Free radicals and reactive oxygen species are produced during normal metabolism and can cause cellular damage that is counteracted by enzymatic and non-enzymatic antioxidant systems. The balance between oxidant production and antioxidant defense determines the oxidative stress level of the cell.
Free Radicals and Reactive Oxygen Species
Free radicals are molecules with one or more unpaired electrons, making them highly reactive. The superoxide anion radical is formed by the one-electron reduction of molecular oxygen, primarily from leakage of the mitochondrial electron transport chain during oxidative phosphorylation and from NADPH oxidases. Superoxide can inactivate iron-sulfur cluster proteins and release free iron, which participates in Fenton chemistry to generate more damaging species.
Hydrogen peroxide is not a free radical but a two-electron reduction product of oxygen. It diffuses freely across membranes and can generate the hydroxyl radical through Fenton chemistry in the presence of ferrous iron. The hydroxyl radical is the most reactive of the ROS, reacting at diffusion-limited rates with virtually any biomolecule.
Peroxynitrite is formed by the reaction of superoxide with nitric oxide. It is a potent oxidant and nitrating agent that modifies proteins, lipids, and DNA. Singlet oxygen and hypochlorous acid, produced by myeloperoxidase in neutrophils, are additional non-radical oxidants that contribute to pathogen killing but also cause tissue damage.
Sources of ROS
The mitochondrial electron transport chain is the primary endogenous source of ROS. Complex I and complex III leak electrons that reduce oxygen to superoxide. Under normal conditions, about 1 to 2% of consumed oxygen is converted to superoxide. This increases when the electron transport chain is damaged or when mitochondrial membrane potential is high.
NADPH oxidases are dedicated ROS-producing enzymes that generate superoxide for signaling and immune defense. NOX family enzymes are expressed in phagocytes, endothelial cells, and other tissues. Other sources include peroxisomal oxidases, cytochrome P450 enzymes, and xanthine oxidase, which produces superoxide during purine degradation.
Exogenous sources of ROS include UV and ionizing radiation, which generate hydroxyl radicals through water radiolysis. Air pollutants, cigarette smoke, and certain drugs and toxins also increase ROS production. Transition metals such as iron and copper catalyze free radical formation.
Oxidative Damage
Lipid peroxidation is a chain reaction initiated by hydroxyl radical abstraction of a hydrogen atom from polyunsaturated fatty acids. The resulting lipid radical reacts with oxygen to form a lipid peroxyl radical, which propagates the chain. Malondialdehyde and 4-hydroxynonenal are toxic byproducts. Lipid peroxidation damages cell membranes and generates reactive aldehydes that modify proteins and DNA.
Protein oxidation modifies amino acid side chains, particularly cysteine, methionine, and aromatic residues. Disulfide bond formation, protein carbonylation, and nitration of tyrosine residues alter protein function and can target proteins for degradation.
DNA oxidation produces 8-oxoguanine as the most common lesion, causing G-to-T transversion mutations if unrepaired. DNA damage by ROS contributes to mutagenesis, aging, and cancer.
Enzymatic Antioxidant Defenses
Superoxide dismutase, which requires metal cofactors, catalyzes the dismutation of superoxide to hydrogen peroxide and oxygen. Three isoforms exist: cytosolic CuZn-SOD, mitochondrial Mn-SOD, and extracellular SOD. SOD is essential for aerobic life, and Mn-SOD knockout mice die shortly after birth.
Catalase converts hydrogen peroxide to water and molecular oxygen. It is concentrated in peroxisomes and has one of the highest turnover rates of any enzyme. Glutathione peroxidase reduces hydrogen peroxide and organic hydroperoxides using reduced glutathione as an electron donor. It contains selenocysteine at the active site. Thioredoxin reductase and peroxiredoxins provide additional peroxide-reducing capacity.
Non-Enzymatic Antioxidants
Glutathione is the most abundant intracellular thiol and the major non-enzymatic antioxidant. It maintains protein thiols in the reduced state and serves as the electron donor for glutathione peroxidase. Reduced glutathione is regenerated by glutathione reductase using NADPH.
Vitamin E is the primary lipid-soluble chain-breaking antioxidant in membranes. It donates a hydrogen atom to lipid peroxyl radicals, terminating lipid peroxidation. The resulting tocopheroxyl radical is recycled by vitamin C. Vitamin C is a water-soluble antioxidant that scavenges ROS directly and regenerates vitamin E. Uric acid, bilirubin, and ubiquinol are additional endogenous antioxidants.
Oxidative Stress in Disease
Oxidative stress contributes to many diseases. In atherosclerosis, LDL oxidation promotes foam cell formation and plaque development. In neurodegeneration, oxidative damage accumulates in Alzheimer, Parkinson, and Huntington diseases. In cancer, ROS promote mutagenesis and tumor progression but can also induce cell death. In diabetes, hyperglycemia drives ROS production through multiple mechanisms, contributing to vascular complications. Ischemia-reperfusion injury features a burst of ROS upon reoxygenation, causing additional tissue damage beyond the ischemic insult.
