Free Radicals & Oxidative Stress
Pathology · General Pathology · lean revision notes
Free Radicals & Oxidative Stress
A free radical is any chemical species with one or more unpaired electrons in its outer orbital, making it extremely unstable and reactive. Oxidative stress is the imbalance between free-radical generation and antioxidant defences, and it is a central, recurring mechanism of cell injury, ageing, chemical toxicity and carcinogenesis in NEET PG pathology.
Definition & basic concepts
Free radicals are produced normally during oxidative metabolism but are kept in check by antioxidant systems. When generation outstrips defence, the radicals attack lipids, proteins and nucleic acids, producing reversible or irreversible cell injury.
Key terminology you must distinguish:
- Reactive oxygen species (ROS): oxygen-derived radicals and their reactive non-radical derivatives (e.g., hydrogen peroxide, H₂O₂, which has no unpaired electron but generates radicals).
- Reactive nitrogen species (RNS): nitric oxide (NO·) and peroxynitrite (ONOO⁻).
- Oxidative stress: state of excess ROS relative to antioxidant capacity.
High-yield: A free radical, by definition, has an unpaired electron. H₂O₂ and singlet oxygen are not radicals but are still ROS because they readily generate radicals.
Classification of important reactive species
| Species | Symbol | Radical? | Key feature / source |
|---|---|---|---|
| Superoxide anion | O₂·⁻ | Yes | First ROS formed; from electron leak in mitochondrial ETC, NADPH oxidase |
| Hydrogen peroxide | H₂O₂ | No | From dismutation of O₂·⁻ by SOD; crosses membranes; substrate for Fenton |
| Hydroxyl radical | OH· | Yes | Most reactive & most damaging; from Fenton/Haber-Weiss |
| Singlet oxygen | ¹O₂ | No | Energised oxygen; photosensitisation |
| Nitric oxide | NO· | Yes | From NO synthase; vasodilator, also reacts with O₂·⁻ |
| Peroxynitrite | ONOO⁻ | No (RNS) | NO· + O₂·⁻; highly cytotoxic |
| Hypochlorous acid | HOCl | No | Myeloperoxidase in neutrophils; potent microbicidal |
High-yield: The hydroxyl radical (OH·) is the most reactive and most injurious free radical in biological systems and the principal mediator of radiation injury (radiolysis of water).
Sources & generation of free radicals
Free radicals arise from both physiological and pathological processes.
Physiological / endogenous sources
- Mitochondrial electron transport chain – the largest source; ~1–3% of O₂ undergoes incomplete reduction, leaking single electrons to form O₂·⁻.
- Reduction–oxidation reactions during normal metabolism.
- Phagocytic respiratory (oxidative) burst – NADPH oxidase in neutrophils/macrophages generates O₂·⁻ for microbial killing.
- Nitric oxide synthase – produces NO·.
- Peroxisomal oxidases & cytochrome P450 metabolism.
- Transition metals (Fe²⁺, Cu⁺) donating/accepting electrons.
Pathological / exogenous sources
- Ionising radiation (radiolysis of water → OH·)
- UV light
- Chemicals & drugs metabolised to radicals (CCl₄, paracetamol overdose, doxorubicin, paraquat, bleomycin)
- Cigarette smoke, air pollutants, hyperoxia
- Inflammation and reperfusion of ischaemic tissue
Two named radical-generating reactions
Fenton reaction: H₂O₂ + Fe²⁺ → Fe³⁺ + OH⁻ + OH·
Haber–Weiss reaction: O₂·⁻ + H₂O₂ → O₂ + OH⁻ + OH· (iron-catalysed)
High-yield: Both reactions converge on the hydroxyl radical, and both require free transition metal (iron/copper). This explains why iron overload (haemochromatosis, repeated transfusions) accelerates oxidative tissue damage, and why iron-chelators such as deferoxamine are protective.
Mechanisms of cellular damage (pathophysiology)
Free radicals injure cells through three principal targets. Remember the triad "Lipids, Proteins, DNA".
1. Lipid peroxidation of membranes (most important) Radicals attack the polyunsaturated fatty acids of membrane lipids, initiating an autocatalytic chain reaction that propagates membrane damage. End-products include malondialdehyde (MDA) and 4-hydroxynonenal, and the volatile breath marker pentane/ethane. Lipid peroxidation destroys plasma, mitochondrial and lysosomal membranes.
2. Protein modification & cross-linking Oxidation of amino-acid side chains (especially sulfhydryl groups), protein–protein cross-linking, fragmentation of the polypeptide backbone, and enhanced proteasomal degradation. This inactivates enzymes and structural proteins.
3. DNA damage Reaction with thymine and the sugar–phosphate backbone causes single-strand breaks. The signature oxidative lesion is 8-hydroxydeoxyguanosine (8-OHdG) — a biomarker of oxidative DNA damage implicated in ageing and carcinogenesis (malignant transformation).
The stepwise injury cascade:
Trigger → ROS generation → lipid peroxidation + protein/DNA damage → membrane & organelle dysfunction → ↑ cytosolic Ca²⁺ → mitochondrial permeability transition → ATP depletion & apoptosis/necrosis.
