Generation of Free Radicals

Slide 1 · Generation of

Free Radicals

Cell injury · oxygen and nitrogen redox chemistry

Dr Abu Suraih Sakhri

MBBS, MD (Pathology)

www.abusuraihsakhri.com

Slide 2 · Learning objectives: Sources, species, reactions

Classify oxygen- and nitrogen-derived reactive intermediates.

Explain sequential reduction of molecular oxygen.

Identify enzymatic, inflammatory, physical and chemical sources.

Trace Fenton chemistry and the formation of peroxynitrite.

Connect generation mechanisms to selected clinical scenarios.

Slide 3 · Oxidative stress belongs to the core mechanisms of cell injury

The context

Physiologic oxidant formation

Rate of formation exceeds control

Reactive species accumulate

Cellular oxidative stress

Oxidative stress with chemical injury, radiation and reperfusion.

The same chemistry participates in host antimicrobial defense.

Slide 4 · What makes a molecule a free radical?

Unpaired electron

A free radical has at least one unpaired electron. That electron contributes to chemical reactivity.

Reaction propagation

Transfer of an electron or hydrogen atom can generate a new radical, sometimes sustaining a chain reaction.

Not every biologically important oxidant contains an unpaired electron.

Slide 5 · Free radical is a chemical category—not a synonym for ROS

Superoxide

O₂•⁻

Oxygen-derived radical

Hydrogen peroxide

H₂O₂

Reactive oxygen species; Not a radical

Hydroxyl radical

•OH

Very reactive oxygen-derived radical

Nitric oxide

NO•

Nitrogen-centered free radical

Peroxynitrite

ONOO⁻

Reactive nitrogen species; Not a radical

Slide 6 · ROS and RNS are overlapping, but distinct, concepts

Reactive oxygen species (ROS)

Include oxygen-centered radicals and nonradicals.

Examples: O₂•⁻, H₂O₂ and •OH.

Chemical behavior differs markedly between species.

Reactive nitrogen species (RNS)

Involve nitrogen-centered or nitrogen-containing chemistry.

NO• is a free radical.

ONOO⁻ is reactive but is not a free radical.

Slide 7 · Six principal routes to reactive species production

Basal metabolism

Mitochondria · ER · cytosol · peroxisomes · lysosomes

Activated host defense

Phagocyte NADPH oxidase

Physical and chemical

Radiation · xenobiotic metabolism

Catalytic chemistry

Fe²⁺ / Cu⁺ cycling · NO + O₂•⁻

Slide 8 · Normal oxygen reduction uses four electrons

O₂ + 4e⁻ + 4H⁺ → 2H₂O

Complete reduction

This is the net chemistry of oxygen reduction to water.

Biologic respiratory enzymes coordinate the electron-transfer process.

Incomplete reduction gives rise to reactive intermediates.

Slide 9 · Partial oxygen reduction generates reactive intermediates

O₂

Oxygen substrate

O₂•⁻

One-electron radical

H₂O₂

Nonradical peroxide

•OH

Fenton-derived radical

Partial reduction

Dismutation / oxidases

Fe²⁺ + H₂O₂

Separate complete reduction: O₂ + 4e⁻ + 4H⁺ → 2H₂O

These are possible generating routes—not obligatory stages of respiration.

Slide 10 · One-electron reduction produces superoxide

O₂ + e⁻ → O₂•⁻

First electron

Superoxide is a radical anion.

It forms during incomplete oxygen reduction and through oxidase activity.

Its local abundance depends on production and dismutation.

Slide 11 · Two-electron reduction produces hydrogen peroxide

O₂ + 2e⁻ + 2H⁺ → H₂O₂

Peroxide formation

Hydrogen peroxide contains no unpaired electron.

It can arise through superoxide dismutation or direct oxidase reactions.

It can feed further chemistry, especially metal-catalyzed •OH generation.

Slide 12 · Hydroxyl radical: High reactivity, short range

Formation

Ionizing radiation can generate •OH from water. Fe²⁺ can generate •OH from hydrogen peroxide via Fenton chemistry.

Consequence of reactivity

The hydroxyl radical reacts rapidly close to where it forms; The local generation site matters greatly.

