Pathologic Effects of Free Radicals

Slide 1 · Pathologic Effects

of Free Radicals

Mechanisms of oxidative cellular injury

Dr Abu Suraih Sakhri

MBBS, MD (Pathology)

www.abusuraihsakhri.com

Slide 2 · Learning objectives

Identify the three principal macromolecular targets of free radical injury.

Explain how lipid peroxidation amplifies damage through chain propagation.

Relate protein and DNA oxidation to cellular dysfunction and outcomes.

Integrate antioxidant defenses with representative clinical injury mechanisms.

Slide 3 · Three major targets account for diverse effects

Membrane lipids undergo peroxidation, threatening membrane integrity.

Proteins acquire oxidative changes that alter structure, activity and turnover.

DNA develops breaks, cross-links and adducts with potential long-term effects.

Excess reactive species

Membrane lipids

Peroxidation

Proteins

Oxidation

DNA

Strand lesions

Slide 4 · A radical is defined by its electron configuration

A free radical contains an unpaired electron in an outer orbital.

The unpaired electron can favor reaction with nearby cellular molecules.

In some reactions, the attacked molecule becomes reactive and propagates injury.

Slide 5 · Reactive species are not all free radicals

Superoxide (O₂•⁻) and hydroxyl (•OH) are oxygen-centered radicals.

Hydrogen peroxide (H₂O₂) lacks an unpaired electron but is an important ROS.

Peroxynitrite (ONOO⁻) is a reactive nitrogen/oxygen species; Terminology should distinguish reactivity from radical status.

O₂•⁻

Superoxide

Radical

H₂O₂

Hydrogen peroxide

Non-radical ROS

•OH

Hydroxyl radical

Radical

ONOO⁻

Peroxynitrite

Reactive N/O species

Slide 6 · Oxidant production and clearance are normally balanced

Cells generate some ROS during ordinary metabolism.

Intracellular antioxidants and enzymes limit accumulation and damage.

An excess of production over scavenging results in oxidative stress.

ROS generation

ROS removal

Steady-state: Limited damage

Oxidative stress arises when generation exceeds removal

Slide 7 · Oxidative stress is a mechanism, not one diagnosis

An oxidant imbalance can contribute to chemically induced or radiation-induced injury.

It also participates in ischemia-reperfusion injury and cellular aging.

The tissue outcome depends on the target molecules and other concurrent injury pathways.

Slide 8 · ROS generation occurs in several settings

Endogenous metabolism generates partially reduced oxygen intermediates.

Radiant energy can produce radicals from water.

Inflammatory leukocytes intentionally generate ROS, while metals and chemicals can intensify radical reactions.

Metabolism

ROS / radicals

Radiation

ROS / radicals

Leukocytes

ROS / radicals

Metals / toxins

ROS / radicals

Slide 9 · Normal respiration generates reactive intermediates

Molecular oxygen is normally reduced during cellular respiration.

Partial oxygen reduction produces intermediates, including superoxide and hydrogen peroxide.

These species are continuously managed by enzymatic scavengers.

Normal oxidative metabolism

Partial O₂ reduction

Reactive oxygen intermediates

Slide 10 · Radiation can generate radicals from water

Radiant energy is one source of free radical generation.

Ionizing radiation can produce hydroxyl and hydrogen radicals by radiolysis of water.

Radicals can then interact with lipids, proteins and nucleic acids.

Ionizing radiation

Water

•OH and H• radicals

Slide 11 · The respiratory burst has a physiological purpose

Activated neutrophils and macrophages generate abundant reactive oxygen species.

A membrane-associated NADPH oxidase complex controls this response.

The products help destroy microbes, but excessive or misdirected oxidant production can contribute to tissue injury.

Activated leukocyte

Inflammation

NADPH oxidase

Controlled ROS burst

Microbial killing

Desired function

Slide 12 · Other oxidases and xenobiotics generate radicals

Intracellular oxidases such as xanthine oxidase can generate superoxide.

Enzymatic metabolism of certain foreign chemicals generates reactive radical intermediates.

