Removal of Free Radicals

Slide 1 · Removal of Free Radicals

Cellular antioxidant defenses and oxidative stress

Mechanisms of cell injury

Dr Abu Suraih Sakhri

MBBS, MD (Pathology)

www.abusuraihsakhri.com

Slide 2 · Learning objectives

Identify the chemical species that must be controlled during oxidative stress.

Explain nonenzymatic antioxidants and sequestration of iron and copper.

Trace superoxide disposal through SOD, catalase, and glutathione peroxidase.

Predict cellular consequences when production exceeds antioxidant capacity.

Slide 3 · Why cells remove free radicals

Removing oxidants preserves cellular macromolecules

Normal physiology

Reactive species arise during routine metabolism and regulated cell functions.

Injury prevention

Scavenging prevents excess oxidants from damaging membranes, proteins, and DNA.

Slide 4 · Free radicals are not identical to ROS

Free radical

Contains an unpaired electron

High chemical reactivity can modify nearby molecules

Examples: O₂•⁻ and •OH

Reactive oxygen species (ROS)

Broader oxygen-derived oxidant category

Includes free radicals and nonradical oxidants

H₂O₂ is an ROS but not itself a free radical

Slide 5 · Principal reactive species in cell injury

Species

Formation / identity

Why it matters

O₂•⁻ (superoxide)

Partial reduction of oxygen

SOD converts it to H₂O₂

H₂O₂ (peroxide)

SOD product; cellular oxidases

Detoxified by catalase or GPx

•OH (hydroxyl radical)

Radiation or Fenton reaction

Very reactive; no dedicated enzyme shown

ONOO⁻ (peroxynitrite)

Reaction of superoxide with NO

Reactive nitrogen/oxygen species; damages macromolecules

Species

Formation / identity

Why it matters

Slide 6 · Oxidative stress is a balance problem

ROS production

Respiration, oxidases, radiation, chemicals

ROS control

Scavengers and metal-binding proteins

Cellular outcome

Low ROS with control; Injury when excess persists

Oxidative stress occurs if oxidant generation exceeds removal capacity.

Slide 7 · Why a single radical can start wider injury

Radical reacts

A reactive species attacks a neighboring molecule

New radical forms

Target molecule can become reactive itself

Propagation

Additional reactions spread damage through cellular structures

Chain reactions make timely scavenging and prevention important.

Slide 8 · Oxidant sources are diverse

Endogenous metabolism

Mitochondrial respiration partially reduces oxygen

Intracellular oxidases also form superoxide

Some ROS formation is a normal by-product

Inflammation / external insults

Activated phagocytes deliberately produce ROS

Ionizing radiation can generate •OH from water

Chemicals and transition metals may amplify radical formation

Slide 9 · Mitochondrial oxygen metabolism

Oxygen use

Cells normally reduce O₂ during energy production

Partial reduction

Reactive oxygen intermediates can arise

Antioxidant need

Local ROS removal limits damage during routine respiration

This source explains why mitochondrial SOD is strategically localized.

Slide 10 · Activated leukocytes generate ROS

Inflammation

Phagocytes are activated near microbes

NADPH oxidase

Regulated rapid oxidant generation

Microbial killing

ROS contribute to destruction of microbes and unwanted material

The aim is controlled antimicrobial activity, not zero ROS.

Slide 11 · Ionizing radiation produces hydroxyl radicals

Radiant energy

X-rays can interact with cellular water

Water radiolysis

•OH and other radicals are generated

Cellular injury

Reactive species can damage biological macromolecules

Slide 12 · Transition metals accelerate oxidant chemistry

Free iron and copper can catalyze radical formation

Risk

Unbound transition metals participate in redox cycling and oxidative reactions.

Control

Storage and transport proteins restrict the reactive free-metal pool.

Slide 13 · Fenton chemistry generates hydroxyl radicals

H₂O₂ + Fe²⁺ → Fe³⁺ + •OH + OH⁻

The substrate

Hydrogen peroxide becomes dangerous when reactive ferrous iron is available.

The critical product

•OH is highly reactive and can rapidly modify adjacent molecules.

