REACTIVE OXYGEN AND NITROGEN SPECIES
Reactive oxygen species are generated by many different systems in the cell. However, mitochondria is the major site of free radical production and also the target of the reactive oxygen and nitrogen species generated by many different systems.

Superoxide (O₂^.)
During respiration, superoxide is produced in Complex I (NADH-ubiquinone-oxidoreductase) and Complex III (ubiquinol-cytochrome c-oxidoreductase), which can be enhanced by inhibition of the respiratory chain owing to a lack of oxygen. Other potentially important sources of superoxide are the meabolism of arachidonic acid through the cyclooxygenase and lipooxygenase pathways, xanthine oxidase and NADPH oxidase.

Hydrogen peroxide (H₂O₂)
About 1-5 % of mitochondrial oxygen consumption leads to H₂O₂ production. H2O2 acts upon mitochondria, causing a disruption of mitochondrial membran potential and the release of cytochrome-c, and cause upregulation of Fas/FasL system and modulation of transcription factors such as NFkB resulting in altered gene expression. Furthermore, H2O2 suppresses both the activation and activity of caspases, possibly through modulation of the redox status of the cell and the oxidation of cystein-residues. In the presence of transition metals such as copper and iron, H2O2 is converted to a highly reactive species, hydroxyl radical.

Nitric oxide (NO)
Nitric oxide is involved in several signaling pathways related to a diverse array of cell functions. Under physiological condition, endothelial nitric oxide synthase (eNOS) plays a physiological role in regulation of vasodilatation, and a low level of NO is produced as a neurotransmitter in a subpopulation of neurons by nNOS, which requires Ca2+/calmodulin binding for activation. Under stress conditions such as ischemia/reperfusion injury glutamate-mediated Ca2+ influx promotes NO production by both eNOS and nNOS. In addition, inflammatory processes upregulate inducible forms of NOS (iNOS) in activated microglia, infiltrating leukocytes and macrophages. Moreover, reactive astrocytic response may upregulate the synthesis of iNOS, which leads to increased production of NO.

Peroxynitrite (ONOO^-)
A continuous NO generation in the presence of prolonged increase of superoxide anion generation results in the formation of peroxynitrite. Peroxynitrite can directly produce damage or be converted to other strong oxidants including the highly reactive hydroxyl radical and nitrogen dioxide. Peroxynitrite-mediated protein nitrosylation may disrupt signaling pathways that use tyrosine phosphorilation and dephosphorilation. The targets of peroxynitrite in mitochondria include the major enzyme mitochondrial complexes of the electron transport chain and ATP synthetase, witch could directly impair respiration. Furthermore, inhibit aconitase and creatine kinase, superoxide dismutase, increases the proton leak in isolated mitochondria, and leads to opening of MPT pore. MPT pore opening associated with cytochrome-c release leading to a breakdown of mitochondrial electron flow downstream of the ubiquinone site, in turn resulting in increased generation of superoxide anions and derived reactive oxygen and nitrogen species. Inhibition of NO production is controversial.


Figure 1. Generation of free radicals. During mitochondrial respiration some electrons go directly to oxygen forming superoxide anion (•O₂-) that leads to the production of other free radicals such as peroxynitrite (ONOO^-) through reaction with nitric oxide (NO); hydrogen peroxide (H₂O₂) through enzymatic dismutation by superoxide dismutase (SOD); and hydroxyl radical (•OH). Nitric oxide can undergo autooxidation leading to production of other nitorgen species such as nitric dioxide (NO₂) and dinitric trioxide (N₂O₃).

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TARGETS
Free radicals attack bases in nucleic acids, amino acid side chains in proteins and double-bonds in unsaturated fatty acids.
Hydrogen peroxide can damage proteins directly by the oxidation of -SH groups.
Peroxynitrite and the hydroxyl radical can react directly with proteins and other macromolecules to produce carbonyls (aldehydes and ketones), cross-linking and lipid peroxidation.
Hydroxyl radical is highly reactive and can cause covalent cross-linking or free-radical propagation in a wide variety of biological molecules.