Not every injurious radical is oxygen-derived: The trichloromethyl radical is one example.

Slide 13 · Iron can amplify radical formation

Transition metals catalyze electron-transfer reactions associated with radical formation.

The Fenton reaction uses hydrogen peroxide and ferrous iron to generate hydroxyl radicals.

Superoxide can facilitate iron reduction, allowing radical-generating processes to interact.

H₂O₂ + Fe²⁺

Fe³⁺ + •OH + OH⁻

Transition-metal availability influences radical formation

Slide 14 · Nitric oxide connects oxygen and nitrogen chemistry

Nitric oxide is formed by several cell types, including endothelium and leukocytes.

Its reactions may generate highly reactive nitrogen-containing products, including peroxynitrite.

Reactive nitrogen and oxygen species participate in injury, particularly during reperfusion.

Slide 15 · Different sources converge on common macromolecules

The source of a reactive species does not uniquely determine the injured molecule.

Membrane lipids, proteins and DNA are all potential oxidative targets.

The site and severity of oxidant generation influence the pattern of injury.

Excess reactive species

Membrane lipids

Peroxidation

Proteins

Oxidation

DNA

Strand lesions

Slide 16 · Lipid peroxidation affects plasma and organelle membranes

Membranes are lipid-rich structures that contain susceptible fatty-acid chains.

Oxidant reactions can occur in plasma membranes and membranes of intracellular organelles.

Damage to membrane components can compromise membrane function.

Membrane phospholipid bilayer — schematic

Unsaturated lipid tails provide targets for radical attack

Slide 17 · Unsaturated fatty acids are vulnerable targets

Oxidative attack involves the double bonds of unsaturated fatty acids within membrane lipids.

Reaction of these lipids with radicals generates chemically reactive intermediates.

The architecture of the lipid bilayer makes widespread damage functionally important.

Membrane phospholipid bilayer — schematic

Unsaturated lipid tails provide targets for radical attack

Slide 18 · Initiation begins with radical attack on a lipid

An oxygen-derived radical interacts with an unsaturated membrane lipid.

The attacked lipid becomes part of a reactive sequence rather than remaining inert.

This initiation step permits subsequent reactions with neighboring lipids.

Radical attack

Lipid reaction

Reactive peroxide

Further reactions

Autocatalytic propagation can extend injury

Slide 19 · Propagation turns a local reaction into a chain

Lipid–radical interactions yield peroxides that are themselves reactive and unstable.

These reactive products support further lipid oxidation.

The propagation process can extend damage beyond the initial point of attack.

Initial attack

Reactive lipid products

New lipid attack

Chain propagation

Autocatalytic propagation can extend injury

Slide 20 · The chain reaction can amplify membrane damage

Lipid peroxidation is described as autocatalytic because products can sustain additional reactions.

A discrete initial radical reaction may therefore evolve into more extensive injury.

The ultimate pathological consequence is loss of membrane integrity or function.

Radical attack

Lipid radicals

Reactive peroxides

Further lipid oxidation

Autocatalytic propagation can extend injury

Slide 21 · Lipid peroxides are not chemically inert products

Formation of a lipid peroxide does not necessarily terminate damage.

Instability and reactivity of peroxidation products contribute to chain propagation.

This distinguishes peroxidation from a single isolated covalent modification.

Slide 22 · Plasma membrane injury compromises cell integrity

Peroxidation of the plasma membrane is one biochemical route to membrane damage.

Loss of normal selective permeability is an important event in cell injury.

Severe membrane defects contribute to irreversible injury and cell death.

Lipid peroxidation

Membrane dysfunction

Cellular injury

Slide 23 · Oxidative injury also affects organellar membranes

Lipid peroxidation need not be restricted to the cell surface.

Organellar membranes contain lipids vulnerable to oxidative reactions.

Disruption of mitochondrial membranes can cooperate with impaired ATP production and cell-death pathways.

Lipid peroxidation

Membrane dysfunction

Cellular injury

Slide 24 · Membrane damage is mechanistically heterogeneous

ROS-induced lipid peroxidation is one mechanism of membrane injury.

