Free radicals

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

Indian Journal of Clinical Biochemistry, 2014 

Free radicals are products of 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 capable of independent existence. 

The odd number of electrons of a free radical makes it unstable, short lived and highly reactive, can abstract electrons from other compounds to attain stability. 

Thus, the attacked molecule loses its electron, becomes a free radical itself, beginning a chain reaction cascade which finally damages the living cell. 

Both ROS and RNS collectively constitute the free radicals and other non-radical reactive species play a twofold job as both beneficial and toxic compounds. 

At moderate or low levels ROS/RNS have beneficial effects and involve in various physiological functions such as in immune function as defence against pathogenic microorganisms in cellular signalling pathways in mitogenic response, in redox regulation.

At higher concentration, both ROS, RNS generate oxidative stress and nitrosative stress causing potential damage to the biomolecules. 

The oxidative stress and nitrosative stress are developed when there is excess production of ROS/RNS and a deficiency of enzymatic and non-enzymatic antioxidants. 

ROS, reactive oxygen species is produced from endogenous or exogenous sources.

The endogenous sources of ROS include cellular organs where the oxygen consumption is high.

Excess ROS damages the integrity of biomolecules.

The process is integral to oxidative phosphorylation, where ATP is synthesized, but can also result in the formation of harmful ROS. 

Free radical generation:

Radicals are molecular species containing at least one unpaired electron in their outer electron shells, rendering highly reactive, capable of independent existence.

1.Initiation of reactive oxygen species (ROS) formation:

Electron leakage during oxidative phosphorylation leads to incomplete reduction of O2, results in formation of superoxide radicals, primary reactive oxygen species. 

2.Enzymatic generation of ROS:

Enzymatic reaction within cells can also generate ROS.

Superoxide radicals are byproducts of metabolic activities of xanthine oxidase and cytochrome P450.

Formation of superoxide radicals (O2 ·−):

Superoxide radicals are formed through either the direct auto-oxidation of molecular oxygen or enzymatic processes, and they can catalyse chain reactions, superoxide anion radical is a reduced form of molecular oxygen created by receiving an electron

Conversion of hydrogen peroxide (H2O2):

Superoxide dismutase (SOD) catalyses the conversion of superoxide radicals to hydrogen peroxide

Formation of hydroxyl radicals (·OH):

Generated via the Fenton reaction between hydrogen peroxide and metal ions, iron (Fe2+), or through the radiolysis of water in the presence of ionizing radiation.

Summary of radical ROS species formation:

Superoxide (O2 ·−) generated by direct auto-oxidation of O2 during mitochondrial electron transport.

Oxygen radical (O∙∙2) one electron reduction by molecular oxygen, mechanism by enzymatic rection, autoxidation, non enzymatic electron transfer.

Hydroxyl (OH∙) formed in Fenton reaction, water radiolysis and reaction of H2O2 with (Fe2+) / metal ions.

Haber–Weiss reaction generates hydroxyl radicals from hydrogen peroxide and superoxide anions.

Hydroxyl anions (OH−) when water molecules dissociate/lose hydrogen ion (H⁺) as self-ionization, when metal hydroxides dissolve in water. 

Alkoxy radical (RO∙) by decomposition of alkyl peroxides.

Peroxyl radical (ROO∙) derived from oxygen formed by the decomposition of hydroperoxides (ROOH), results in formation of peroxyl (ROO·), alkoxyl (RO·) radicals.

Hydroperoxyl (per hydroxyl) radical (HOO·) formation of hydroperoxyl is by superoxide protonation.

Carbonate radical anion (CO3•−) produced by radiolysis of bicarbonate / carbonate in aqueous solution.

Non-radical species, while not free radicals themselves, have the potential to undergo redox reactions and contribute to oxidative stress in living systems

RNS generated by enzymatic, non-enzymatic process.

RNS, reactive nitrogen species mechanism involves their generation, reactions, and biological effects. RNS are highly reactive molecules derived from nitric oxide (·NO) and other nitrogenous compounds, and play dual roles in physiological signalling, pathological damage. 

Nitric oxide (·NO) synthesis and Enzymatic pathway:

Nitric oxide synthase (NOS) enzymes catalyse the conversion of l-arginine to ·NO and l-citrulline using NADPH and O₂.

eNOS (endothelial NOS): Involved in vascular homeostasis. Require intracellular calcium (Ca2+)

nNOS (neuronal NOS): Facilitates neurotransmission.

iNOS (inducible NOS): Produces large amounts of ·NO during inflammation.

Summary of non-radical ROS species formation:

Hydrogen peroxyl (HO2) by superoxide anion radical protonation or by hydroperoxide decomposition.

Hydrogen peroxide (H2O2) enzyme reaction, by the dismutation of O2 or by the direct reduction of O2

Hydrogen peroxide can be formed by a two-electron reduction of dioxygen.