Slide 14 · Physiologic functions versus injury

Controlled ROS generation

Phagocyte antimicrobial reactions

Regulated cellular signaling at low oxidant levels

Oxidants are normally counterbalanced by scavengers

Excessive oxidative burden

Accumulation of ROS exceeds detoxification

Lipid, protein, and DNA injury develops

Necrosis and apoptosis may follow severe damage

Slide 15 · Cellular defenses act at three levels

Prevent formation

Metal-binding proteins limit radical-generating catalysis

Intercept oxidants

Small-molecule antioxidants scavenge reactive species

Enzymatic disposal

SOD, catalase, and glutathione peroxidase clear key oxidants

The layers are complementary, not interchangeable.

Slide 16 · Superoxide can dismutate spontaneously

2 O₂•⁻ + 2 H⁺ → H₂O₂ + O₂

Spontaneous reaction

Superoxide is unstable and can decay in aqueous surroundings.

Important limitation

The reaction leaves H₂O₂, which still requires cellular control.

Slide 17 · Superoxide clearance is a two-stage problem

Superoxide

O₂•⁻ is generated near oxygen-reducing systems

Dismutation

Spontaneous or SOD-mediated conversion

Peroxide disposal

Catalase or glutathione peroxidase removes H₂O₂

Lower superoxide concentration does not by itself establish complete detoxification.

Slide 18 · Nonenzymatic defense categories

Defense

Textbook examples

Protective principle

Lipid-soluble antioxidant

Vitamins E and A

Antioxidant action within lipid-associated environments

Water-associated antioxidants

Ascorbic acid; glutathione

Interception of oxidants in cellular aqueous compartments

Metal-binding proteins

Ferritin, transferrin, lactoferrin, ceruloplasmin

Limit catalytically available Fe / Cu

Defense

Textbook examples

Protective principle

Slide 19 · Vitamin E and vitamin A are lipid-soluble

Lipid-soluble antioxidants contribute to radical control

Vitamin E

Named by Robbins among antioxidants capable of limiting free radical reactions.

Vitamin A

Also listed among lipid-soluble antioxidant defenses.

The source lists these vitamins; It does not quantify their relative clinical effects.

Slide 20 · Ascorbate and glutathione are small antioxidants

Ascorbic acid

Listed among cellular nonenzymatic antioxidants

Participates in the broader small-molecule defense against oxidants

Source does not specify a disease-specific treatment effect

Glutathione (GSH)

Present in cytosolic antioxidant defenses

Also used as the reducing substrate by glutathione peroxidase

Its oxidized form is GSSG

Slide 21 · Two ways an antioxidant can protect cells

Before radical formation

Block conditions that favor radical generation

Bind catalytic metals and limit electron transfer opportunities

Acts upstream of cellular damage

After a radical has formed

Scavenge or inactivate reactive molecules

Interrupt the opportunity for additional target reactions

Acts after formation but before widespread injury

Slide 22 · Metal sequestration protects without scavenging ROS

Iron and copper

Metals may donate or accept electrons

Protein binding

Transport/storage proteins reduce reactive metal availability

Less catalytic chemistry

Fewer opportunities for new ROS generation

This defense prevents radical formation rather than metabolizing H₂O₂.

Slide 23 · Transferrin and ferritin

Transferrin

Iron transport protein listed as a protective metal binder

Reduces the freely reactive metal pool

Works upstream of hydroxyl radical formation

Ferritin

Intracellular iron-storage protein listed by Robbins

Sequesters iron away from redox catalysis

Does not substitute for peroxide-removing enzymes

Slide 24 · Lactoferrin and ceruloplasmin

Lactoferrin

Named among proteins that restrain metal reactivity

Illustrates protective metal binding

Distinct from enzymatic removal of superoxide

Ceruloplasmin

Named among metal-handling proteins

Helps minimize free-metal-mediated chemistry

Included in the preventative antioxidant layer

Slide 25 · Unbound versus protein-bound metal

Reactive free metal

May participate in reactions that form reactive oxygen intermediates

Available Fe²⁺ supports Fenton chemistry

Oxidant burden may increase

Protected protein-associated metal

Sequestration limits access to catalytic reactions

Transport / storage becomes chemically safer

Protection is indirect but mechanistically important

Slide 26 · The enzyme defense system is compartmentalized

Mitochondria

Mn-SOD is close to mitochondrial oxidant generation

Cytoplasm

Cu/Zn-SOD and GSH-based defenses operate in cells

Peroxisomes

Catalase breaks down H₂O₂

Location helps explain why oxidant control is organized, not random.

