻기 위해

전자를 기증하거나 다른 전자를 얻는 경향이 있는

짝을 이루지 않은 전자가 하나 존재하기 때문입니다.

비라디칼 종에는

과산화수소(H2O2),

차아염소산(HOCl),

차아브롬산(HOBr),

오존(O3),

단일 산소(1O2),

아질산(HNO2),

니트로실 양이온(NO+)이 포함됩니다,

The non radical species include

hydrogen peroxide (H2O2),

hypochlorous acid (HOCl),

hypobromous acid (HOBr),

ozone (O3),

singlet oxygen (1O2),

nitrous acid (HNO2),

nitrosyl cation (NO+),

nitroxyl anion (NO−),

dinitrogen trioxide (N2O3),

dinitrogen tetraoxide (N2O4),

nitronium (nitryl) cation (NO2+),

organic peroxides (ROOH),

aldehydes (HCOR) and

peroxynitrite (ONOOH)

니트로실 음이온(NO-), 삼산화이질소(N2O3), 사산화이질소(N2O4), 니트로늄(니트릴) 양이온(NO2+), 유기 과산화물(ROOH), 알데히드(HCOR), 과산화아질산염(ONOOH) [16, 17].

이러한

비라디칼 종은

자유 라디칼은 아니지만

살아있는 유기체에서

자유 라디칼 반응을 쉽게 일으킬 수 있습니다 [18].

Table 1

List of ROS and RNS produced during metabolism [16, 17, 19]

Free radicalSymbolHalf-life

Reactive oxygen species-ROS
Radicals
Superoxide	O2 •−	10−6 s
Hydroxyl	OH•	10−10 s
Alkoxyl radical	RO•	10−6
Peroxyl Radical	ROO•	17 s
Non radicals
Hydrogen peroxide	H2O2	Stable
Singlet oxygen	1O2	10−6 s
Ozone	O3	s
Organic peroxide	ROOH	Stable
Hypochlorous acid	HOCl	Stable (min)
Hypobromous acid	HOBr	Stable (min)
Reactive nitrogen species-RNS
Radicals
Nitric oxide	NO•	sa
Nitrogen dioxide	NO2•	s
Non radicals
Peroxynitrite	ONOO−	10−3 s
Nitrosyl cation	NO+	s
Nitroxyl anion	NO−	s
Dinitrogen trioxide	N2O3	s
Dinitrogen tetraoxide	N2O4	s
Nitrous acid	HNO2	s
Peroxynitrous acid	ONOOH	Fairly stable
Nitryl chloride	NO2Cl	s

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a The half life of some radicals depends on the environmental medium, for example the half life of NO• in an air saturated solution may be few minutes. S seconds, min minutes

Properties of Some Free Radicals

Superoxide Ion Radical (O∙−2) 

Superoxide anion radical is the most important widespread ROS formed by the enzymatic process, autooxidation reaction and by a nonenzymatic electron transfer reactions in which an electron is transferred to molecular oxygen [20]. It is mostly produced within the mitochondria and its reactivity with the biomolecules is low. The enzymes that can produce superoxide include xanthine oxidase [21], lipooxygenase, cyclooxygenase [22, 23] and NADPH dependent oxidase. It can exist in two forms such as O2•− or hydroperoxyl radical (HO2) at low pH [24]. The hydroperoxyl radical is the most important form and can easily enter the phospholipid bilayer than the charged form (O2•−). Under physiological pH the most occurring form is superoxide. It can act as reducing agent and it reduces iron complexes such as cytochrome-c and ferric-ethylene diaminetetraacetic acid (Fe+-EDTA), in which Fe+3 is reduced to Fe+2. It can also act as oxidizing agent and oxidize ascorbic acid and tocopherol.

일부 자유 라디칼의 특성
슈퍼옥사이드 이온 라디칼(O∙-2)

슈퍼옥사이드 음이온 라디칼은

효소 과정,

자가 산화 반응 및 전자가 분자 산소로 전달되는

비효소적 전자 전달 반응에 의해 형성되는

가장 중요한 광범위 ROS입니다[20].

주로

미토콘드리아 내에서 생성되며

생체 분자와의 반응성이 낮습니다.

슈퍼옥사이드를 생성할 수 있는 효소로는

크산틴 산화효소[21],

리포옥시게나제,

사이클로옥시게나제[22, 23] 및

NADPH 의존성 산화효소 등이 있습니다.

슈퍼옥사이드는

낮은 pH에서 O2 또는 하이드로페록실 라디칼(HO2)과 같은

두 가지 형태로 존재할 수 있습니다[24].

하이드로페록실 라디칼은

가장 중요한 형태이며

하전된 형태(O2--)보다 인지질 이중층에 쉽게 들어갈 수 있습니다.

생리적 pH에서 가장 많이 발생하는 형태는

슈퍼옥사이드입니다.

환원제로 작용할 수 있으며,

시토크롬-c 및

철-에틸렌 디아미네트라 아세트산(Fe+-EDTA)과 같은

철 복합체를 환원시켜

Fe+3을 Fe+2로 환원시킵니다.

또한

산화제로 작용하여

아스코르브산과

토코페롤을 산화시킬 수 있습니다.

O2+ e−→O∙−2

O2+Fe+2→Fe+3+O∙−2(auto-oxidation)

Superoxide radical react with another superoxide radical in a dismutation reaction (Eq. 1), in which one radical is oxidized to oxygen and other is reduced to hydrogen peroxide [25].

과산화물 라디칼은

다른 과산화물 라디칼과 변이 반응(식 1)을 통해 반응하는데,

이때 한 라디칼은 산소로 산화되고

다른 라디칼은 과산화수소로 환원됩니다[25].

O2+O∙−2+2H2O→Cu,Zn,Mn−SODH2O2+O2

1

Hydroxyl Radical (OH∙) 

Hydroxyl radical is the neutral form of hydroxide ion and is a highly reactive free radical [26]. It can strongly react with both organic and inorganic molecules including DNA, proteins, lipids, and carbohydrates and cause severe damage to the cells than any other ROS can do [27]. It is formed in a Fenton reaction (Eq. 2), in which H2O2 react with metal ions (Fe+2 or Cu+), often bound in complex with different proteins such as ferritin (an intracellular protein that stores iron) and ceruloplasmin (plasma copper carrying protein) or other molecules [28]. Under stress conditions, an excess of O2•−releases free iron from ferritin and the released free iron participates in Fenton reaction to form OH•. It is also formed by the reaction between superoxide radical and H2O2 in a reaction called Haber–Weiss reaction (Eq. 3) [29].