•OH is the radical species; OH⁻ is a distinct hydroxide ion.

Slide 13 · Do not conflate superoxide, peroxide and hydroxyl radical

O₂•⁻

Radical anion; One-electron product

H₂O₂

Nonradical; Precursor for •OH

•OH

Highly reactive radical

These species are connected by chemistry but are not interchangeable.

Naming the particular oxidant improves mechanistic interpretation.

Slide 14 · Radical generation depends on electron-transfer context

Sequential mechanism

Electron donor

Redox enzyme / metal

O₂ or peroxide substrate

Reactive intermediate

Both the substrate and the catalytic setting determine the product.

Compartmentalization directs which biomolecules can be exposed.

Slide 15 · Reactive species formation also occurs in healthy cells

Basal redox metabolism produces small quantities of reactive intermediates.

Formation is not synonymous with tissue injury.

Cells normally maintain a dynamic balance between production and removal.

Oxidative stress denotes an imbalance in favor of oxidants.

Normal generation is expected; Pathologic accumulation is context-dependent.

Slide 16 · Oxygen-derived species arise in several compartments

CELL: Multiple oxidant-generating compartments

MITOCHONDRION

Respiratory redox

ENDOPLASMIC

RETICULUM

PEROXISOME

LYSOSOME

CYTOSOL: Soluble oxidases

Slide 17 · Mitochondria link respiration to superoxide production

Unintended side products of respiratory electron transfer

Electron transport

O₂ receives partial electron transfer

O₂•⁻ forms locally

Further ROS chemistry

Mitochondrial respiration is a normal source of oxygen-derived intermediates.

The central distinction is complete versus incomplete O₂ reduction.

Slide 18 · Electron leakage is a route to mitochondrial ROS

Ordered electron transfer

Respiration channels electrons toward four-electron reduction of oxygen to water.

Side transfer to O₂

Premature, single-electron reduction can generate O₂•⁻ before complete water formation.

Slide 19 · Mitochondrial generation is local but has broader consequences

Superoxide is produced near the electron-transfer source.

Related oxidants may be formed as superoxide is converted.

H₂O₂ can influence sites beyond its initial production compartment.

The production–removal balance determines net exposure.

Slide 20 · Cytosolic oxidases are additional ROS sources

Cytosol

Soluble oxidoreductases

Substrate

O₂ accepts electrons

Products

O₂•⁻ or H₂O₂

Cytosolic oxidative enzymes among sources of intermediates.

Enzyme identity and substrate availability govern product formation.

Slide 21 · Endoplasmic-reticulum redox reactions may generate ROS

Sequential mechanism

ER enzyme-mediated oxidation

Electron transfer to O₂

Partial reduction products

The ER is part of the intracellular oxidative-enzyme network.

Oxidative drug metabolism offers an important related clinical example.

Slide 22 · Peroxisomal oxidases contribute to hydrogen peroxide

Sequential mechanism

Peroxisomal oxidase

O₂ accepts electrons

H₂O₂ generated

Peroxisomes contain enzymes that create H₂O₂ during substrate oxidation.

Peroxide production and peroxide detoxification coexist within the organelle.

Slide 23 · Lysosomes are included among cellular redox compartments

The source chapter lists lysosomal oxidative enzymes among cellular sources.

The lysosomal environment is relevant to degradative cell functions.

Do not assume all lysosomal ROS originate from mitochondrial leakage.

Exact enzyme-specific pathways require separate mechanistic evidence.

Slide 24 · Xanthine oxidase is a named intracellular oxidant source

Oxidase-mediated production

Purine metabolic substrates

Xanthine oxidase activity

Transfer of electrons to O₂

Superoxide formation

Robbins explicitly identifies xanthine oxidase as an intracellular O₂•⁻-generating oxidase.

The source illustrates enzyme-driven generation beyond mitochondrial respiration.

Slide 25 · Basal metabolic ROS versus deliberate immune-cell ROS

Ordinary metabolism

Redox enzymes function continuously.

Small reactive intermediate quantities may appear.

Exposure is usually constrained by cellular defenses.