Slide 27 · Superoxide dismutase: The substrate and product

2 O₂•⁻ + 2 H⁺ → H₂O₂ + O₂

Substrate

Two superoxide anions are converted during dismutation.

Products

Hydrogen peroxide and oxygen; Peroxide requires downstream removal.

Slide 28 · Two SOD systems

Enzyme

Primary localization

Relevant protective action

Mn-SOD

Mitochondria

Disposes of mitochondrial superoxide

Cu/Zn-SOD

Cytoplasm

Converts superoxide formed in cytoplasmic settings

Enzyme

Primary localization

Relevant protective action

Slide 29 · Manganese superoxide dismutase

Mn-SOD protects the mitochondrial compartment

Why mitochondria?

Mitochondrial oxygen metabolism can form partially reduced oxygen species.

What Mn-SOD does

It promotes superoxide dismutation where this oxidant arises.

Slide 30 · Copper–zinc superoxide dismutase

Cu/Zn-SOD provides cytoplasmic superoxide removal

Substrate

Cytoplasmic O₂•⁻ is converted into H₂O₂ and oxygen.

Next step

Peroxide must subsequently be metabolized by appropriate scavenging systems.

Slide 31 · SOD is necessary but not the endpoint

Step 1

Superoxide falls following enzymatic dismutation

Step 2

Hydrogen peroxide remains as a chemically reactive oxidant

Step 3

Catalase / glutathione peroxidase completes downstream disposal

SOD and peroxide-metabolizing enzymes form a functional sequence.

Slide 32 · Catalase decomposes hydrogen peroxide

2 H₂O₂ → 2 H₂O + O₂

Localization

Catalase is present in peroxisomes.

Protective role

Decomposition of H₂O₂ limits oxidant accumulation.

Slide 33 · Peroxisomes contain catalase

Peroxisomal catalase converts H₂O₂ into stable products

Input

Hydrogen peroxide generated by cellular oxidase reactions or SOD-dependent conversion.

Output

Water and molecular oxygen; Catalytic removal of peroxide.

Slide 34 · Glutathione peroxidase uses reduced glutathione

H₂O₂ + 2 GSH → GSSG + 2 H₂O

Electron donor

GSH supplies reducing equivalents during detoxification.

Oxidized product

GSSG is oxidized glutathione; The reaction reduces peroxide to water.

Slide 35 · Glutathione cycles between redox states

Reduced form

GSH participates in peroxide detoxification

GPx reaction

Two GSH are converted into one GSSG

Oxidized form

GSSG accumulates relative to GSH when the cellular redox balance shifts

GSH/GSSG status as a useful indicator of oxidative state.

Slide 36 · Reduced versus oxidized glutathione

GSH: Reduced form

A reactant in the glutathione-peroxidase reaction

Represents reducing capacity available for H₂O₂ breakdown

Used up as substrate during this reaction

GSSG: Oxidized form

Product formed when glutathione is oxidized

Read with GSH to understand cellular oxidation status

An increased GSSG/GSH ratio indicates a more oxidizing state

Slide 37 · The glutathione redox ratio is an indicator

GSSG/GSH reflects the oxidative state of the cell

Higher oxidized fraction

A relative shift toward GSSG indicates a more oxidized glutathione pool.

Interpretation limit

The ratio is an indicator of redox state, not a stand-alone diagnosis of a particular disease.

Slide 38 · Integrated pathway: SOD followed by catalase

O₂•⁻

SOD catalyzes dismutation

H₂O₂

Catalase decomposes peroxide

H₂O + O₂

Products of peroxide detoxification

Two enzymes manage sequential oxidants produced by oxygen metabolism.

Slide 39 · Integrated pathway: SOD followed by GPx

O₂•⁻

SOD generates H₂O₂

H₂O₂ + GSH

GPx uses reduced glutathione

H₂O + GSSG

Peroxide is detoxified while glutathione is oxidized

The GSH/GSSG balance supplies information about the redox burden.