하이드록실 라디칼(OH∙)

하이드록실 라디칼은

수산화 이온의 중성 형태이며

반응성이 매우 높은 자유 라디칼입니다[26].

DNA, 단백질, 지질, 탄수화물을 포함한

유기 및 무기 분자 모두와 강력하게 반응할 수 있으며

다른 어떤 ROS보다 세포에 심각한 손상을 일으킬 수 있습니다[27].

이는 H2O2가

금속 이온(Fe+2 또는 Cu+)과 반응하는 펜톤 반응(식 2)에서 형성되며,

종종 페리틴(철을 저장하는 세포 내 단백질),

세룰로플라즈민(혈장 구리 운반 단백질) 또는

다른 분자와 같은 다른 단백질과 복합적으로 결합합니다[28].

스트레스 조건에서

과량의 O2--는 페리틴에서 유리 철을 방출하고

방출된 유리 철은 펜톤 반응에 참여하여

OH-를 형성합니다.

또한

하버-바이스 반응(식 3)이라는 반응에서

슈퍼옥사이드 라디칼과

H2O2의 반응에 의해 형성됩니다[29].

Fe+2 +  H2O2  →  Fe+3 +  OH∙ +  OH-(Fenton reaction)

2

O∙−2+H2O2→O2+OH∙+OH−(Haber-Weiss reaction)

3

Peroxyl Radical (ROO∙) 

It is derived from oxygen in living systems. The simplest form of peroxyl radical is perhydroxyl radical (HOO•) which is formed by the protonation of superoxide [30]. About 0.3 % of the total O2•− in the cytosol of a typical cell is in the protonated form. It initiates fatty acid peroxidation and also can promote tumor development [31].

퍼옥실 라디칼(ROO∙)

생명체의 산소로부터 파생됩니다.

가장 단순한 형태의 퍼옥실 라디칼은

과산화물의 양성자화에 의해 형성되는

퍼하이드록실 라디칼(HOO-)입니다[30].

일반적인 세포의 세포질에 있는

총 O2--의 약 0.3%가

양성자화된 형태입니다.

이는

지방산 과산화를 시작하고

종양 발생을 촉진할 수 있습니다 [31].

Hydrogen Peroxide (H2O2) 

Hydrogen peroxide is formed in vivo in a dismutation reaction catalyzed by the enzyme superoxide dismutase (SOD) (Eq. 1). It is not a free radical but it can cause damage to the cell at relatively low concentration (10 μM), but at higher levels, the cellular energy producing enzymes such as glyceraldehhyde-3-phosphate dehydrogenase are inactivated. It can easily penetrate the biological membranes. H2O2 has no direct effect on DNA but can damage DNA by producing hydroxyl radical (OH−) in the presence of transition metal ions [32]. The major antioxidant enzymes that can eliminate the H2O2 include catalase, glutathione peroxidase and peroxiredoxins [33, 34].

과산화수소(H2O2)

과산화수소는

생체 내에서 슈퍼옥사이드 디스뮤타제(SOD) 효소에 의해 촉매되는

돌연변이 반응으로 형성됩니다(식 1).

활성산소는 아니지만

비교적 낮은 농도(10μM)에서는 세포를 손상시킬 수 있지만,

높은 농도에서는 글리세랄데히드-3-포스페이트 탈수소효소와 같은

세포 에너지 생성 효소가 비활성화됩니다.

생체막을

쉽게 투과할 수 있습니다.

H2O2는

DNA에 직접적인 영향을 미치지는 않지만

전이 금속 이온이 있는 경우 하이드 록실 라디칼 (OH-)을 생성하여

DNA를 손상시킬 수 있습니다 [32].

H2O2를 제거할 수 있는 주요 항산화 효소로는

카탈라아제, 글루타치온 퍼옥시다제, 퍼옥시레독신 등이 있습니다[33, 34].

Singlet Oxygen (1O2) 

It is an electronically high excited, meta-stable state of molecular oxygen and is a highly reactive toxic reactive oxygen species [35]. Upon activation, the molecular oxygen is excited to first state 1Δg and then to next higher excited singlet state, 1εg. The first excited state, 1Δg, has two electrons with opposite spins in the same π* orbital whereas, the second excited state, 1εg, has one electron in each degenerated π* orbital with opposite spins [36]. The 1Δg state is extremely reactive, and compared to the other electronically excited states [36]. It is produced in vivo by the activation of neutrophils (Eq. 4) [37] and eosinophils [38]. It is also formed by some of the enzymatic reactions catalyzed by enzymes such as lipoxygenases [39], dioxygenases [40], and lactoperoxidase [41]. It is a highly potent oxidizing agent that can cause DNA damage [42] and tissue damage [38].

HOCl +  H2O2  →  1O2 +  H2O +  Cl-

4

Ozone (O3) 

Ozone is a powerful oxidant may be produced in vivo by antibody catalyzed water oxidation pathway which plays an important role in inflammation [43]. It can form free radicals and other reactive intermediates by oxidizing the biological molecules. It can cause lipid peroxidation [44] and oxidize different functional groups, for example, amine, alcohol, aldehyde and sulphydryl, present in proteins [45, 46] and nucleic acids [47]. It can also cause chromosomal aberrations which may be due to direct attack by O3 or by the free radicals generated by it [48].

Hypochlorous Acid (HOCl) 

It is a major oxidant produced by the activated neutrophils at the site of inflammation from hydrogen peroxide and chloride in a reaction catalyzed by the enzyme myeloperoxidase [49].

H2O2 +  Cl-  →  HOCl +  OH·

HOCl is a strong reactive species involved in oxidation and chlorination reactions. It can oxidize thiols and other biological molecules including, ascorbate, urate, pyridine nucleotides, and tryptophan [50, 51]. HOCl chlorinates several compounds such as amines to give chloramines; tyrosyl residues to give ring chlorinated products, cholesterol and unsaturated lipids to give chlorohydrins, and it can also chlorinate DNA [52].