Leukocyte respiratory burst

Activation rapidly elevates oxidant production.

A multiprotein NADPH oxidase complex is involved.

Oxidants support microbial killing.

Slide 26 · Activated phagocytes intentionally generate oxidants

Sequential mechanism

Pathogen recognition / activation

Oxidase activation

Rapid O₂•⁻ generation

Microbicidal chemistry

Inflammation recruits neutrophils and macrophages that produce reactive oxygen species.

The burst is a controlled host-defense program, not simply metabolic leakage.

Slide 27 · NADPH oxidase is a membrane-associated electron-transfer system

Extracellular fluid / phagosomal lumen

O₂

O₂•⁻

NADPH

Oxidase (NOX2)

CYTOSOL

NADPH → NADP⁺

Electrons transferred across membrane

E⁻

Slide 28 · NADPH supplies reducing equivalents for phagocyte oxidase

NADPH + 2O₂ → NADP⁺ + H⁺ + 2O₂•⁻

NOX reaction — simplified

The oxidase transfers electrons from cytosolic NADPH to molecular oxygen.

Superoxide is generated on the membrane-opposite side of electron donation.

This schematic omits regulatory subunits and proton-coupled steps.

Slide 29 · Respiratory burst connects superoxide to other oxidants

Sequential mechanism

NOX-generated O₂•⁻

Dismutation

H₂O₂

Subsequent oxidant chemistry

H₂O₂ is a downstream product of superoxide conversion.

Specific microbial killing pathways depend on phagocyte enzymes and local conditions.

Slide 30 · The location of oxidase assembly matters

Phagosomal membrane

Electron transfer can create oxidants in the phagosomal lumen.

Reactive intermediates confront ingested microbes.

Cell surface membrane

Activated oxidase can release oxidants extracellularly.

Surrounding host tissue may be exposed during inflammation.

Slide 31 · Leukocyte oxidant formation is controlled and inducible

Inflammatory activation precedes the rapid increase in oxidant production.

A multiprotein NADPH oxidase system mediates the response.

The respiratory burst is separable from mitochondrial respiration.

Defects in this pathway compromise a major antimicrobial mechanism.

Slide 32 · Defective superoxide production: Chronic granulomatous disease

Sequential mechanism

NADPH oxidase defect

Impaired superoxide burst

Reduced oxidative microbial killing

Recurrent infections

Chronic granulomatous disease as the classic clinical consequence.

Not all leukocyte antimicrobial functions are lost.

Slide 33 · Clinical application: Failure of respiratory burst

CASE / APPLICATION

A child has recurrent bacterial and fungal infections. Neutrophils phagocytose organisms, but the oxidative burst is defective.

Which reaction is primarily compromised?

A. NADPH oxidase–mediated O₂•⁻ production

B. Peroxisomal catalase breakdown of H₂O₂

C. Fe²⁺-mediated peroxide cleavage

Slide 34 · Radiant energy can initiate radical chemistry

Energy input

Radiant energy absorbed

Primary target

Water and other molecules

Reactive products

Radical intermediates

Ultraviolet light and x-rays among relevant radiant energy sources.

For ionizing radiation, radiolysis of water is a well-established route to •OH.

Slide 35 · Radiolysis of water produces reactive radical fragments

H₂O + ionizing radiation → •OH + H•

Simplified radiolysis

This is an illustrative net representation, not a full radiation-chemistry reaction set.

Hydroxyl radicals formed locally can attack nearby cellular targets.

Slide 36 · Ionizing radiation and water radiolysis

Sequential mechanism

Ionizing radiation

Water ionization / excitation

Reactive intermediates

Nearby molecular reactions

Water is abundant in cells and serves as a major radiation target.

Short-lived radicals can mediate an indirect component of radiation injury.

Slide 37 · UV irradiation and x-rays are not mechanistically identical

Ultraviolet light

Can initiate photochemical cellular damage.

Wavelength and target determine the pathway.

Not every UV effect is explained by water radiolysis.

X-rays / ionizing radiation

Can ionize water and generate free radicals.

•OH is among important products.

Radical and direct ionization effects may coexist.