ATP depletion can reduce phospholipid synthesis.

Calcium-dependent phospholipases accelerate phospholipid breakdown; Cytoskeletal defects also weaken membrane stability.

Slide 25 · Oxidative lipid injury interacts with other pathways

Impaired energy production can make membrane maintenance more difficult.

Calcium dysregulation can activate phospholipid-degrading enzymes.

Free radical injury may therefore act alongside rather than replace ATP depletion and calcium-mediated injury.

Lipids → membrane disruption

Proteins → altered cell function

DNA → genomic lesions

Slide 26 · Protein oxidation comprises several distinct reactions

Oxidative modification can involve amino acid side chains.

Free radicals can produce covalent cross-links between protein molecules.

The protein backbone is another site of oxidative modification.

Side-chain oxidation

Protein cross-links

Backbone oxidation

Conformational change

Slide 27 · Amino acid side-chain oxidation changes protein chemistry

Protein side chains contain chemical groups that can be modified by oxidants.

Side-chain oxidation can alter interactions necessary for protein structure and function.

The effect depends on the location and role of the modified residue.

Side-chain oxidation

Protein cross-links

Backbone oxidation

Conformational change

Slide 28 · Covalent cross-links can alter protein organization

Oxidative stress can promote protein–protein covalent cross-linking.

Disulfide bond formation is an example cited in the source.

Inappropriate cross-linking may alter the conformation and behavior of cellular proteins.

Protein oxidation

Covalent cross-links

Altered folding

Functional disturbance

Slide 29 · Oxidation may involve the protein backbone

Free radical reactions are not limited to amino acid side chains.

Oxidation of the protein backbone can disturb protein integrity.

This expands the range of possible structural and biochemical consequences.

Side-chain oxidation

Protein cross-links

Backbone oxidation

Conformational change

Slide 30 · Oxidized enzyme active sites may lose function

The catalytic activity of an enzyme depends on correct active-site structure.

Oxidative modification of active sites can disrupt that structure.

Impaired enzyme function can propagate metabolic disturbances throughout the cell.

Native enzyme

Oxidative active-site change

Impaired catalytic function

Slide 31 · Structural proteins depend on stable conformation

Structural proteins contribute to cellular organization and mechanical integrity.

Oxidative modification may change their three-dimensional conformation.

Such changes can compromise normal interactions and structural roles.

Structural protein

Oxidative change

Conformation altered

Altered cellular function

Slide 32 · Oxidative injury increases the burden on proteostasis

Oxidized proteins can unfold or misfold.

Proteasomal degradation of abnormal proteins may increase.

Protein oxidation therefore disrupts both function and protein-quality control.

Oxidative modification

Misfolding / unfolding

Proteasomal degradation

Slide 33 · Protein injury extends beyond individual molecules

Oxidative changes can affect enzymes, structural proteins and protein-turnover pathways simultaneously.

The resulting consequences can be biochemical, structural and regulatory.

Global cellular dysfunction can emerge without a single uniquely diagnostic protein lesion.

Lipids → membrane disruption

Proteins → altered cell function

DNA → genomic lesions

Slide 34 · DNA is a major molecular target of oxidants

Free radicals react with nucleic acids as well as proteins and lipids.

Strand breaks, cross-linking and adduct formation as major DNA lesions.

These processes can have immediate and persistent biological consequences.

Single-strand break

Cross-link

Adduct

Slide 35 · Single- and double-strand breaks are distinct lesions

A single-strand break interrupts one DNA strand.

A double-strand break affects both strands of the double helix.

Both forms are included among the DNA lesions induced by free radicals.

Single-strand break

One strand interrupted

Double-strand break

Both strands interrupted

Cross-link / adduct

DNA chemistry altered

Slide 36 · DNA cross-linking creates abnormal covalent connections

Free radicals can produce cross-linking involving DNA strands.

Such lesions chemically link components that are normally able to separate.

Cross-linking is mechanistically different from direct strand breakage.