High-yield: Malondialdehyde (MDA) is the classic laboratory marker of lipid peroxidation; 8-OHdG is the classic marker of oxidative DNA damage.
Antioxidant defence mechanisms
The body neutralises radicals through enzymatic and non-enzymatic systems. A clean two-line memory aid: "SOD-Catalase-Glutathione" for enzymes; "A, C, E + selenium" for the non-enzymatic vitamins/cofactors.
| Antioxidant | Type | Action / reaction | Cofactor |
|---|---|---|---|
| Superoxide dismutase (SOD) | Enzyme | 2 O₂·⁻ + 2H⁺ → H₂O₂ + O₂ | Cu/Zn (cytosol), Mn (mitochondria) |
| Catalase | Enzyme | 2 H₂O₂ → 2 H₂O + O₂ (in peroxisomes) | Iron (haem) |
| Glutathione peroxidase | Enzyme | H₂O₂/lipid peroxide + 2GSH → GSSG + H₂O | Selenium |
| Vitamin E (α-tocopherol) | Non-enzymatic | Major lipid/membrane-phase chain-breaking antioxidant | — |
| Vitamin C (ascorbate) | Non-enzymatic | Aqueous-phase; regenerates vitamin E | — |
| Glutathione (GSH) | Non-enzymatic | Major intracellular thiol buffer | — |
| Others | — | β-carotene, ceruloplasmin (Cu), transferrin/ferritin (Fe sequestration) | — |
High-yield: SOD is the first-line enzyme — it converts superoxide to H₂O₂, which is then cleared by catalase (peroxisomes) and glutathione peroxidase (cytosol/mitochondria, selenium-dependent). Glutathione peroxidase handles low/physiological H₂O₂ loads; catalase handles high concentrations.
High-yield: Vitamin E is the principal membrane-localised, lipid-soluble, chain-breaking antioxidant; vitamin C works in the aqueous phase and regenerates oxidised vitamin E.
Storage and transport proteins (transferrin, ferritin, lactoferrin, ceruloplasmin) act as antioxidants by sequestering free iron and copper, denying these metals to the Fenton/Haber–Weiss reactions.
Clinical conditions driven by oxidative stress
1. Ischaemia–reperfusion (I-R) injury — exam favourite
Paradoxically, restoring blood flow to ischaemic tissue causes additional damage. On reperfusion, re-oxygenation produces a burst of ROS from injured parenchymal/endothelial cells and infiltrating neutrophils, along with complement activation and calcium overload. The enzyme xanthine oxidase (formed from xanthine dehydrogenase during ischaemia) generates O₂·⁻ on reperfusion.
High-yield: In ischaemia–reperfusion injury, reperfusion (re-oxygenation) generates the ROS burst that worsens injury; allopurinol (xanthine oxidase inhibitor) is experimentally protective. Clinically relevant in MI thrombolysis/PCI, stroke, organ transplantation and free-flap surgery.
2. Chemical toxicity
- Carbon tetrachloride (CCl₄): Cytochrome P450 (CYP2E1) converts it to the trichloromethyl radical CCl₃·, causing hepatocyte lipid peroxidation → centrilobular fatty change and necrosis. A classic experimental model of free-radical injury.
- Paracetamol overdose: depletes glutathione; reactive metabolite NAPQI causes centrilobular hepatic necrosis. N-acetylcysteine (replenishes GSH) is the antidote.
- Paraquat (lung), doxorubicin (heart), bleomycin (lung fibrosis): redox cycling generates ROS.
3. Oxygen toxicity
- Retinopathy of prematurity (retrolental fibroplasia) and bronchopulmonary dysplasia in neonates given high inspired oxygen.
- Adult respiratory distress syndrome and oxygen-toxicity pneumonitis.
4. Ageing and degenerative disease
The free-radical theory of ageing attributes cumulative oxidative damage to mitochondrial DNA and proteins as a driver of senescence. Implicated in atherosclerosis (oxidised LDL), neurodegeneration (Parkinson's, Alzheimer's, ALS), cataract, and type-2 diabetes complications.
5. Carcinogenesis
Oxidative DNA damage (8-OHdG, strand breaks, point mutations) contributes to malignant transformation — the link concept between cell injury and neoplasia frequently tested in NEET PG.
6. Inflammation and the respiratory burst
Neutrophil NADPH oxidase produces O₂·⁻ → H₂O₂ → (via myeloperoxidase) HOCl for microbial killing. Loss of this system causes chronic granulomatous disease (CGD) — recurrent catalase-positive infections, diagnosed by negative nitroblue tetrazolium (NBT) test or abnormal dihydrorhodamine (DHR) flow cytometry.
Diagnosis & investigations (biomarkers)
There is no single bedside test; oxidative stress is assessed by surrogate markers:
| Marker | Reflects |
|---|---|
| Malondialdehyde (MDA) / TBARS | Lipid peroxidation |
| 4-hydroxynonenal | Lipid peroxidation |
| 8-OHdG | Oxidative DNA damage |
| Protein carbonyls | Protein oxidation |
| GSH : GSSG ratio | Redox status (low ratio = stress) |
| Exhaled pentane/ethane | Lipid peroxidation (non-invasive) |
| NBT / DHR test | Functional NADPH-oxidase assay (CGD) |
High-yield: The NBT (nitroblue tetrazolium) test is the classic screen for chronic granulomatous disease; the more sensitive modern test is dihydrorhodamine-123 flow cytometry.