Slide 38 · Drug and chemical metabolism can generate non-ROS radicals

Sequential mechanism

Exogenous compound

Enzymatic biotransformation

Reactive organic radical

Subsequent molecular reactions

Free radicals are not limited to oxygen-centered species.

Carbon tetrachloride as a chemical example.

Slide 39 · Carbon tetrachloride: A classic radical-forming xenobiotic

CCl₄ + e⁻ → •CCl₃ + Cl⁻

Reductive bioactivation — schematic

Reductive metabolism forms the trichloromethyl radical (•CCl₃).

Further reactions can propagate damage to cellular membranes.

An organic radical can initiate downstream ROS-linked injury.

Slide 40 · Radical formation and oxidative damage are not the same step

Initiation

CCl₄ is converted enzymatically to •CCl₃.

The newly formed radical is not itself an ROS.

Propagation

Reactive intermediates can attack neighboring molecules.

A radical chain can amplify the initial chemical event.

Slide 41 · Chemical exposure: Identify the initiating species

CASE / APPLICATION

A halogenated solvent undergoes enzymatic reductive metabolism in hepatocytes, generating a carbon-centered free radical.

What is the principal initiating product in the Robbins example?

A. Trichloromethyl radical (•CCl₃)

B. Superoxide (O₂•⁻) as the only product

C. Peroxynitrite (ONOO⁻)

Slide 42 · Transition metals catalyze radical production

Sequential mechanism

Redox-active Fe / Cu

Electron donation or acceptance

Peroxide chemistry

Reactive radical generation

Iron and copper can move between oxidation states during redox reactions.

Metal availability changes which peroxide reactions proceed.

Slide 43 · Fenton chemistry generates hydroxyl radicals

Fe²⁺ + H₂O₂ → Fe³⁺ + •OH + OH⁻

Fenton reaction

Ferrous iron (Fe²⁺) participates directly.

Hydrogen peroxide is converted into hydroxyl radical plus hydroxide.

Ferric iron (Fe³⁺) is the oxidized iron product.

Slide 44 · Fe³⁺ must be reduced to Fe²⁺ to sustain Fenton chemistry

Fe²⁺ + H₂O₂

Electron donation / peroxide cleavage

Fe³⁺ + •OH + OH⁻

Highly reactive hydroxyl radical

Superoxide can facilitate Fe³⁺ → Fe²⁺ recycling

O₂•⁻ present

Fe³⁺ reduction

Fe²⁺ regenerated

Further Fenton chemistry

Slide 45 · Superoxide can support Fe³⁺ to Fe²⁺ recycling

O₂•⁻ + Fe³⁺ → O₂ + Fe²⁺

Conceptual metal cycling

This electron-transfer reaction replenishes ferrous iron.

Superoxide and peroxide may therefore cooperate in •OH production.

The Fenton reaction itself consumes Fe²⁺ and generates Fe³⁺.

Slide 46 · Redox-active copper provides a related catalytic route

Metal redox flexibility

Copper also alternates between oxidation states while donating or accepting electrons.

Biologic implication

The availability of poorly bound, reactive metal ions can accelerate oxidant-generating reactions.

The core Robbins passage identifies both iron and copper as redox-active catalysts.

Slide 47 · Metal-binding proteins restrict the reactive metal pool

Iron binding

Ferritin / transferrin

Copper binding

Ceruloplasmin

Inflammatory sites

Lactoferrin

Tightly bound metal is less available to catalyze free-radical formation.

This is preventive context; Removal of radicals is treated in the next Robbins topic.

Slide 48 · Why peroxide plus labile iron is a hazardous combination

CASE / APPLICATION

A cell accumulates hydrogen peroxide and an expanded pool of redox-active ferrous iron. Antioxidant buffering is overwhelmed.

Which species is directly generated by the Fenton reaction?

A. Hydroxyl radical (•OH)

B. Nitric oxide (NO•)

C. Molecular oxygen (O₂) alone

Slide 49 · Nitric oxide is a free radical generated by several cell types

Endothelium

NO supports vascular signaling

Macrophages

NO participates in host defense

Neurons

NO functions as a signaling mediator

NO as an important chemical mediator and a free radical.