Nitric Oxide or Nitrogen Monoxide (NO•) 

It is a small molecule generated in tissues by different nitric oxide synthases (NOS)s which convert L-arginine to L-citrulline [53]. In this reaction one of the terminal guanido nitrogen atoms undergo oxidation and produces NO.. Three types of isoforms of NOS such as neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS) are involved in the formation of the NO radical.

L−Arginine+O2+NADPH→NOSL−Citrulline+NO∙+NADP+

It is both aqueous and lipid soluble and therefore it readily diffuses through cytoplasm and plasma membrane [54]. The NO• is an important intracellular second messenger stimulates guanylate cyclase and protein kinases and helps in smooth muscle relaxation in blood vessels. It is identical to endothelium derived relaxing factor (EDRF) produced by vascular endothelial cells which is an important mediator of vascular responses [55]. It can also act as an important cellular redox regulator [56] and regulate enzymatic activity by nitrosylating the proteins [57]. Since it is involved in many biological activities like blood pressure regulation, smooth muscle relaxation, neurotransmission, defensive mechanisms and immune regulation, this molecule was regarded as molecule of the year 1992 [58].

Peroxynitrite (OONO−) and Other Reactive Nitrogen Species 

Peroxynitrite (OONO−) is formed by the reaction between O∙−2 and NO∙. It is highly toxic [59] and can directly react with CO2 to form other highly reactive nitroso peroxo carboxylate (ONOOCO2−) or peroxynitrous acid (ONOOH). The ONOOH further undergo homolysis to form both OH• and NO2 or rearrange to form NO3. OONO- can oxidize lipids, oxidize methionine and tyrosine residues in proteins and oxidizes DNA to form nitroguanine [60]. The nitrotyrosine residues are considered as marker of peroxynitrite induced cellular damage [61].

NO reacts with O2 and water to form nitrate and nitrite ions. One electron oxidation of NO• results in nitrosonium cation (NO∙+2) while on electron reduction results in nitroxyl anion (NO−). These two ions can react with NO and form N2O and OH∙. NO∙ can react with a variety of radicals such as H2O2 and HOCl to form N2O3, NO2− and NO3− [62].

Sources of Free Radicals

The ROS can be produced from either endogenous or exogenous sources. The endogenous sources of ROS include different cellular organs such as mitochondria, peroxisomes and endoplasmic reticulum, where the oxygen consumption is high.

Mitochondria 

Most of the intracellular ROS are derived from mitochondria (Fig. 1). The superoxide radicals are produced at two major sites in the electron transport chain, namely complex I (NADH dehydrogenase) and complex III (ubiquinone cytochrome c reductase). The transfer of electrons from complex I or II to coenzyme Q or ubiquinone (Q) results in the formation of reduced form of coenzyme Q (QH2). The reduced form QH2 regenerates coenzyme Q via an unstable intermediate semiquinone anion (∙Q-) in the Q-cycle. The formed ∙Q- immediately transfers electrons to molecular oxygen leading to the formation of superoxide radical. The generation of superoxide is non-enzymatic and therefore higher the metabolic rate, the greater is the production of the ROS [63].

자유 라디칼의 공급원

활성산소는

내인성 또는 외인성 소스에서 생성될 수 있습니다.

활성 산소의 내인성 공급원에는

산소 소비량이 많은 미토콘드리아,

퍼옥시좀 및 소포체와 같은 다양한 세포 기관이 포함됩니다.

미토콘드리아
세포 내 ROS의 대부분은

미토콘드리아에서 파생됩니다(그림 1).

슈퍼옥사이드 라디칼은

전자 전달 사슬의 두 가지 주요 부위,

즉 복합체 I(NADH 탈수소효소)과

복합체 III(유비퀴논 시토크롬 C 환원효소)에서 생성됩니다.

복합체 I 또는 II에서

코엔자임 Q 또는 유비퀴논(Q)으로 전자가 이동하면

환원된 형태의 코엔자임 Q(QH2)가 형성됩니다.

환원된 형태의 QH2는

Q-사이클에서 불안정한 중간 세미퀴논 음이온(∙Q-)을 통해

코엔자임 Q를 재생성합니다.

형성된 ∙Q-는

즉시 전자를 분자 산소로 전달하여

슈퍼옥사이드 라디칼을 형성합니다.

슈퍼옥사이드의 생성은

비효소적이기 때문에

대사율이 높을수록 ROS의 생성량도 증가합니다 [63].

Fig. 1

Mitochondrial ROS production

The superoxide anion is converted to hydrogen peroxide by the action of mitochondrial superoxide dismutase (MnSOD). H2O2 can be detoxified by the Catalase (CAT) and glutathione peroxidase (GPx).

The other mitochondrial components which contribute to the formation of ROS include monoamino oxidase, α-ketoglutarae dehydrogenase, glycerol phosphate dehydrogenase and p66shc [64].

The p66Shc is an important member of the ShcA protein family, which contains another two more proteins, p46Shc and p52 Shc. The mammalian p66Shc is a 66-kDa isoform of the growth factor adaptor protein involved in apoptosis. It mediates the production of ROS in mitochondria. Most of the p66Shc is located in cytoplasm with a small fraction localized in the mitochondrial intermembrane space. Upon oxidative stress, p66Shc translocates to mitochondrial intermembrane space, where it associates with cytochrome-c, thus inducing ROS generation [65].

미토콘드리아 ROS 생성

과산화물 음이온은

미토콘드리아 슈퍼옥사이드 디스뮤타제(MnSOD)의 작용에 의해

과산화수소로 전환됩니다.

H2O2는

카탈라아제(CAT)와 글루타치온 퍼옥시다제(GPx)에 의해 해독될 수 있습니다.

ROS 형성에 기여하는 다른 미토콘드리아 성분으로는 모노아미노 옥시다아제, α-케토글루타래 탈수소효소, 글리세롤 인산염 탈수소효소 및 p66shc가 있습니다 [64].

p66Shc는 ShcA 단백질 계열의 중요한 구성원으로, 다른 두 가지 단백질인 p46Shc와 p52Shc를 더 포함하고 있습니다. 포유류의 p66Shc는 세포 사멸에 관여하는 성장 인자 어댑터 단백질의 66-kDa 동형 단백질입니다. 이 단백질은 미토콘드리아에서 ROS 생성을 매개합니다. 대부분의 p66Shc는 세포질에 존재하며 소량은 미토콘드리아 막간 공간에 국한되어 있습니다. 산화 스트레스를 받으면 p66Shc는 미토콘드리아 막간 공간으로 이동하여 사이토크롬-c와 결합하여 ROS 생성을 유도합니다[65].