Slide 40 · Defense distribution across cellular locations

Location

Source-aligned antioxidant system

Chemical task

Mitochondria

Manganese-SOD; glutathione peroxidase

Restrict superoxide and peroxide

Cytoplasm

Copper–zinc-SOD; GSH-related systems

Limit cytoplasmic oxidative stress

Peroxisomes

Catalase

Break down H₂O₂

Storage / transport compartments

Iron- and copper-binding proteins

Restrict metal-catalyzed radical formation

Location

Source-aligned antioxidant system

Chemical task

Slide 41 · Compare the major removal pathways

Mechanism

Acts primarily on

Main outcome

Spontaneous dismutation

Superoxide (O₂•⁻)

H₂O₂ + O₂

Superoxide dismutase

Superoxide (O₂•⁻)

H₂O₂ + O₂

Catalase

Hydrogen peroxide

H₂O + O₂

Glutathione peroxidase

Hydrogen peroxide + GSH

H₂O + GSSG

Antioxidants / metal binding

Radicals or their upstream formation

Intercept oxidants / limit new ROS

Mechanism

Acts primarily on

Main outcome

Slide 42 · Antioxidant protection has finite capacity

Generation increases

More reactive molecules appear per unit time

Removal is outpaced

Free radicals persist or propagate reactions

Macromolecular injury

Membrane, protein, and DNA damage may occur

This imbalance as oxidative stress.

Slide 43 · Oxidative stress: Increased production

Stimulus

Radiation, toxins, reperfusion, inflammation

ROS load

Increased production overwhelms clearance

Injury

Oxidants attack biomolecular targets

Slide 44 · Oxidative stress: Decreased removal

Defense deficit

Lower effective scavenging capacity

Oxidant persistence

Normal or elevated ROS is inadequately cleared

Injury

Oxidative modification proceeds despite continued generation

Slide 45 · Uncontrolled ROS can peroxidize membrane lipids

Initiation

Radicals attack unsaturated lipid components

Propagation

Lipid peroxides extend chain reactions

Membrane injury

Permeability and membrane integrity become impaired

This is a core downstream consequence of insufficient free-radical removal.

Slide 46 · Membranes are high-value antioxidant targets

Lipid peroxidation compromises cell and organelle membranes

Mechanistic link

Membrane phospholipid modification destabilizes barriers and organelles.

Possible consequences

Membrane dysfunction contributes to worsening cellular injury and necrosis.

Slide 47 · Free radicals chemically modify proteins

Oxidative attack

Amino-acid side chains / backbones are modified

Structural effects

Cross-linking and unfolding can follow

Functional loss

Enzymes and structural proteins may malfunction

Slide 48 · Proteins: Immediate and downstream effects

Immediate oxidative changes

Side-chain oxidation

Covalent interprotein cross-links

Oxidation of protein backbones

Cell-level consequences

Altered enzyme active sites

Misfolded or unfolded protein accumulation

Enhanced proteasomal degradation

Slide 49 · Reactive species can damage nuclear DNA

DNA oxidation

ROS alter DNA chemical structure

Lesions

Strand breaks, cross-links, and adducts

Consequences

Damage responses; Potential mutation if not corrected

Slide 50 · DNA damage initiates protective decisions

DNA lesions

Persistent oxidative and other DNA damage

Damage response

P53-associated cell-cycle arrest and repair

If repair fails

Apoptotic elimination of severely damaged cells

The source connects DNA damage, p53 responses, and apoptosis.

Slide 51 · ROS injury can lead to multiple death pathways

Necrosis

Extensive cellular and membrane injury can culminate in necrosis

Oxidative destruction contributes to loss of cell integrity

Apoptosis

ROS can also initiate programmed cell-death pathways

DNA damage signaling is one established route toward apoptosis

Slide 52 · Ischemia-reperfusion: A setting for oxidative injury

Ischemia

Tissue has experienced inadequate perfusion

Reperfusion

Restoration of blood supply can increase ROS production

Oxidative injury

Reactive species add to cell and membrane damage

Slide 53 · External causes illustrate distinct ROS routes

Radiation injury

Radiation can split cellular water

•OH radicals are generated

DNA and other cell components are vulnerable

Chemical injury

Metabolism of some chemicals generates radicals

CCl₄ can yield •CCl₃ radicals

Not all toxic radicals are oxygen-derived

Slide 54 · Inflammatory ROS: Useful and potentially harmful

Host defense

NADPH oxidase deliberately produces oxidants

Phagocytes use ROS to kill microbes

Production is spatially and temporally regulated

Collateral injury

Inflammatory ROS can contribute to surrounding tissue damage

Protection depends on effective oxidant control

Excess oxidative stress may injure host cells

Slide 55 · Oxidative stress is implicated in chronic disease

A persistent oxidant imbalance has broad pathologic relevance

Textbook-associated processes

Cell injury, aging, cancer, and selected degenerative disorders.

