Reactive oxygen species

In chemistry and biology, reactive oxygen species (ROS) are highly reactive chemicals formed from diatomic oxygen (O₂), water, and hydrogen peroxide. Some prominent ROS are hydroperoxide (H₂O₂), superoxide (O₂⁻),^[1] hydroxyl radical (OH^.), and singlet oxygen(¹O₂).^[2] ROS are pervasive because they are readily produced from O₂, which is abundant. ROS are important in many ways, both beneficial and otherwise. ROS function as signals, that turn on and off biological functions. They are intermediates in the redox behavior of O₂, which is central to fuel cells. ROS are central to the photodegradation of organic pollutants in the atmosphere. Most often however, ROS are discussed in a biological context, ranging from their effects on aging and their role in causing dangerous genetic mutations.

ROS are not uniformly defined. All sources include superoxide, singlet oxygen, and hydroxyl radical. Hydrogen peroxide is not nearly as reactive as these species, but is readily activated and is thus included.^[3] Peroxynitrite and nitric oxide are reactive oxygen-containing species as well.

• Fenton reaction
  of hydrogen peroxide with ferrous compounds and related reducing agents:

Fe(II) + H₂O₂ → Fe(III)OH + HO·

In its fleeting existence, the hydroxyl radical reacts rapidly irreversibly with all organic compounds.

• superoxide (O−2) is produced by reduction of O₂.^[4] Several grams are produced per day in the human body within the mitochondria.^[5]

O₂ + e⁻ → O−2

Competing with its formation, superoxide is destroyed by the action of superoxide dismutases, enzymes that catalyze its disproportionation:

2 O−2 + 2H⁺ → O₂ + H₂O₂

• hydrogen peroxide (H₂O₂) is also produced as a side product of respiration.^[4]
• Peroxynitrite (ONO−2) results from the reaction of superoxide and nitric oxide.
• β-carotene arising from the presence of singlet oxygen act as second messengers that can either protect against singlet oxygen induced toxicity or initiate programmed cell death. Levels of jasmonate play a key role in the decision between cell acclimation or cell death in response to elevated levels of this reactive oxygen species.^[6]

In a biological context, ROS are byproducts of the normal metabolism of oxygen. ROS have roles in cell signaling and homeostasis.^{[7][8][9][10]} ROS are intrinsic to cellular functioning, and are present at low and stationary levels in normal cells.^[11] In plants, ROS are involved in metabolic processes related to photoprotection and tolerance to various types of stress.^[12] However, ROS can cause irreversible damage to DNA as they oxidize and modify some cellular components and prevent them from performing their original functions. This suggests that ROS has a dual role; whether they will act as harmful, protective or signaling factors depends on the balance between ROS production and disposal at the right time and place.^[13][8][14] In other words, oxygen toxicity can arise both from uncontrolled production and from the inefficient elimination of ROS by the antioxidant system. ROS were also demonstrated to modify the visual appearance of fish.^[15] This potentially affects their behavior and ecology, such as their temperature control, their visual communication, their reproduction and survival. During times of environmental stress (e.g.,

ROS are produced during the processes of respiration and photosynthesis in organelles such as

If too much damage is present in mitochondria, a cell undergoes apoptosis or programmed cell death.^[26][27]

In addition, ROS are produced in immune cell signaling via the

The hydroxyl radical is extremely reactive and immediately removes electrons from any molecule in its path, turning that molecule into a free radical and thus propagating a chain reaction. However, hydrogen peroxide is actually more damaging to DNA than the hydroxyl radical, since the lower reactivity of hydrogen peroxide provides enough time for the molecule to travel into the nucleus of the cell, subsequently reacting with macromolecules such as DNA.^{[citation needed]}

In plants, the production of ROS occurs during events of abiotic stress that lead to a reduction or interruption of metabolic activity. For example, the increase in temperature, drought are factors that limit the availability of CO₂ due to

Superoxide dismutases (SOD) are a class of enzymes that catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide. As such, they are an important antioxidant defense in nearly all cells exposed to oxygen. In mammals and most chordates, three forms of superoxide dismutase are present. SOD1 is located primarily in the cytoplasm, SOD2 in the mitochondria and SOD3 is extracellular. The first is a dimer (consists of two units), while the others are tetramers (four subunits). SOD1 and SOD3 contain copper and zinc ions, while SOD2 has a manganese ion in its reactive centre. The genes are located on chromosomes 21, 6, and 4, respectively (21q22.1, 6q25.3 and 4p15.3-p15.1).^{[citation needed]}

The SOD-catalysed

where M = Cu (n = 1); Mn (n = 2); Fe (n = 2); Ni (n = 2). In this reaction the oxidation state of the metal cation oscillates between n and n + 1.