Peroxisomes 

In peroxisomes the respiratory pathway involves the transfer of electrons from various metabolites to the oxygen leads to H2O2 formation [66], but is not coupled to oxidative phosphorylation to produce ATP instead free energy is released in the form of heat. The other free radicals produced in peroxisomes include H2O2, O2•− OH• and NO•. The β-oxidation of fatty acids is the major metabolic process producing H2O2 in the peroxisomes. As reviewed elsewhere, the different peroxisomal enzymes such as acyl CoA oxidases, D-amino acid oxidase, L-α-hydroxy oxidase, urate oxidase, xanthine oxidase, D-aspartate oxidase have been shown to produce different ROS [67] [see Table 2].

퍼옥시좀

퍼옥시좀에서 호흡 경로는

다양한 대사 산물에서 산소로 전자가 전달되어

H2O2가 형성되지만[66],

산화 인산화와 결합하여 ATP를 생성하는 대신

자유 에너지가 열의 형태로 방출됩니다.

퍼옥시좀에서 생성되는

다른 자유 라디칼로는

H2O2, O2-- OH- 및 NO-가 있습니다.

지방산의 β-산화는

퍼옥시좀에서 H2O2를 생성하는 주요 대사 과정입니다.

다른 곳에서 검토한 바와 같이,

아실 CoA 산화효소,

D-아미노산 산화효소,

L-α-하이드록시 산화효소,

요산 산화효소,

크산틴 산화효소,

D-아스파르트산염 산화효소 등

다양한 퍼옥시솜 효소가

서로 다른 ROS를 생성하는 것으로 나타났습니다[67] [표 2 참조].

Table 2

ROS producing enzymes in peroxisomes

EnzymeSubstrateROS

Acyl CoA-oxidases (enzymes of β-oxidation)	Fatty acids	H2O2
D-amino acid oxidase	d-proline	H2O2
L-α-hydroxy oxidase	Glycolate	H2O2
Urate oxidase	Uric acid	H2O2
d-aspartate oxidase	d-aspartate	H2O2
Xanthine oxidase	Xanthine	O2•−, H2O2

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Endoplasmic Reticulum 

The enzymes of endoplasmic reticulum such as cytochrome p-450 and b5 enzymes and diamine oxidase contribute to the formation of ROS [68]. Another important thiol oxidase enzyme, Erop1p catalyses the transfer of electrons from dithiols to molecular oxygen results in the formation of H2O2 [69].

The other endogenous sources of ROS include prostaglandin synthesis, auto-oxidation of adrenalin, phagocytic cells, reduced riboflavin, FMNH2, FADH2, cytochrome P 450, immune cell activation, inflammation, mental stress, excessive exercise, infection, cancer, aging, ischemia etc. [68].

On the other hand, ROS are also produced in the biological systems by various exogenous sources shown in Table 3 [10].

소포체

사이토크롬 p-450 및 b5 효소와

디아민 산화 효소와 같은 소포체의 효소는

ROS의 형성에 기여합니다 [68].

또 다른 중요한 티올 산화 효소인

Erop1p는

디티올에서 분자 산소로의 전자의 이동을 촉매하여 H2O2를 형성합니다[69].

ROS의 다른 내인성 공급원으로는

프로스타글란딘 합성,

아드레날린의 자가 산화,

식세포,

리보플라빈 감소,

FMNH2, FADH2, 사이토크롬 P 450, 면역 세포 활성화, 염증, 정신적 스트레스, 과도한 운동, 감염, 암, 노화, 허혈 등이 있습니다. [68].

한편, 표 3에 표시된 다양한 외인성 소스에 의해 생물학적 시스템에서도 ROS가 생성됩니다 [10].

Table 3

ROS generated from exogenous sources

Air & water pollution	Ultraviolet light
Alcohol	Cooking (smoked meat, used oil, fat) Drugs such as Halothene, Paracetamol, Bleomycine, Doxorubicin, Metrenidazole, Ethanol. CCl4
Tobacco smoke
Transition metals- Cd, Hg, Pb, As
Heavy metals- Fe, Cu, Co, Cr
Industrial solvents
Pesticides
High temperature

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Molecular targets of free radicals 

When there is an imbalance between the free radical production (ROS/RNS) and antioxidant defenses, the former will be produced in higher concentrations leading to oxidative stress and nitrosative stress. Since these free radicals are highly reactive, they can damage all the three important classes of biological molecules including nucleic acids, proteins, and lipids [70].

Deoxyribonucleic Acid (DNA) 

Both ROS/RNS can oxidatively damage the nucleic acids. The mitochondrial DNA is more vulnerable to the ROS attack than the nuclear DNA, because it is located in close proximity to the ROS generated place. ROS, most importantly, the OH• radical directly reacts with all components of DNA such as purine and pyrimidine bases, deoxyribose sugar backbone [71] and causes a number of alternations including single and double stranded breaks in DNA. The OH∙ radical abstracts hydrogen atoms to produce a number of modified puine as well as pyrimidine base by-products and DNA- protein cross links. The pyrimidine attack by OH• produces different pyrimidine adducts like thymine glycol, uracil glycol, 5-hydroxydeoxy uridine, 5-hydroxy deoxycytidine, hydantoin and others. The purine adducts formed by hydroxyl radical attack include, 8-hydroxydeoxy guanosine, 8-hydroxy deoxy adenosine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine. The other free radical induced adducts of DNA bases include, 5-formyl uracil, cytosine glycol, 5,6-dihydrothyronine, 5-hydroxy-6-hydro-cytosine, 5-hydroxy-6-hydro uracil, uracil glycol, and alloxan [72]. The major free radical induced adducts of the sugar moiety in DNA include glycolic acid, 2-deoxytetrodialdose, erythrose, 2-deoxypentonic acid lactone, 2-deoxypentose-4-ulose [72]. 8-hydroxy deoxyguanosine is considered as the biomarker of oxidative DNA damage and is involved in mutagenesis, carcinogenesis and ageing. The levels of 8-OHdG are higher in mitochondrial DNA than in nuclear DNA [73].