Scientific caution

An association with oxidative stress does not by itself establish a specific therapy or causal contribution in every case.

Slide 56 · The aim is controlled redox physiology

Adequate ROS removal—not complete ROS elimination

Protective purpose

Detoxification prevents excessive damage to lipids, proteins, and DNA.

Physiologic requirement

Some ROS are useful in host defense and cellular signaling.

Slide 57 · Checkpoint: Which pathway removes H₂O₂?

A cell has converted superoxide to hydrogen peroxide. Which reaction directly detoxifies the remaining peroxide?

A. Superoxide dismutase generates more H₂O₂

B. Catalase yields H₂O and O₂

C. Iron-dependent Fenton chemistry produces •OH

D. Free iron is released from storage proteins

Slide 58 · Checkpoint: Mechanistic answer

B. Catalase decomposes H₂O₂ into water and oxygen.

SOD processes superoxide, but its reaction produces peroxide rather than finishing removal.

Fenton chemistry generates hydroxyl radicals; Unbound iron therefore increases oxidative hazard.

Slide 59 · Application: Mitochondrial oxidant burden

Consider a hypothetical cell with excess mitochondrial O₂•⁻. Which enzyme is directly positioned to accelerate its dismutation?

A. Catalase in peroxisomes

B. Manganese superoxide dismutase

C. Copper–zinc superoxide dismutase in cytoplasm

D. Transferrin-mediated iron transport

Slide 60 · Application: Match location to reaction

B. Manganese-SOD acts in mitochondria.

Mitochondrial respiration can form superoxide; Mn-SOD converts O₂•⁻ into H₂O₂ and O₂.

Cytoplasmic Cu/Zn-SOD performs analogous chemistry elsewhere; The peroxide still needs removal.

Slide 61 · Application: Transition-metal catalysis

Consider two otherwise similar systems containing H₂O₂. One has more freely reactive Fe²⁺. What is the expected chemical risk?

A. Less hydroxyl-radical formation

B. Greater potential for Fenton-generated •OH

C. Direct conversion of H₂O₂ into GSSG without enzymes

D. No change because iron cannot participate in ROS formation

Slide 62 · Application: Explain the iron effect

B. More available Fe²⁺ permits more Fenton chemistry.

Fe²⁺ reacts with H₂O₂ to generate Fe³⁺, hydroxyl radical, and hydroxide.

Binding proteins such as ferritin and transferrin lower metal availability for radical-forming reactions.

Slide 63 · Frequent conceptual errors

Incorrect inference

More accurate interpretation

H₂O₂ is a free radical

H₂O₂ is ROS, but lacks an unpaired electron

SOD finishes oxidant detoxification

SOD forms H₂O₂; catalase or GPx is still needed

Metal-binding proteins are ROS-degrading enzymes

They prevent metal-catalyzed ROS formation

Every ROS molecule is harmful

Controlled ROS participates in microbial killing and signaling

GSSG alone identifies a particular disease

GSSG/GSH indicates cellular redox state, not disease specificity

Incorrect inference

More accurate interpretation

Slide 64 · Integrating the complete antioxidant defense

Formation / amplification

Respiration, inflammation, radiation, reactive metals

Control / removal

Metal binding; Antioxidants; SOD → catalase or GPx

Pathologic threshold

Persistent radicals damage lipids, proteins, and DNA

Protective control is multilayered and coordinated.

Slide 65 · Core principles to retain

Free radicals are limited by both spontaneous decay and active cellular defenses.

Nonenzymatic antioxidants and metal-binding proteins reduce the effective oxidant burden.

SOD generates H₂O₂ from O₂•⁻; Catalase and glutathione peroxidase dispose of H₂O₂.

Excess formation or inadequate removal permits oxidative damage to membranes, proteins, and DNA.

Slide 66 · How to reason through an oxidative-injury problem

Identify which reactive species is accumulating and where it is generated.

Ask whether the deficit lies in production control, radical scavenging, or peroxide disposal.

Match each enzyme to its substrate, reaction product, and cellular location.

Connect the unresolved oxidant to a specific macromolecular target and cellular consequence.