Single-strand break

One strand interrupted

Double-strand break

Both strands interrupted

Cross-link / adduct

DNA chemistry altered

Slide 37 · Adduct formation chemically modifies DNA

An adduct is a chemical modification formed when another group becomes attached to DNA.

Oxidative reactions can create DNA adducts in addition to strand breaks.

The specific downstream effect depends on how the lesion is processed by the cell.

Single-strand break

One strand interrupted

Double-strand break

Both strands interrupted

Cross-link / adduct

DNA chemistry altered

Slide 38 · DNA damage activates protective cell responses

In the surrounding Robbins discussion, DNA damage activates p53-dependent responses.

Cell-cycle arrest and DNA repair help prevent propagation of damaged genetic material.

When damage is not successfully corrected, p53 can initiate apoptotic signaling.

DNA damage

P53 response

Cell-cycle arrest / repair

Apoptosis if damage persists

Slide 39 · Oxidative DNA injury has been linked to cellular aging

The supplied text implicates oxidative DNA damage in cellular aging.

Accumulated nucleic-acid injury provides one mechanistic connection between stress and age-associated change.

This is an implicated contribution, not a claim that ROS explain all biological aging.

Slide 40 · Oxidative DNA injury has oncogenic implications

Oxidative DNA damage is implicated in malignant transformation.

Persistent or improperly resolved genetic lesions are relevant to abnormal cell survival and proliferation.

The relationship is mechanistic; It does not mean that every oxidatively injured cell becomes malignant.

Slide 41 · Apoptosis can prevent persistence of damaged DNA

The parent discussion emphasizes cell-cycle control and DNA repair after injury.

If damage cannot be corrected, apoptosis removes cells carrying potentially dangerous DNA changes.

This protective response illustrates why DNA injury can result in cell death rather than mutation survival.

DNA damage

P53 response

Cell-cycle arrest / repair

Apoptosis if damage persists

Slide 42 · Lipid, protein and DNA injury differ in consequences

Lipid injury primarily threatens membrane properties.

Protein oxidation can impair catalysis, architecture and turnover.

DNA lesions threaten genomic integrity and engage damage-response pathways.

Excess reactive species

Membrane lipids

Peroxidation

Proteins

Oxidation

DNA

Strand lesions

Slide 43 · The three macromolecular targets often coexist

A given oxidant exposure can affect more than one class of cellular macromolecule.

Membrane failure, protein dysfunction and DNA lesions may interact in severe injury.

Understanding the dominant target explains how radical chemistry becomes cellular pathology.

Lipids → membrane disruption

Proteins → altered cell function

DNA → genomic lesions

Slide 44 · Oxidative injury can contribute to necrosis

Historically, free radical injury was commonly linked to necrotic cell death.

Extensive membrane injury is especially relevant to loss of cellular integrity.

Oxidative injury frequently operates alongside ATP depletion and other necrosis-promoting processes.

Excessive ROS

Necrosis

Apoptosis

Depending on the injury context, oxidative damage can participate in both pathways

Slide 45 · Free radicals can also trigger apoptosis

Robbins explicitly identifies apoptosis as another possible consequence of radical injury.

DNA damage and mitochondrial pathways offer routes from injurious stress to apoptotic responses.

Thus necrosis is not the only fate of cells exposed to excessive reactive species.

Excessive ROS

Necrosis

Apoptosis

Depending on the injury context, oxidative damage can participate in both pathways

Slide 46 · Reactive species are not uniformly pathological

Free radicals produced under controlled conditions may serve physiological signaling roles.

Their effects are influenced by dose, cellular context and local regulation.

The distinction is regulated signaling versus damaging oxidative excess.

Controlled, low-level ROS

Receptor / pathway signaling

Physiologic function is context-dependent

Slide 47 · Cellular outcome reflects context rather than ROS alone

Controlled generation can support signaling or microbial killing.

Excessive generation or inadequate detoxification promotes macromolecular injury.

The severity and reversibility of the associated cellular disturbance shape final outcomes.