On the other hand, the RNS, most importantly, peroxynitrite (OONO−) interacts with guanine to produce nitrative and oxidative DNA lesions such as 8-nitroguanine and 8-oxodeoxyguanosine respectively [74]. 8-nitroguanine formed is unstable and can be spontaneously removed, resulting in the formation of an apurininc site [75]. Conversely adenine can be paired with 8-nitroguanine during DNA synthesis resulting in a G-T transversions [76]. Accordingly 8-nitroguanine is a mutagenic DNA lesion involved in carcinogenesis.

Ribonucleic acid (RNA) 

ROS can attack different RNAs produced in the body. The RNA is more prone to oxidative damage than DNA, due to its single stranded nature, lack of an active repair mechanism for oxidized RNA, less protection by proteins than DNA and moreover these cytoplasmic RNAs are located in close proximity to the mitochondria where loads of ROS are produced. Indeed, RNA is subjected to more oxidative damage than DNA in humans [77]. 7, 8-dihydro-8-oxo-guanosine (8-oxoG) is the most extensively studied RNA damage product and its levels are raised in various pathological conditions like Alzheimer’s disease [78], Parkinson’s disease [79], atherosclerosis [80], hemochromastosis [81] and myopathies [82].

Lipids 

The membrane lipids, especially the polyunsaturated fatty acid residues of phospholipids are more susceptible to oxidation by free radicals [83]. The lipid peroxidation is very important in vivo because of its involvement in various pathological conditions. The lipid peroxidation results in the loss of membrane functioning, for example, decreased fluidity, inactivation of membrane bound enzymes and receptors [84]. The lipid peroxidation is initiated, when any free radical attacks and abstracts hydrogen from a methylene groups (CH2) in a fatty acid (LH) which results in the formation of a carbon centered lipid radical (L•). The lipid radical can react with molecular oxygen to form a lipid peroxyl radical (LOO•). The resultant lipid peroxyl radical (LOO•) undergo rearrangement via a cyclisation reaction to form endoperoxides, which finally form malondialdehyde (MDA) and 4-hydroxyl nonenal (4-HNA), the toxic end products of lipid peroxidation that cause damage to the DNA and proteins [85]. These lipid peroxyl radicals can further propagate the peroxidation process by abstracting hydrogen atoms from the other lipid molecules. Isoprostanes (prostaglandin like substances produced by in the body by the esterfication of arachidonic acid) constitute the important product of lipid peroxidation of arachidonic acid and are considered as the makers of the oxidative lipid damage [86].

Proteins 

The protein oxidation can be induced by radical species such as O2•−, OH•, peroxyl, alkoxyl, hydroperoxyl as well as by the non radical species such as H2O2, O3, HOCl, singlet oxygen, OONO- [87]. ROS oxidize different amino acids present in the proteins, causing formation of protein–protein cross linkages, results in the denaturing and loss of functioning of proteins, loss of enzyme activity, loss of function of receptors and transport proteins [88]. The sulphur containing amino acids such as methionine and cysteine are more susceptible to oxidation by ROS and are converted to disulphides and methionine sulphoxide [89, 90] respectively. However in biological systems, only these two oxidized forms of proteins can be converted back to their native form by two different enzymes namely disulfide reductases and methionine sulfoxide reductases respectively [91–94].

The ROS mediated attack of different amino acids results in the formation of different oxidation products such as, tryptophan forms nitrotryptophan, kynurenine, formylkynurinine; Phenylalanine forms 2,3-Dihydroxyphenylalanine, 2-, 3-, and 4-hydroxyphenylalanine; Tyrosine forms 3,4-Dihydroxyphenylalanine, tyrosine–tyrosine cross-linkages, Tyr-O-Tyr, cross-linked nitrotyrosine; Histidine forms 2-Oxohistidine, asparagine, aspartic acid; Arginine forms glutamic semialdehyde; Lysine forms a-Aminoadipic semialdehyde; Proline forms 2-Pyrrolidone, 4- and 5-hydroxyproline pyroglutamic acid, glutamic semialdehyde; threonine forms 2-Amino-3-ketobutyric acid; leucine and valine residues form hydroxyl residues [91].

The ROS induced oxidative damage of amino acid residues such as lysine, proline, threonine and arginine yields carbonyl derivatives. The presence of carbonyl groups in proteins has been considered as the marker of ROS mediated protein oxidation [95]. The other specific markers of protein oxidation are O-tyrosine (a marker for hydroxyl radical) and 3-nitrotyrosine (a marker for RNS). An increase in the levels of protein carbonyls is observed in a number of pathological conditions such as, Alzheimer’s disease [96], parkinson’s disease [97], muscular dystrophy [98], cataractogenesis [99], Rheumatoid Arthritis [100], diabetes [101], progeria, atherosclerosis, respiratory dystrous syndrome, Werner’s syndrome [91], and ageing [96, 102].

Free Radicals and Diseases

Free radicals are involved in many pathological conditions such as many types of diabetes, neurodegenerative diseases, cardiovascular diseases (CVDs), cancer, cataracts, asthma, rheumatoid arthritis, inflammation, burns, intestinal tract diseases, progerias and ischemic and post-ischemic pathologies. The role of free radicals in some of the important disease conditions (see Fig. 2) is discussed in this section.

Fig. 2

Pathological role of free radicals

Diabetes Mellitus

Diabetes mellitus is heterogeneous group of chronic disorders characterized by enhanced blood glucose levels (hyperglycemia) resulting from defective insulin secretion (in type I diabetes), resistance to insulin action (in type II diabetes) or both [103]. The major symptoms are thirst, hunger, emaciation, and weakness, eventually lead to coma. DM is associated with the increased production of free radicals or decreased activity of the antioxidant systems, which leads to development of oxidative stress [104, 105]. The hyperglycemic condition induces increased free radical production via four different routes namely, 1) increased glycolysis, results in increased ratio between the rate of oxidation of G3P to 1, 3-DPG, following increased NADH/NAD+ ratio (redox imbalance) 2) activated sorbitol (or polyol) pathway, causes the accumulation of both sorbitol and fructose, results in decreased reduced GSH and increased NADH/NAD+ ratio. 3) autoxidation of glucose, results in the generation of different free radicals such as H2O2, OH•, O2•− and ketoaldehydes and 4) non enzymatic protein glycation, results in the formation of AGEs which upon interacting with RAGEs generate oxidative stress [106].