${\displaystyle {\begin{aligned}&{\ce {2H2O2->[{\text{catalase}}]2H2O{}+O2}}\\&{\ce {2GSH{}+H2O2->[][{\text{glutathione}} \atop {\text{peroxidase}}]GS-SG{}+2H2O}}\end{aligned}}}$

Effects of ROS on cell metabolism are well documented in a variety of species.

In general, the harmful effects of reactive oxygen species on the cell are the damage of DNA or RNA, oxidation of polyunsaturated fatty acids in lipids (lipid peroxidation), oxidation of amino acids in proteins, and oxidative deactivation of specific enzymes by oxidation co-factors.^[38]

When a plant recognizes an attacking pathogen, one of the first induced reactions is to rapidly produce superoxide (O⁻
₂) or hydrogen peroxide (H
₂O
₂) to strengthen the cell wall. This prevents the spread of the pathogen to other parts of the plant, essentially forming a net around the pathogen to restrict movement and reproduction.

In the mammalian host, ROS is induced as an antimicrobial defense.^[28] To highlight the importance of this defense, individuals with chronic granulomatous disease who have deficiencies in generating ROS, are highly susceptible to infection by a broad range of microbes including Salmonella enterica, Staphylococcus aureus, Serratia marcescens, and Aspergillus spp.

Studies on the homeostasis of the Drosophila melanogaster's intestines have shown the production of ROS as a key component of the immune response in the gut of the fly. ROS acts both as a bactericide, damaging the bacterial DNA, RNA and proteins, as well as a signalling molecule that induces repair mechanisms of the epithelium.^[39] The uracil released by microorganism triggers the production and activity of DUOX, the ROS-producing enzyme in the intestine. DUOX activity is induced according to the level of uracil in the gut; under basal conditions, it is down-regulated by the protein kinase MkP3. The tight regulation of DUOX avoids excessive production of ROS and facilitates differentiation between benign and damage-inducing microorganisms in the gut.^[40]

The manner in which ROS defends the host from invading microbe is not fully understood. One of the more likely modes of defense is damage to microbial DNA. Studies using Salmonella demonstrated that DNA repair mechanisms were required to resist killing by ROS. A role for ROS in antiviral defense mechanisms has been demonstrated via Rig-like helicase-1 and mitochondrial antiviral signaling protein. Increased levels of ROS potentiate signaling through this mitochondria-associated antiviral receptor to activate interferon regulatory factor (IRF)-3, IRF-7, and nuclear factor kappa B (NF-κB), resulting in an antiviral state.^[41] Respiratory epithelial cells induce mitochondrial ROS in response to influenza infection. This induction of ROS led to the induction of type III interferon and the induction of an antiviral state, limiting viral replication.^[42] In host defense against mycobacteria, ROS play a role, although direct killing is likely not the key mechanism; rather, ROS likely affect ROS-dependent signalling controls, such as cytokine production, autophagy, and granuloma formation.^[43][44]

Reactive oxygen species are also implicated in activation,

In mice, the story is somewhat similar. Deleting antioxidant enzymes, in general, yields shorter lifespan, although overexpression studies have not (with some exceptions) consistently extended lifespan.[51] Study of a rat model of premature aging found increased oxidative stress, reduced antioxidant enzyme activity and substantially greater DNA damage in the brain neocortex and hippocampus of the prematurely aged rats than in normally aging control rats.^[52] The DNA damage 8-OHdG is a product of ROS interaction with DNA. Numerous studies have shown that 8-OHdG increases with age^[53] (see DNA damage theory of aging).

ROS are constantly generated and eliminated in the biological system and are required to drive regulatory pathways.^[54] Under normal physiological conditions, cells control ROS levels by balancing the generation of ROS with their elimination by scavenging systems. But under oxidative stress conditions, excessive ROS can damage cellular proteins, lipids and DNA, leading to fatal lesions in the cell that contribute to carcinogenesis.

Cancer cells exhibit greater ROS stress than normal cells do, partly due to oncogenic stimulation, increased metabolic activity and mitochondrial malfunction. ROS is a double-edged sword. On one hand, at low levels, ROS facilitates cancer cell survival since cell-cycle progression driven by growth factors and receptor tyrosine kinases (RTK) require ROS for activation^[55] and chronic inflammation, a major mediator of cancer, is regulated by ROS. On the other hand, a high level of ROS can suppress tumor growth through the sustained activation of cell-cycle inhibitor^[56][57] and induction of cell death as well as senescence by damaging macromolecules. In fact, most of the chemotherapeutic and radiotherapeutic agents kill cancer cells by augmenting ROS stress.^[58][59] The ability of cancer cells to distinguish between ROS as a survival or apoptotic signal is controlled by the dosage, duration, type, and site of ROS production. Modest levels of ROS are required for cancer cells to survive, whereas excessive levels kill them.