ROS generation

ROS removal

Steady-state: Limited damage

Oxidative stress arises when generation exceeds removal

Slide 48 · Cells have overlapping antioxidant defenses

Free radicals may decay spontaneously and can be scavenged by specific molecules.

Cells also limit participation of redox-active metals in harmful reactions.

Specialized enzymes detoxify superoxide and hydrogen peroxide near their sources.

Antioxidants

Limit oxidant injury

Metal binding

Limit oxidant injury

SOD

Limit oxidant injury

Catalase / GPx

Limit oxidant injury

Slide 49 · Small-molecule antioxidants limit radical injury

The text lists vitamin E, vitamin A, ascorbic acid and glutathione among protective antioxidants.

These agents can block radical formation or inactivate reactive species.

Protection is one component of the broader enzymatic and metal-binding network.

Slide 50 · Metal-binding proteins reduce catalytic availability

Free iron and copper can participate in radical-forming reactions.

Proteins including transferrin, ferritin, lactoferrin and ceruloplasmin sequester reactive metals.

Metal sequestration limits the opportunity for radical amplification.

H₂O₂ + Fe²⁺

Fe³⁺ + •OH + OH⁻

Transition-metal availability influences radical formation

Slide 51 · Superoxide dismutase converts superoxide

Superoxide dismutase (SOD) catalyzes conversion of superoxide to hydrogen peroxide and oxygen.

Manganese-SOD is localized in mitochondria; Copper-zinc-SOD occurs in the cytoplasm.

Hydrogen peroxide still requires additional detoxification.

2 O₂•⁻ + 2 H⁺

H₂O₂ + O₂

Catalase / GPx then remove hydrogen peroxide

Slide 52 · Catalase detoxifies hydrogen peroxide

Catalase is localized in peroxisomes.

It decomposes hydrogen peroxide into water and oxygen.

This limits hydrogen peroxide accumulation and its participation in further oxidant reactions.

2 H₂O₂

2 H₂O + O₂

Catalase acts in peroxisomes

Slide 53 · Glutathione peroxidase consumes reducing capacity

Glutathione peroxidase detoxifies hydrogen peroxide using reduced glutathione (GSH).

Oxidized glutathione (GSSG) is produced as hydrogen peroxide is converted into water.

The GSSG-to-GSH relationship reflects the cellular oxidative state.

H₂O₂ + 2 GSH

2 H₂O + GSSG

Reduced glutathione is consumed as oxidants are detoxified

Slide 54 · Antioxidant systems form a coordinated sequence

SOD converts superoxide into hydrogen peroxide.

Catalase and glutathione peroxidase help remove hydrogen peroxide.

Metal sequestration and molecular antioxidants act through complementary mechanisms.

Antioxidants

Limit oxidant injury

Metal binding

Limit oxidant injury

SOD

Limit oxidant injury

Catalase / GPx

Limit oxidant injury

Slide 55 · Oxidative stress occurs when protection is overwhelmed

Increased ROS generation can exceed the capacity of scavenging systems.

Alternatively, weakened or depleted defenses can increase susceptibility to injury.

The relevant abnormality is the imbalance between the two processes.

ROS generation

ROS removal

Steady-state: Limited damage

Oxidative stress arises when generation exceeds removal

Slide 56 · Reperfusion can both rescue and injure tissue

Restoring blood flow can recover reversibly ischemic cells.

Reoxygenation can also initiate new injurious processes in vulnerable tissue.

Ischemia-reperfusion injury is especially relevant to myocardial and cerebral infarction.

Ischemic tissue

Reoxygenation

ROS / RNS generation

Additional cell injury

Slide 57 · ROS and RNS contribute to reperfusion injury

Reoxygenated leukocytes, endothelial cells and parenchymal cells can generate reactive species.

Antioxidant defenses may already be compromised by preceding ischemia.

The resulting oxidative stress is one component of reperfusion-associated cell loss.

Ischemic tissue

Reoxygenation

ROS / RNS generation

Additional cell injury

Slide 58 · Reperfusion injury involves interacting pathways

Oxidative stress may coexist with intracellular calcium overload.