Both mitochondrial and non-mitochondrial derived ROS contribute to oxidative stress during DM. Under normal conditions, the electron transport chain complexes I and III are the key sites of superoxide production [107]. However, the increased glucose levels in DM lead to increased glycolysis resulting in the augmented generation of pyruvate, thus raising the inner mitochondrial membrane potential upwards, followed by mitochondrial dysfunction and increased ROS production at electron transport chain complex II [108].

Neurodegenrative Diseases 

The central nervous system (CNS) is particularly susceptible to the oxidants due to the presence of high lipid content, high consumption of oxygen, and low levels of antioxidant enzymes, for example, SOD is localized primarily in neurons, and GSH and GPx are localized in astrocytes [109]. The lipid peroxidation by ROS leads to progressive loss of membrane fluidity, decreases membrane potential, and increases permeability to ions such as Ca2+. The regions of the brain such as hippocampus, substantia nigra, and the striatum are particularly susceptible to attack by free radicals [110, 111]. The oxidative-stress state has been also implicated in several neurodegenerative diseases such as Alzheimer’s [112], Parkinson’s [113], Huntington’s, lateral amyotrophic sclerosis [114], and multiple Sclerosis [115].

Parkinson’s Disease (PD) 

Parkinson’s disease (PD) is characterized by the loss of dopaminergic neurons (involve in learning, memory and motor control), especially in the midbrain area called the substantia nigra, accompanied by deposition of inclusion bodies (Lewy bodies) of α-synuclein. The redox imbalance causes oxidative damage to these neurons and begins to alter the synthesis and metabolic pathway of dopamine leads to a further increase in oxidative stress because of quinine formation [111]. The characteristic clinical symptoms of PD include, jerky movements, trembling of the hands and lips, and tremors [116]. Dopamine, a neurotransmitter, can also act as a metal chelator, has the ability to generate H2O2 via Fenton reaction. Ceruloplasmin (an extracellular ferroxidase required for regulating cellular iron load and transport) oxidation results in the decreased ferroxidase activity followed by the accumulation of intracellular iron in neurons in PD. The increased levels of Fe+3 mediate the production of hydroxyl radicals, results in the damage of dopaminergic neurons in PD [117].

Alzheimer’s Disease (AD) 

Alzheimer’s disease (AD) is characterized by the accumulation of amyloid protein plaques (formed from the improper folding and processing of amyloid β precursor protein-ABPP) [168] and intracellular neurofibrillary tangles made up of abnormal and hyperphosphorylated tau protein [118]. The hyperphosphorylated tau protein aggregates binds to Fe3+, results in the production of neurofibrillary tangles [119]. The Amyloid-β peptide (Aβ) can chelate with transition metal ions (Cu2+, Zn2+ and Fe3+), and produce H2O2 via transition metal mediated catlysis and finally gives toxic OH˙ radical [120]. The lipid peroxidation is also extensive in AD patients, which can induce neuronal death by multiple mechanisms such as impairment of function of ion pumps (both Na+/K+-ATPase and Ca+2-ATPase), glucose transporters and glutamate transporters. The other oxidative markers of protein damage such as protein carbonyls and 3-nitrotyrosine have been also observed in AD patients [118].

Multiple Sclerosis (MS) 

MS is an autoimmune neuronal disorder characterized by impaired nerve conduction due to demylination of central nervous system (CNS). The activated microglia/macrophages initiate the MS by the generation of ROS [121] that can induce lipid peroxidation, results in the demylination and damage of neurons. Elevated TBARS levels and reduced protein SH groups, the representatives of protein oxidation and slightly reduced SOD was reported in MS patients [122]. Apart from ROS generation, an impaired iron metabolism has been also considered to play a major role in pathogenesis of disease.

Cancer

It is one of the leading causes of death in humans. Free radicals cause different types of chemical changes in DNA, thus they could be mutagenic and involved in the etiology of cancer [123, 124]. Cancer cells in particular, in comparison to normal cells, have higher levels of ROS and are more susceptible to mitochondrial dysfunction due to their higher metabolic rate [125]. Cancer cells display elevated levels of oxidative stress due to activation of oncogenes and loss of tumor suppressors [126]. ROS by altering the growth signals and gene expression‎ cause continuous proliferation of cancer cells [127]. ROS can damage DNA by inducing base modifications, deletions, strand breakage, chromosomal rearrangements and hyper- and hypo-methylation of DNA [128].

Colorectal Cancer 

Colorectal cancer (CRC) is the third most common cancer worldwide, accounting for 608,000 deaths per year [129]. The gastrointestinal tract, particularly the colon and rectum, is continuously exposed to ROS originating from both endogenous and exogenous sources [130]. Colon cancer originates from the epithelial cells that line the bowel. These cells divide rapidly and have a high metabolic rate [131]. Since the intestinal mucosa is constantly confronting with diet and bacterial-derived oxidants and carcinogens, an unrestrained production of free radicals, redox imbalance, and DNA damage occurs, finally leads to an altered intestinal metabolic homeostasis with cancer as an endpoint [133]. The human colorectal tumors have increased levels of nitric oxide (NO) [132], 8-oxodG in DNA [133], and lipid peroxides [134]. Suzuki et al. 2004 [135] have reported increased serum levels of oxidized low density lipoprotein in patients with CRC compared to healthy individuals.

Breast Cancer 

Damage to the breast epithelium by ROS can lead to fibroblasts proliferation, hyperplasia of epithelium, cellular atypia and breast cancer [136]. In majority of breast carcinomas the oxidative stress can be induced by the over expression‎ of thymidine phosphorylase enzyme which catabolizes thymidine to thymine and 2-deoxy-D-ribose-1-phosphate; the latter is a powerful reducing sugar that rapidly glycates proteins, generating oxygen radicals within the carcinoma cell [137]. Another breast specific mechanism of oxidative stress induction involves a mammary gland specific lactoperoxidase enzyme catalyzed one electron oxidation of 17-β-oestradiol to a reactive phenoxyl radical [138].