Metabolic adaptation in tumours balances the cells' need for energy with equally important need for macromolecular building blocks and tighter control of redox balance. As a result, production of

Most risk factors associated with cancer interact with cells through the generation of ROS. ROS then activate various transcription factors such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), activator protein-1 (AP-1), hypoxia-inducible factor-1α and signal transducer and activator of transcription 3 (STAT3), leading to expression of proteins that control inflammation; cellular transformation; tumor cell survival; tumor cell proliferation; and invasion, angiogenesis as well as metastasis. And ROS also control the expression of various tumor suppressor genes such as p53, retinoblastoma gene (Rb), and phosphatase and tensin homolog (PTEN).[61]

ROS-related oxidation of DNA is one of the main causes of mutations, which can produce several types of DNA damage, including non-bulky (8-oxoguanine and formamidopyrimidine) and bulky (cyclopurine and etheno adducts) base modifications, abasic sites, non-conventional single-strand breaks, protein-DNA adducts, and intra/interstrand DNA crosslinks.^[62] It has been estimated that endogenous ROS produced via normal cell metabolism modify approximately 20,000 bases of DNA per day in a single cell. 8-oxoguanine is the most abundant among various oxidized nitrogeneous bases observed. During DNA replication, DNA polymerase mispairs 8-oxoguanine with adenine, leading to a G→T transversion mutation. The resulting genomic instability directly contributes to carcinogenesis. Cellular transformation leads to cancer and interaction of atypical PKC-ζ isoform with p47phox controls ROS production and transformation from apoptotic cancer stem cells through blebbishield emergency program.^[63][64]

Uncontrolled proliferation is a hallmark of cancer cells. Both exogenous and endogenous ROS have been shown to enhance proliferation of cancer cells. The role of ROS in promoting tumor proliferation is further supported by the observation that agents with potential to inhibit ROS generation can also inhibit cancer cell proliferation.^[61] Although ROS can promote tumor cell proliferation, a great increase in ROS has been associated with reduced cancer cell proliferation by induction of G2/M cell cycle arrest; increased phosphorylation of ataxia telangiectasia mutated (ATM), checkpoint kinase 1 (Chk 1), Chk 2; and reduced cell division cycle 25 homolog c (CDC25).^[65]

A cancer cell can die in three ways: apoptosis, necrosis, and autophagy. Excessive ROS can induce apoptosis through both the extrinsic and intrinsic pathways.^[66] In the extrinsic pathway of apoptosis, ROS are generated by Fas ligand as an upstream event for Fas activation via phosphorylation, which is necessary for subsequent recruitment of Fas-associated protein with death domain and caspase 8 as well as apoptosis induction.^[61] In the intrinsic pathway, ROS function to facilitate cytochrome c release by activating pore-stabilizing proteins (Bcl-2 and Bcl-xL) as well as inhibiting pore-destabilizing proteins (Bcl-2-associated X protein, Bcl-2 homologous antagonist/killer).^[67] The intrinsic pathway is also known as the caspase cascade and is induced through mitochondrial damage which triggers the release of cytochrome c. DNA damage, oxidative stress, and loss of mitochondrial membrane potential lead to the release of the pro-apoptotic proteins mentioned above stimulating apoptosis.^[68] Mitochondrial damage is closely linked to apoptosis and since mitochondria are easily targeted there is potential for cancer therapy.^[69]

The cytotoxic nature of ROS is a driving force behind apoptosis, but in even higher amounts, ROS can result in both apoptosis and necrosis, a form of uncontrolled cell death, in cancer cells.^[70]

Numerous studies have shown the pathways and associations between ROS levels and apoptosis, but a newer line of study has connected ROS levels and autophagy.^[71] ROS can also induce cell death through autophagy, which is a self-catabolic process involving sequestration of cytoplasmic contents (exhausted or damaged organelles and protein aggregates) for degradation in lysosomes.^[72] Therefore, autophagy can also regulate the cell's health in times of oxidative stress. Autophagy can be induced by ROS levels through many pathways in the cell in an attempt to dispose of harmful organelles and prevent damage, such as carcinogens, without inducing apoptosis.^[73] Autophagic cell death can be prompted by the over expression of autophagy where the cell digests too much of itself in an attempt to minimize the damage and can no longer survive. When this type of cell death occurs, an increase or loss of control of autophagy regulating genes is commonly co-observed.^[74] Thus, once a more in-depth understanding of autophagic cell death is attained and its relation to ROS, this form of programmed cell death may serve as a future cancer therapy. Autophagy and apoptosis are distinct mechanisms for cell death brought on by high levels of ROS. Aautophagy and apoptosis, however, rarely act through strictly independent pathways. There is a clear connection between ROS and autophagy and a correlation seen between excessive amounts of ROS leading to apoptosis.^[73] The depolarization of the mitochondrial membrane is also characteristic of the initiation of autophagy. When mitochondria are damaged and begin to release ROS, autophagy is initiated to dispose of the damaging organelle. If a drug targets mitochondria and creates ROS, autophagy may dispose of so many mitochondria and other damaged organelles that the cell is no longer viable. The extensive amount of ROS and mitochondrial damage may also signal for apoptosis. The balance of autophagy within the cell and the crosstalk between autophagy and apoptosis mediated by ROS is crucial for a cell's survival. This crosstalk and connection between autophagy and apoptosis could be a mechanism targeted by cancer therapies or used in combination therapies for highly resistant cancers.