Calcium excess promotes mitochondrial dysfunction and further cellular injury.

Inflammatory-cell recruitment and complement activation also contribute to tissue damage.

Slide 59 · Leukocyte ROS have both benefit and risk

Phagocytes deliberately generate ROS to kill microbes and clear damaged material.

ROS also contribute to collateral tissue injury in some inflammatory contexts.

The pathologic significance depends on localization, regulation and tissue susceptibility.

Activated leukocyte

Inflammation

NADPH oxidase

Controlled ROS burst

Microbial killing

Desired function

Slide 60 · Carbon tetrachloride illustrates radical-mediated toxicity

Cytochrome P-450 metabolizes carbon tetrachloride (CCl₄) to a reactive trichloromethyl radical (•CCl₃).

That radical initiates lipid peroxidation and damage to cellular structures.

It demonstrates how chemical metabolism can generate radical-mediated cell injury.

CCl₄

Cytochrome P-450

•CCl₃ radical

Lipid peroxidation / injury

Slide 61 · Reactive metabolites explain one form of chemical injury

Many toxic compounds must first undergo enzymatic conversion to reactive metabolites.

Radical formation and subsequent lipid peroxidation are major mechanisms for some chemicals.

Other toxic mechanisms, including direct covalent binding, may coexist.

CCl₄

Cytochrome P-450

•CCl₃ radical

Lipid peroxidation / injury

Slide 62 · Lipofuscin is a morphologic clue to lipid oxidation

Lipofuscin is a yellow-brown cytoplasmic pigment related to lipid peroxidation products.

It is particularly evident in aging or chronically stressed tissues such as heart and liver.

The pigment itself is not considered injurious; It indicates prior free radical injury.

Membrane lipid peroxidation

Lipofuscin accumulation

Indicator, not injurious pigment

Slide 63 · Do not infer ROS injury from morphology alone

Lipid peroxidation, protein oxidation and DNA lesions are biochemical processes.

A nonspecific pattern of cell injury does not identify a unique free radical mechanism.

Interpret oxidative stress in the context of the cause, tissue response and competing injury pathways.

Slide 64 · Clinical application: Tissue injury after reperfusion

A patient undergoes restoration of coronary blood flow after prolonged ischemia.

Some jeopardized cells recover; Additional injury may nevertheless occur after reperfusion.

Which source of ROS and which antioxidant vulnerability help explain this paradox?

Ischemic tissue

Reoxygenation

ROS / RNS generation

Additional cell injury

Slide 65 · Reperfusion: Mechanistic interpretation

Reactive oxygen and nitrogen species are generated during reoxygenation.

Leukocytes and previously injured endothelial and parenchymal cells can contribute.

Compromised antioxidant defenses amplify the impact; Calcium overload and inflammation add injury.

Slide 66 · Mechanism exercise: Identify the primary target

Loss of membrane function after an autocatalytic chain reaction points to lipid peroxidation.

Inactivated enzymes and increased abnormal-protein turnover point to protein oxidation.

DNA strand breaks, cross-links and adducts point to nucleic-acid damage.

Excess reactive species

Membrane lipids

Peroxidation

Proteins

Oxidation

DNA

Strand lesions

Slide 67 · Integrative model of free radical pathology

A shift in oxidant production relative to detoxification establishes oxidative stress.

Reactive species modify membrane lipids, proteins and DNA through distinct chemical reactions.

Combined macromolecular damage can trigger dysfunction, necrosis or apoptosis; Controlled ROS also signal physiologically.

Excess reactive species

Membrane lipids

Peroxidation

Proteins

Oxidation

DNA

Strand lesions

Slide 68 · Primary reference

Robbins, Cotran & Kumar

Pathologic Basis of Disease · 11th edition (2026)

Chapter 2: Cell Injury, Cell Death, and Adaptations

Relevant sections: oxidative stress, generation and removal of free radicals,

pathologic effects, membrane and DNA injury, and selected clinical correlations.