Prostate Cancer 

The ROS produced are responsible for the cellular proliferation of prostate cancer cells [139]. Overexpression‎ of NADPH oxidase 1 (Nox1) protein is an early event in the development of prostate cancer. Prostrate Tumors have considerably higher levels of ROS and Nox1 levels [140]. The superoxide produced (by NOX) in prostate cancer cells facilitates cellular immortality through resistance to programmed cell death which results in cancer-promoting effect [141]. Kumar et al. 2008 [142] have reported elevated ROS levels in prostate cancer cells compared with normal cells. Veeramani et al. 2008 [143] have reported that elevated levels of ROS might also be formed by the simultaneous increased levels of p66Shc protein in prostate cancer cells.

Lung Cancer 

Lung cancer has been the most commonly diagnosed cancer and is the central cause of cancer death in men worldwide [144]. Lung cancer mortality account 30 % of all cancer related deaths. Oxidative stress plays an important role in lung inflammation and lung cancer [145]. Cigarette Smoking is the most crucial environmental risk factor in lung cancer etiology. It is estimated that smoking accounts for ~80 % of global lung cancer burden in males and 50 % in females [146]. Cigarette smoke particulate matter contains complex mixture of numerous carcinogens and stable ROS with very long half-lives. These ROS can damage the tissues resulting in progressive transformation of cells into the malignant form, which leads to increased frequency of mutations by the oxidative damage to DNA and, eventually leading to lung cancer [147]. Smokers develop lung cancer a 10-fold higher than non-smokers. Lung cancer (LC) and chronic obstructive pulmonary disease (COPD) commonly coexist in smokers, and the presence of COPD increases the risk of developing LC [148].

Bladder Cancer 

Bladder cancer is one of the most common cancers across the world, ranking the fourth and tenth in men and women, respectively [149]. The most common risk factors for bladder cancer are cigarette smoking, exposure to industrial carcinogens (aromatic amines), high levels of arsenic intake and diet [150]. Oxidative stress critically contributes to the development of bladder cancer [151]. Various lines of evidence reported an increased oxidative stress in patients with breast cancer [152, 153]. Increased NO levels have been reported in bladder cancer patients [154]. This NO stimulates matrix metalloproteinases (MMPs), especially prolidase activity, which is involved in the terminal step of collagen degradation. Significantly higher serum prolidase activities were reported in patients with bladder cancer than healthy controls [155]. Therefore, increased prolidase activity may, in part, play a role in the pathogenesis of bladder cancer.

Epidemiological studies reveal that low levels of antioxidants are associated with an increased risk of cancer. Significant increase in total oxidant status levels and decrease in total antioxidant status were observed in patients with bladder cancer [156]. Significantly lower levels of plasma protein, total thiol groups and protein-bound thiol groups and elevated levels of Protein carbonyl groups were observed in bladder cancer patients than in healthy controls [157].

Cardiovascular Diseases (CVDs) 

Cardiovascular diseases are a class of pathologies involving the heart and blood vessels (arteries, capillaries, and veins). They include cardiac diseases, vascular diseases of the brain and kidney, and peripheral arterial disease. Most of the people are dying due to CVDs compared to other diseases [158].

Atherosclerosis 

Atherosclerosis is a condition commonly referred to as hardening of the arteries. Hyperlipidemia is a major risk factor for atherosclerosis. Elevated levels of oxidized low density lipoprotein (LDL), glucose and free fatty acids are found in patients with atherosclerosis, T2D, and obesity [159]. A profound imbalance of oxidants and antioxidants resulting in oxidative stress is observed in atherosclerosis. In the vessel wall, endothelial cells, smooth muscle cells (SMCs) and macrophages are sources of free radicals [160]. Endothelial dysfunction leads to increased endothelial permeability, up regulation of endothelial adhesion molecules, and inflammatory cell infiltration into the arterial wall [160]. A substantial data has been shown that ROS are involved in endothelial injury, dysfunction, and lesion progression [161]. The ROS dependent activation of the MMPs results in the degradation of intimal extracellular matrices and promotes smooth muscle cell migration [162]. Cigarette smoking contain large amount of free radicals and may down-regulate key exogenous and endogenous antioixdants such as vitamin-D, carotenes, GPx and SOD and can lead to the dysfunction of monocytes and vascular smooth muscle cells [163]. The proatherogenic agents such as oxidaised lipids, high glucose and cigarette constituents give rise to increased free radical production.

The endothelial nitric oxide synthase (eNOS) derived NO. plays an important role in maintaining vascular tone and vasoreactivity, vasodialation, platelet aggregation and in maintaining balance between smooth muscle cell growth and differentiation. Decreased NO bioavailabilty is the one of the major feature of the CVDs [249]. During reduced availability of BH4 or L-Arg, eNOS becomes uncoupled from a NO∙ to a O∙−2 state [164]. The O2•− formed can interact with NO• to produce ONOO-, a damaging and cytotoxic free radical with potential to disturb cardiovascular function. ONOO- reduces the bioavailability of NO leading to reduced endothelial vascular regulatory capacity and increased vascular dysfunction. ONOO− also inactivates the BH4 cofactor effectively amplifying the damaging effects of eNOS uncoupling the endothelium [164]. Mitochondrial DNA damage is frequently observed in human atherosclerosis in both circulating and vessel wall cells [165]. Oxidative stress mediated damaged mitochondrial DNA that escape autophagy induces a potent inflammatory response in atherosclerosis [166]. Malfunction of DNA repair leads to defects in cell proliferation, apoptosis, and mitochondrial dysfunction, which in turn leads to ketosis, hyperlipidemia, and increased fat storage, promoting atherosclerosis and the metabolic syndrome [167].

Hypertension (HT) 

Hypertension (HT) is a major health problem worldwide account 40 % of the total adult population [168]. Persons with hypertension are at an increased risk for stroke, heart disease, kidney failure, and premature mortality. Free radical induced oxidative stress in part contributes to endothelial dysfunction and development of hypertension [169]. Increased ROS generation eliminates NO• by forming ONOO-, thus reducing NO• bioavailability which leads to decreased endothelium-dependent vasodilation resulting in hypertension [170]. A decrease in NO bioavailability and an increase in oxidative stress are present in human hypertension [171]. Oxidation-induced impairment of NO also results in reduced opposition to the vasoconstrictive and hypertensive effects of angiotensin II. Angiotensin II decreases NO bioavailability by promoting oxidative stress [172].