Hypochlorous acid (HOCl) produced by the activated neutrophils, generated by hydrogen peroxide and chloride anion in a catalysed reaction.

In biological systems, HOCl formed in process of phagocytosis catalysed by myeloperoxidase-mediated peroxidation of chloride anions (Cl−) using H202.

Hypobromous acid (HOBr) through the action of heme peroxidases, such as eosinophil peroxidase (EPO) and myeloperoxidase (MPO) which catalyze the reaction between bromide ions (Br-), hydrogen peroxide (H2O2). 

Ozone (O3) produced by antibody, water oxidation pathway, by oxidizing biological molecules. 

Singlet oxygen (1O2) produced by photodynamic reaction, electronically excited formed during photosensitization, unpaired electrons from triplet oxygen enter different orbitals, results oxidant named singlet oxygen. 

Summary of non-radical RNS species:

Nitric oxide (nitrogen monoxide) (NO∙) generated in tissues by different nitric oxide synthases (NOS)

A small, uncharged, labile, and lipid-permeable molecule containing an unpaired electron. 

Nitrogen dioxide (NO∙2) formed by the reaction of the peroxyl radical and NO

Nitrous acid (HNO2)

Nitrosyl cation (NO+)

Nitroxyl anion (NO−)

Dinitrogen trioxide (N2O3)

Dinitrogen tetra oxide (N2O4)

Nitronium (nitryl) cation (NO2+)

Organic peroxides (ROOH)

Aldehydes (HCOR)

Peroxynitrite (ONOO−) nitric oxide inactivation with superoxide radical, formation of Peroxynitrite.

Organic derivatives of sulphur can form thiols, disulfides, sulfenic acids or sulfoxides, Sulfinic acid and sulfonic acids. By analogy with ROS and RNS, these identified as reactive sulphur species (RSS). Thiols can generate free radicals. In the presence of traces of transition metal ions, thiol compounds are oxidized to thiyl radicals and reactive oxygen species. Reactive Sulphur Species (RSS) can be both radical and non-radical.

Endogenous Sources of ROS

Mitochondria: 

Superoxide anion radical (O₂•−), Hydroxyl radical radical (HO•), Hydrogen Peroxide radical (H2O2)

Endoplasmic reticulum: 

Hydrogen peroxide radical (H2O2)

NADPH oxidase: 

Superoxide radical (O₂•−)

Xanthine oxidoreductase (XOR):

Superoxide radical (O₂•−), Hydrogen peroxide radical (HO•)

Peroxisomes the respiratory pathway: 

Hydrogen peroxide radical (H2O2), Singlet oxygen (1O2)

Hydroxyl radical radical (HO•), Nitric oxide (NO·)

Endoplasmic reticulum: 

Hydrogen peroxide (H2O2)

Myeloperoxidase (MPO): 

Hypochlorous acid (HOCl)

Endothelial nitric oxide synthase (eNOS): 

Nitric oxide (NO)

Exogenous sources of ROS

Ionizing radiation: 

Nitric oxide (NO·)

UV radiation: 

Singlet oxygen (1O2), Hydroxyl radical (HO•), Hydrogen peroxide radical (H2O2)

Smoking: 

Nitric oxide (NO·), Hydrogen peroxide (H2O2), Hydroxyl radical (HO•)

Alcohol consumption: 

Mitochondrial electron transport chain (ETC), Hydroxyl radical (HO•)

Natural contaminant: 

Nitrogen oxide, ozone, Sulphur oxide

Drugs: 

Superoxide radical (O₂•−)

Targets of Free Radicals

Deoxyribonucleic Acid (DNA): Hydroxyl radical (•OH), Peroxynitrite (ONOO−)

Ribonucleic acid (RNA): ROS attacks, close proximity to mitochondria where loads of ROS is produced

Membrane lipids: Peroxyl radical (RO2•)

Proteins by radical species: Oxygen radical (O∙∙2), Hydroxyl radical (•OH), Peroxyl radical (RO2•), Alkoxyl radical (RO•)

Proteins by non-radical species: Hydrogen peroxide (H2O2), Hypochlorous acid (HOCl), Peroxynitrite (ONOO−), Singlet oxygen (1O2)

Molecular Targets of Free Radicals

Hydrogen peroxide (H2O2), Nitric oxide (NO•), Superoxide (O2•−) react promptly with some molecules.

Hydroxyl radical (•OH) reacts with almost everything.

Peroxyl radical (RO2•), Nitrate radical (NO3•), Alkoxyl radical (RO•), Hypochlorous acid (HOCl), Hypobromous acid (HOBr), Carbonate (CO3•−), Carbon dioxide radical (CO2•−), Peroxynitrite (ONOO−), Nitrogen dioxide (NO2•−), Ozone (O3) have intermediate reactivities.

Reference Links:

https://www.researchgate.net/search, https://www.mdpi.com/, https://pubmed.ncbi.nlm.nih.gov/, https://www.sciencedirect.com/