After growth factor stimulation of RTKs, ROS can trigger activation of signaling pathways involved in cell migration and invasion such as members of the mitogen activated protein kinase (MAPK) family – extracellular regulated kinase (ERK), c-jun NH-2 terminal kinase (JNK) and p38 MAPK. ROS can also promote migration by augmenting phosphorylation of the focal adhesion kinase (FAK) p130Cas and paxilin.^[75]

Both in vitro and in vivo, ROS have been shown to induce transcription factors and modulate signaling molecules involved in angiogenesis (MMP, VEGF) and metastasis (upregulation of AP-1, CXCR4, AKT and downregulation of PTEN).^[61]

Experimental and epidemiologic research over the past several years has indicated close associations among ROS, chronic inflammation, and cancer.^[61] ROS induces chronic inflammation by the induction of COX-2, inflammatory cytokines (TNFα, interleukin 1 (IL-1), IL-6), chemokines (IL-8, CXCR4) and pro-inflammatory transcription factors (NF-κB).^[61] These chemokines and chemokine receptors, in turn, promote invasion and metastasis of various tumor types.

Both ROS-elevating and ROS-eliminating strategies have been developed with the former being predominantly used. Cancer cells with elevated ROS levels depend heavily on the antioxidant defense system. ROS-elevating drugs further increase cellular ROS stress level, either by direct ROS-generation (e.g. motexafin gadolinium, elesclomol) or by agents that abrogate the inherent antioxidant system such as SOD inhibitor (e.g. ATN-224, 2-methoxyestradiol) and GSH inhibitor (e.g. PEITC, buthionine sulfoximine (BSO)). The result is an overall increase in endogenous ROS, which when above a cellular tolerability threshold, may induce cell death.^[76] On the other hand, normal cells appear to have, under lower basal stress and reserve, a higher capacity to cope with additional ROS-generating insults than cancer cells do.^[77] Therefore, the elevation of ROS in all cells can be used to achieve the selective killing of cancer cells.

Radiotherapy also relies on ROS toxicity to eradicate tumor cells. Radiotherapy uses X-rays, γ-rays as well as heavy particle radiation such as protons and neutrons to induce ROS-mediated cell death and mitotic failure.^[61]

Due to the dual role of ROS, both prooxidant and antioxidant-based anticancer agents have been developed. However, modulation of ROS signaling alone seems not to be an ideal approach due to adaptation of cancer cells to ROS stress, redundant pathways for supporting cancer growth and toxicity from ROS-generating anticancer drugs. Combinations of ROS-generating drugs with pharmaceuticals that can break the redox adaptation could be a better strategy for enhancing cancer cell cytotoxicity.^[61]

James Watson^[78] and others^[79] have proposed that lack of intracellular ROS due to a lack of physical exercise may contribute to the malignant progression of cancer, because spikes of ROS are needed to correctly fold proteins in the endoplasmatic reticulum and low ROS levels may thus aspecifically hamper the formation of tumor suppressor proteins.^[79] Since physical exercise induces temporary spikes of ROS, this may explain why physical exercise is beneficial for cancer patient prognosis.^[80] Moreover, high inducers of ROS such as 2-deoxy-D-glucose and carbohydrate-based inducers of cellular stress induce cancer cell death more potently because they exploit the cancer cell's high avidity for sugars.^[81]

ROS are critical in memory formation.^[84][85] ROS also have a central role in epigenetic DNA demethylation, which is relevant to learning and memory^[86][87]

In mammalian nuclear DNA, a methyl group can be added, by a

The thousands of CpG sites being demethylated during memory formation depend on ROS in an initial step. The altered protein expression in neurons, controlled in part by ROS-dependent demethylation of CpG sites in gene promoters within neuron DNA, are central to memory formation.[88]

• Antioxidant effect of polyphenols and natural phenols
• Iodide
• Melanin
• Mitohormesis
• Oxidative stress
• Oxygen toxicity
• Pro-oxidant
• Reactive nitrogen species
• Reactive sulfur species
• Reactive carbonyl species
• Reactive oxygen species production in marine microalgae