Its chemistry becomes particularly important in the presence of superoxide.

Slide 50 · Nitric oxide differs chemically from peroxynitrite

Nitric oxide: NO•

Nitrogen-centered radical.

Synthesized in multiple cell types.

Acts as a mediator and reacts with O₂•⁻.

Peroxynitrite: ONOO⁻

Formed from NO• and O₂•⁻.

Reactive nitrogen-containing oxidant.

Not itself a free radical.

Slide 51 · Superoxide reacts with nitric oxide to form peroxynitrite

NO• + O₂•⁻ → ONOO⁻

ROS–RNS coupling

The reaction links sources of nitric oxide with superoxide production.

Peroxynitrite is a highly reactive product.

The location and coincidence of both reactants matter.

Slide 52 · NO-derived chemistry includes nitrite and nitrate

Sequential mechanism

NO• generated

Oxidation / reaction pathways

NO₂⁻ (nitrite) and NO₃⁻ (nitrate)

These compounds are nitrogen oxide metabolites.

They should not be depicted as identical to short-lived radical intermediates.

Slide 53 · Generation network: Oxygen radicals meet nitrogen chemistry

Sequential mechanism

Mitochondria / NOX → O₂•⁻

SOD / dismutation → H₂O₂

Fe²⁺ → •OH

NO• + O₂•⁻ → ONOO⁻

This is a source-to-product map; Not every arrow is a single uninterrupted pathway.

The terminal chemistry depends on which reactants coexist.

Slide 54 · Source-to-species crosswalk

Principal superoxide pathways

Mitochondrial partial O₂ reduction

Phagocyte NADPH oxidase

Xanthine oxidase and other oxidases

Possible Fe³⁺ reduction by O₂•⁻

Other reactive species pathways

Radiolysis → •OH and H•

Fenton reaction → •OH

CCl₄ metabolism → •CCl₃

NO• + O₂•⁻ → ONOO⁻

Slide 55 · Oxidant production and oxidative stress are not equivalent

Sequential mechanism

Low / controlled generation

Reactive species removal

Steady state

An increase in production can cause oxidant accumulation.

A decrease in clearance can yield a similar imbalance.

Free-radical formation also performs physiologic signaling and host defense.

Slide 56 · Clinicopathologic contexts have different initiation routes

Chemical / physical insults

X-rays → water radiolysis

CCl₄ → trichloromethyl radical

Reactive metal pool → Fenton chemistry

Cellular / inflammatory sources

Mitochondrial respiration → O₂•⁻

Activated phagocytes → NOX burst

NO• with O₂•⁻ → peroxynitrite

Slide 57 · Application: Match the initiating route with its product

CASE / APPLICATION

During inflammation, nitric oxide and superoxide are produced in the same microenvironment. Both react before superoxide is cleared.

Which reactive product is most directly expected?

A. Peroxynitrite (ONOO⁻)

B. Trichloromethyl radical (•CCl₃)

C. Hydroxide ion (OH⁻) as the defining oxidant

Slide 58 · Mechanistic synthesis: Six experimentally distinguishable origins

Redox enzymes

Mitochondria / oxidases

Host defense

NADPH oxidase burst

External triggers

Radiation / xenobiotics

Iron and copper catalysis and NO-dependent chemistry add further pathways.

Identify the source, immediate reactive species and subsequent reaction separately.

Slide 59 · Essential distinctions for interpreting free-radical mechanisms

O₂•⁻ and •OH are radicals; H₂O₂ and ONOO⁻ are nonradical oxidants.

Physiologic O₂ reduction to water can leak partially reduced intermediates.

NOX-driven leukocyte production is deliberately inducible.

Radiation, chemical metabolism and transition metals generate distinct radical products.

NO• plus superoxide couples oxygen and nitrogen reactive chemistry.

Slide 60 · Primary reference and scope

Robbins, Cotran & Kumar: Pathologic Basis of Disease, 11th edition (2026).

Chapter 2: Cell Injury, Cell Death, and Adaptations.

Sections: Oxidative Stress—Generation of Free Radicals; Table 2.3.

Companion topics in this series address removal of radicals and their pathologic effects.