1. Polyunsaturated fatty acids
   The main targets of reactive oxygen and nitrogen species in mitochondria are the protein component of the membranes and the polyunsaturated fatty acid.
   Hydroxyl radicals can react with molecules (LH) in membranes to produce lipid molecule radicals (alkyl or alkoxyl= L*). These lipid radicals can then react directly with oxygen (autoxidation) forming lipid peroxides or peroxyl radicals (LOO*). Lipid peroxyl radicals can react lipid side chains forming hydroperoxides (LOOH) and alkyl radicals resulting in auto-amplifying chain reaction. The lipid hydroperoxides can promote a Fenton reaction.
   During lipid peroxidation, many damaging aldehydes are formed such as malondialdehyde (MDA) and 4-hydroxy-nonenal (4-HNE). MDA is a major metabolite of arachidonic acid (20:4). MDA, 4-HNE are long-lived and can drift far from membranes, damaging a wide variety of proteins, lipids and nucleic acids. Aldehyde-bridge formation leads to the protein-protein cross-linking associated with lipofuscin formation.
   
   
2. Proteins
   Free radicals affect cystein residues (sulfhydryl group), causing intramolecular cross-linkings and formation of protein aggregates. Sulfhydryl groups in the adenosine nucleotide translocator are important molecular targets of oxidative stress that could be responsible for inducing the mitochondrial permeability transition.
   
   
3. Nucleic acids
   Because of the absence of mitochondrial DNA-protecting proteins, the low efficiency reparation mechanisms and the proximity of the respiration chain, mtDNA is a privileged target for free radicals.
   The most frequent oxidative damage to DNA is the 8-hydroxylation/oxidation of the guanine base to 8-hydroxydeoxyguanosine (8-OHdG), a molecule which is equivalent to 8-oxo-7,8-dihydroguanine (8-oxoG) because the hydroxyl hydrogen can easily move to the 7-position leaving a double-bonded oxygen at the 8-position. 8-OHdG is mutagenic because it can be paired with adenosine rather than cytosine during DNA replication leading to GC-to-AT conversion.

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ANTIOXIDANT SYSTEM
To prevent oxidative damage, mammalian cells have developed a complex antioxidant defense system that include enzymatic activities (superoxide dismutase, catalase, glutathion peroxidase) and free radical scavangers (glutathione, thioredoxine, vitamin C and vitamin E). However, under stress conditions, free radical production may overwhelm endogenous protective mechanisms.

Cytoplasmic copper superoxide dismutase (CuSOD) and mitochondrial SOD (MnSOD) catalyze the dismutation of superoxide to H₂O₂, which is further converted to water and oxygen by catalase and glutathion (GSH) peroxidase.

Glutathione is the major antioxidant in cytoplasm composed of the amino acids cysteine, glycine and glutamic acid. Glutathione peroxidase destroys fat peroxides and neutralizes hydrogen peroxide by taking hydrogens from two GSH (reduced form) molecules resulting in GSSG (oxidized form) and H₂O. The enzyme glutathione reductase then regenerates GSH with NADPH.

Vitamin C appears to enhance glutathione peroxidase activity.

Vitamin E is the main free-radical trap in the (lipid) membranes.

Melatonin, a hormone produced by the pineal gland is effective against hydroxyl radicals.

Uric acid (which is mostly formed from purine degradation) protects against free-radical catalysis by binding iron, protects Vitamin C from oxidation by divalent ions (iron and copper) ions.

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DETECTION METHODS
Direct measurement of free radicals is almost impossible because of their very short half-life.
Superoxide can be measured by oxidized hydroethidine (Murakami, 1998), formazam (nitroblue tetrazolium reagent), ferricytochrome-c (Kuthan, 1982) or aconitase activity (Patel, 1996). Hydrogen peroxide can be measured by Amplex red. Other free radicals can be measured by the by-product or derivatives of modified proteins, lipids and nucleic acids.

Modification	Marker	Assay	Caused by
Lipid peroxidation	Malondialdehyde	TBARS, thiobarbituric acid reacting substances	OH.
Protein modification	S-nitrosothiols (RSNO)	Hg2+/Griess reaction, 0.5-100 uM DAN (diaminonaftalen), 50-1000 uM	N2O3
	3-nitrotyrosin	anti-3-nitrotyrosin antibody	ONOO-
	Carbonyl groups (C=O)	dinitrofenilhydrasin	alkyl radical (L.)
DNA damage	8-hidroxy-deoxiguanosine (8-OHdG) 8-oxo-7,8-dihydroxiguanosine (8-oxoG)	anti-8-hidroxy-deoxiguanosine (8-OHdG) antibody	OH.

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Links
American Federation for Aging Research