Reactive oxygen species

Function

In a biological context, ROS are byproducts of the normal metabolism of oxygen. ROS have roles in cell signaling and homeostasis. ROS are intrinsic to cellular functioning, and are present at low and stationary levels in normal cells. In plants, ROS are involved in metabolic processes related to photoprotection and tolerance to various types of stress. 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. In other words, oxygen toxicity can arise both from uncontrolled production and from the inefficient elimination of ROS by the antioxidant system. ROS are intermediates in the redox behavior of O₂, which is central to fuel cells.

During times of environmental stress (e.g., UV or heat exposure), ROS levels can increase dramatically. This may result in significant damage to cell structures. Cumulatively, this is known as oxidative stress. The production of ROS is strongly influenced by stress factor responses in plants, these factors that increase ROS production include drought, salinity, chilling, defense of pathogens, nutrient deficiency, metal toxicity and UV-B radiation. ROS are also generated by exogenous sources such as ionizing radiation generating irreversible effects in the development of tissues in both animals and plants. ROS have been demonstrated to modify the visual appearance of fish. This potentially affects their behavior and ecology, such as their temperature control, their visual communication, their reproduction and survival.

ROS are central to the photodegradation of organic pollutants in the atmosphere.

Sources of ROS production

Endogenous sources

ROS are produced during the processes of respiration and photosynthesis in organelles such as mitochondria, peroxisomes and chloroplasts. During the respiration process the mitochondria convert energy for the cell into a usable form, adenosine triphosphate (ATP). The process of ATP production in the mitochondria, called oxidative phosphorylation, involves the transport of protons (hydrogen ions) across the inner mitochondrial membrane by means of the electron transport chain. In the electron transport chain, electrons are passed through a series of proteins via oxidation-reduction reactions, with each acceptor protein along the chain having a greater reduction potential than the previous. The last destination for an electron along this chain is an oxygen molecule. In normal conditions, the oxygen is reduced to produce water; however, in about 0.1–2% of electrons passing through the chain (this number derives from studies in isolated mitochondria, though the exact rate in live organisms is yet to be fully agreed upon), oxygen is instead prematurely and incompletely reduced to give the superoxide radical (^•O⁻
₂), most well documented for Complex I and Complex III.

Another source of ROS production in animal cells is the electron transfer reactions catalyzed by the mitochondrial P450 systems in steroidogenic tissues. These P450 systems are dependent on the transfer of electrons from NADPH to P450. During this process, some electrons "leak" and react with O₂ producing superoxide. To cope with this natural source of ROS, the steroidogenic tissues, ovary and testis, have a large concentration of antioxidants such as vitamin C (ascorbate) and β-carotene and anti-oxidant enzymes.

If too much damage is present in mitochondria, a cell undergoes apoptosis or programmed cell death.

In addition, ROS are produced in immune cell signaling via the NOX pathway. Phagocytic cells such as neutrophils, eosinophils, and mononuclear phagocytes produce ROS when stimulated.

In chloroplasts, the carboxylation and oxygenation reactions catalyzed by rubisco ensure that the functioning of the electron transport chain (ETC) occurs in an environment rich in O₂. The leakage of electrons in the ETC will inevitably produce ROS within the chloroplasts. ETC in photosystem I (PSI) was once believed to be the only source of ROS in chloroplasts. The flow of electrons from the excited reaction centers is directed to the NADP and these are reduced to NADPH, and then they enter the Calvin cycle and reduce the final electron acceptor, CO₂. In cases where there is an ETC overload, part of the electron flow is diverted from ferredoxin to O₂, forming the superoxide free radical (by the Mehler reaction). In addition, electron leakage to O₂ can also occur from the 2Fe-2S and 4Fe-4S clusters in the PSI ETC. However, PSII also provides electron leakage locations (QA, QB) for O₂-producing O₂-. Superoxide (O₂-) is generated from PSII, instead of PSI; QB is shown as the location for the generation of O₂•-.

Exogenous sources

The formation of ROS can be stimulated by a variety of agents such as pollutants, heavy metals, allergens, cigarette smoke, drugs, insecticides, ozone, pesticides, toxins, UV radiation. In plants, in addition to the action of dry abiotic factors, high temperature, interaction with other living beings can influence the production of ROS.

In a process termed radiolysis, through the interaction of water and ionizing radiation, damaging intermediates can be generated. In the process, a water molecule gets excited and subsequently dissociates into a hydrogen (^•H) and a hydroxyl radical (^•OH), which both are highly reactive. The radicals may attack cells and molecules on the cell surface, and through a chain reaction dozens of molecules can be damaged by the homolysis of a single water molecule.