Management / therapeutic principles
Management is condition-specific but built on three strategies: remove the source, scavenge the radical, support endogenous antioxidants.
- N-acetylcysteine – replenishes glutathione; drug of choice in paracetamol poisoning and used in contrast-induced nephropathy prophylaxis (limited evidence).
- Iron/copper chelation – deferoxamine/deferasirox in iron overload limits Fenton-driven damage.
- Allopurinol – xanthine-oxidase inhibition, protective in experimental reperfusion injury.
- Antioxidant vitamins – vitamin E, C, β-carotene, selenium (dietary; routine megadose supplementation is not proven to reduce mortality and β-carotene may increase lung-cancer risk in smokers).
- Controlled oxygen therapy in neonates to prevent retinopathy of prematurity.
- SOD/catalase mimetics – experimental.
High-yield: N-acetylcysteine is the antidote for paracetamol toxicity precisely because it restores depleted glutathione, the cofactor for glutathione peroxidase.
Complications & sequelae
Unchecked oxidative stress culminates in:
- Irreversible cell injury → apoptosis (low-grade ROS) or necrosis (overwhelming ROS, ATP collapse).
- Membrane failure, mitochondrial permeability transition, lysosomal rupture.
- Tissue fibrosis (e.g., bleomycin lung, CCl₄ cirrhosis).
- Mutagenesis → malignancy.
- Accelerated ageing and atherogenesis.
Key differentials / concepts often confused
| Confusion point | Clarification |
|---|---|
| ROS vs free radical | All radicals are reactive; not all ROS are radicals (H₂O₂, ¹O₂ are not) |
| SOD product vs catalase substrate | SOD makes H₂O₂; catalase/GPx remove H₂O₂ |
| Catalase vs glutathione peroxidase | Catalase = peroxisomes, high H₂O₂; GPx = selenium-dependent, low H₂O₂ + lipid peroxides |
| Most reactive vs first formed | OH· most reactive; O₂·⁻ formed first |
| Vitamin E vs vitamin C | E = lipid/membrane phase; C = aqueous, regenerates E |
| Apoptosis vs necrosis in ROS | Mild ROS → apoptosis; severe ROS + ATP loss → necrosis |
Recently asked / exam angle
- "Most reactive free radical" → hydroxyl radical (OH·).
- Which is NOT a free radical → H₂O₂ / singlet oxygen (classic odd-one-out MCQ).
- Fenton reaction requires → Fe²⁺ + H₂O₂ → OH·.
- Selenium-dependent antioxidant enzyme → glutathione peroxidase.
- Lipid-soluble chain-breaking antioxidant → vitamin E.
- Marker of lipid peroxidation → malondialdehyde; of oxidative DNA damage → 8-OHdG.
- Reperfusion injury mechanism / enzyme → ROS burst on re-oxygenation; xanthine oxidase; allopurinol protective.
- CCl₄ injury mechanism → CYP P450 → CCl₃· radical → hepatic lipid peroxidation.
- Paracetamol antidote rationale → NAC restores glutathione.
- NBT/DHR test → chronic granulomatous disease (NADPH oxidase defect).
- Retinopathy of prematurity → oxygen free-radical toxicity in neonates.
- Frequently appears as a linking concept across cell injury, inflammation (respiratory burst), and carcinogenesis chapters.
Rapid revision
- Free radical = species with an unpaired electron; H₂O₂ and singlet O₂ are ROS but not radicals.
- O₂·⁻ is formed first, OH· is the most reactive/damaging.
- Mitochondrial ETC is the largest endogenous ROS source (1–3% O₂ leak).
- Fenton: Fe²⁺ + H₂O₂ → OH·; Haber–Weiss: O₂·⁻ + H₂O₂ → OH· — both need free iron/copper.
- Three damage targets: lipid peroxidation (chief), protein cross-linking, DNA (8-OHdG).
- SOD → H₂O₂; cleared by catalase (peroxisome, high load) and glutathione peroxidase (selenium, low load + lipid peroxides).
- Vitamin E = membrane/lipid antioxidant; vitamin C regenerates it in the aqueous phase.
- MDA = lipid peroxidation marker; 8-OHdG = DNA oxidation marker.
- Ischaemia–reperfusion injury: ROS burst occurs on reperfusion; xanthine oxidase involved; allopurinol protective.
- CCl₄ → CCl₃· radical (via CYP450) → fatty liver & necrosis; paracetamol → NAPQI, antidote NAC (restores GSH).
- NADPH oxidase runs the respiratory burst; its defect = chronic granulomatous disease (NBT/DHR test).
- Oxidative stress underlies ageing, atherosclerosis (oxidised LDL), carcinogenesis, and oxygen toxicity (ROP).