Cataract

It is the most common cause of the visual impairment affecting about 25 million people throughout the world, with the highest incidence occurring in developing countries [173, 174]. It is characterized by opacity of the eye lens that reduces the amount of incoming light and results in visual impairment [173]. Although a number of factors such as genetic factors, diabetes, aging, smoking, drugs, malnutrition, radiation (x-rays and UV rays) and alteration in both endocrine and enzymatic equilibrium have been implicated in cataract formation [175], the free radical induced oxidative stress is considered as one of the major underlying mechanism of cataract disorder [176]. Oxidation of proteins, lipids and DNA is seen in cataract lenses. Proteins lose sulfhydryl (–SH) groups become cross linked by non disulfide bonds, form high molecular aggregates and become insoluble [177]. The oxidative stress induced lipid peroxidation toxic product such as HNE induce the fragmentation of lens proteins contributing towards the opacity of the lens [178]. Oxidative stress has been shown to induce lens opacification both in experimental animal models and cultured lens systems [179].

The cornea absorbs the light in the range of above 300 nm results in the activation of tryptophan, to form N-formyl kynurenine, 3-hydroxy kynurenine and other photoproducts [180, 181]. These photoproducts gradually accumulate in the centre of the lens are capable of generating singlet oxygen which induce protein damage leading to the loss of transparency [181]. Cataract lens have an intracellular ionic imbalance (i.e. altered ionic homeostasis) than normal lens. The ROS induced by UV rays in sunlight alters the ionic homeostasis results in the increased levels of Ca+2 and Na+2, coupled with the decreased levels of Mg+2 and K+2 in the lens [182]. The increased calcium activates calpains, a family of calcium dependent non-lysosomal cysteine proteases [175], which degrade lens proteins such as both α and β crystalline proteins results in opaque lens characteristic of cataract [183]. Elevated levels of H2O2 were observed in cataract lenses than normal lenses [178, 184].

Rheumatoid Arthritis (RA)

RA is chronic multisystem disease of unknown cause which affects approximately 1–2 % of the total world population and women are affected more than men [185]. The disease is characterized by synovial and systemic inflammation with joint swelling, morning stiffness, destruction of articular tissues, joint deformities, fatigue, loss of appetite and weakness [186–189]. It is believed to be a T-lymphocyte driven disease in which a sudden influx of T-cells into the affected joints is followed by an increased number of macrophages and fibroblasts drawn the release of cytokines particularly IL-1 and TNF-alpha. This cytokine release and subsequent migration is thought to be responsible for the chronic inflammation and characteristic changes in RA [188].

Several lines of evidence suggest a role for oxidative stress in the pathogenesis of RA. Both ROS and RNS damage cartilage. Tissue injury in inflammation results in NO. production by articular chondrocytes and synovial fibroblasts and elevated levels of NO. are observed in the serum and synovial fluid of RA patients [190]. The free radicals, particularly NO∙ and O2•−, inhibit the synthesis of matrix components like proteoglycans by chondrocytes and also damage the extracellular matrix through activation and up regulation of matrix metalloproteinases [191]. The HOCl, produced by myeloperoxidase (MPO) in neutrophils, chlorinate the tyrosine residues to form 3-chlorotyrosine and damage the collagen, thus implicated in arthritogensis. RA patients have increased plasma MPO concentrations [186].

Elevated levels of MDA, NO•, protein carbonyls, oxidized hyaluronic acid and oxidized LDL have been reported in RA patients [192–195]. These oxidized LDL can be ingested in large quantities by monocytes results in the formation of Foam cells that are present in atherosclerotic plaques of vessles and have also been found in RA synovial fluid [196]. Cigarette smoking is also considered as the most established environmental factor for RA. Both particulate and gaseous phases of smoke contain high concentrations of free radicals that can interact with DNA and could cause genetic mutations and activation responsible for the development of RA [197].

Asthma

Free radicals are involved in various respiratory diseases such as respiratory distress syndrome, chronic obstructive pulmonary disease, chronic bronchitis, asthma [198]. Asthma is the most common disorders of the airways of the lungs and is one of the major global health problems [199]. It is characterized by chronic inflammation of the airways involving variable and recurrent airflow obstruction and bronchial hyperreactivity associated with airway remodelling [200, 201]. Airway remodeling is a dynamic process involving mucous hypersecretion, collagen deposition, wall thickening, myocyte hypertrophy and hyperplasia, myofibroblast hyperplasia, vascular proliferation and alterations in airway elastic fibers, all of which culminate in persistent structural alterations of the airway [202]. NO is endogenously produced in mammalian airways by NOS and is known to regulate many aspects of human asthma, including modulation of airway and vascular smooth muscle tone and the inflammation. Increased production of airway NO is a key factor in the development of airway hyperresponsiveness [203].

ROS are produced both intracellularly by lung parenchymal cells and extracellularly by lung macrophages. Increased generation of oxidants have been reported in asthma patients than in healthy individuals [204] which provoked airway inflammation by inducing diverse pro-inflammatory mediators including macrophages, neutrophils and eosinophils [205]. Numerous studies have suggested that oxidative stress is caused by overproduction of various free radicals or by an insufficient antioxidant defense system in asthma and thus it contributes to the tissue damage which is induced by inflammatory cells [206]. Elevated levels of oxidative stress markers such as H2O2, 8-isoprostane, nitric oxide, and carbon monoxide were reported in exhaled air of asthmatic patients [204]. Increased MDA levels, and Protein carbonyls; decreased protein sulfhydryl and antioxidant activity were observed in plasma, bronchoalveolar lavage (BAL) fluid and exhaled air of asthamatic patients [207–209].

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Conclusion

Free radicals (ROS/RNS) are produced by normal metabolism and are involved in various physiological and pathological conditions. When there is an imbalance between the antioxidants and oxidants, the fee radicals accumulate leading to vigorous damage to macromolecules such as nucleic acids, proteins and lipids. This leads to tissue damage in various disease conditions such as diabetes mellitus, neurodegenerative diseases, cancer, cardiovascular diseases, cataracts, rheumatoid arthritis, asthma etc. and thus severely hastening the disease progression.

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Contributor Information

Alugoju Phaniendra, Email: moc.liamg@a.ardneinahp.

Dinesh Babu Jestadi, Email: moc.limag@idatsej.hsenid.

Latha Periyasamy, Email: ni.oc.oohay@inamavles.ahtal.

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