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 stomatal closure, increasing the production of ROS, such as O₂·- and ¹O₂ in chloroplasts. The production of ¹O₂ in chloroplasts can cause reprogramming of the expression of nucleus genes leading to chlorosis and programmed cell death. In cases of biotic stress, the generation of ROS occurs quickly and weakly initially and then becomes more solid and lasting. The first phase of ROS accumulation is associated with plant infection and is probably independent of the synthesis of new ROS-generating enzymes. However, the second phase of ROS accumulation is associated only with infection by non-virulent pathogens and is an induced response dependent on increased mRNA transcription encoding enzymes.

Antioxidant enzymes

Superoxide dismutase

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).

The SOD-catalysed dismutation of superoxide may be written with the following half-reactions:

${\displaystyle \begin{array}{cc}& {\text{M}}^{(n+1)+}+{\text{O}}_2^{-}\stackrel{SOD}{\to }{\text{M}}^{n+}+{\text{O}}_2^{}\\ & {\text{M}}^{n+}+{\text{O}}_2^{-}+2{\text{H}}^{+}⟶[\text{SOD}]{\text{M}}^{(n+1)+}+{\text{H}}_2^{}{\text{O}}_2^{}\end{array}}$

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.

Catalase, which is concentrated in peroxisomes located next to mitochondria, reacts with the hydrogen peroxide to catalyze the formation of water and oxygen. Glutathione peroxidase reduces hydrogen peroxide by transferring the energy of the reactive peroxides to a sulfur-containing tripeptide called glutathione. The sulfur contained in these enzymes acts as the reactive center, carrying reactive electrons from the peroxide to the glutathione. Peroxiredoxins also degrade H₂O₂, within the mitochondria, cytosol, and nucleus.

${\displaystyle \begin{array}{cc}& 2{\text{H}}_2^{}{\text{O}}_2^{}\stackrel{\text{catalase}}{\to }2{\text{H}}_2^{}\text{O}+{\text{O}}_2^{}\\ & 2\text{GSH}+{\text{H}}_2^{}{\text{O}}_2^{}\underset{\genfrac{}{}{0ex}{}{\text{glutathione}}{\text{peroxidase}}}{\overset{}{\to }}\text{GS}-\text{SG}+2{\text{H}}_2^{}\text{O}\end{array}}$

Damaging effects

Effects of ROS on cell metabolism are well documented in a variety of species. These include not only roles in apoptosis (programmed cell death) but also positive effects such as the induction of host defence genes and mobilization of ion transporters. This implicates them in control of cellular function. In particular, platelets involved in wound repair and blood homeostasis release ROS to recruit additional platelets to sites of injury. These also provide a link to the adaptive immune system via the recruitment of leukocytes.

Reactive oxygen species are implicated in cellular activity to a variety of inflammatory responses including cardiovascular disease. They may also be involved in hearing impairment via cochlear damage induced by elevated sound levels, in ototoxicity of drugs such as cisplatin, and in congenital deafness in both animals and humans. ROS are also implicated in mediation of apoptosis or programmed cell death and ischaemic injury. Specific examples include stroke and heart attack.

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. They play a role in causing dangerous genetic mutations.

Pathogen response

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. 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. 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.

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. 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. 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.

Reactive oxygen species are also implicated in activation, anergy and apoptosis of T cells.

Oxidative damage

In aerobic organisms the energy needed to fuel biological functions is produced in the mitochondria via the electron transport chain. Reactive oxygen species (ROS) with the potential to cause cellular damage are produced along with the release of energy. ROS can damage lipids, DNA, RNA, and proteins, which, in theory, contributes to the physiology of aging.

ROS are produced as a normal product of cellular metabolism. In particular, one major contributor to oxidative damage is hydrogen peroxide (H₂O₂), which is converted from superoxide that leaks from the mitochondria. Catalase and superoxide dismutase ameliorate the damaging effects of hydrogen peroxide and superoxide, respectively, by converting these compounds into oxygen and hydrogen peroxide (which is later converted to water), resulting in the production of benign molecules. However, this conversion is not 100% efficient, and residual peroxides persist in the cell. While ROS are produced as a product of normal cellular functioning, excessive amounts can cause deleterious effects.

Cell death

A cancer cell can die in three ways: apoptosis, necrosis, and autophagy. Excessive ROS can induce apoptosis through both the extrinsic and intrinsic pathways. 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. 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). 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. Mitochondrial damage is closely linked to apoptosis and since mitochondria are easily targeted there is potential for cancer therapy.

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.

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. 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. 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. 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. 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. Autophagy 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. 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.

Cancer therapy

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. 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. Therefore, the elevation of ROS in all cells can be used to achieve the selective killing of cancer cells.

James Watson and others 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 endoplasmic reticulum and low ROS levels may thus aspecifically hamper the formation of tumor suppressor proteins. Since physical exercise induces temporary spikes of ROS, this may explain why physical exercise is beneficial for cancer patient prognosis. 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.