Ferroptosis

Slide 1 · Ferroptosis

Iron-dependent regulated cell death

Membrane lipid peroxidation • oxidative injury • cell-death mechanisms

Dr Abu Suraih Sakhri

MBBS, MD (Pathology)

www.abusuraihsakhri.com

Slide 2 · Learning objectives

Define ferroptosis and distinguish it from other forms of cell death.

Trace the roles of iron, oxidants, membrane lipids and antioxidant defenses.

Interpret ultrastructural findings and disease associations critically.

Use mechanistic evidence to evaluate whether ferroptosis occurred.

Slide 3 · Ferroptosis in the cell-death landscape

Cell injury

Ischemia, oxidants, toxins

Cell death pathways

Necrosis and regulated death

Ferroptosis

Iron + oxidized lipids

Regulated injury pathways are mechanistically distinct even if tissue injury appears necrotic.

Slide 4 · Definition: Iron-dependent lipid injury

A distinct, regulated pathway of cell death.

Excess iron and/or oxidant burden defeats glutathione-dependent protection.

Unchecked membrane lipid peroxidation is central to the fatal process.

Ultimately, selective plasma-membrane barrier function fails.

Slide 5 · Three converging requirements

Catalytic iron

Enables oxidative reactions

Oxidizable lipids

Membrane substrate

Inadequate defense

Antioxidants overwhelmed

Lytic death

Membrane fails

The diagram is a conceptual convergence, not a universal quantitative threshold.

Slide 6 · Regulated mechanism, necrosis-like end point

Regulated process

Specific biochemical vulnerabilities and controls.

Can be suppressed by limiting iron.

Distinct from unregulated chemical membrane destruction.

Necrosis-like result

Barrier breakdown and eventual cell death.

Cell death may be lytic rather than apoptotic.

Final morphology alone does not specify mechanism.

Slide 7 · Where the lethal damage occurs

Oxidative imbalance

Reactive oxidants accumulate

Lipid peroxidation

Bilayer lipids oxidized

Membrane dysfunction

Transport and signaling altered

Loss of integrity

Cell death

Distinguish a mechanistic sequence from a histologic diagnostic criterion.

Slide 8 · Mechanism versus microscopic appearance

Mechanism

Iron-dependent oxidation of phospholipids.

Defective antioxidant control.

A regulated cell-death pathway.

Appearance

Loss of membrane integrity, resembling necrosis.

Characteristic mitochondrial alterations reported.

Conventional H&E is not pathway-specific.

Slide 9 · Reactive oxygen species have normal roles

Low levels arise during ordinary cellular metabolism.

Scavenging pathways normally constrain oxidant accumulation.

Physiologic signaling can use controlled oxidant production.

Ferroptosis concerns destructive oxidation, not ROS presence alone.

Slide 10 · Oxidative stress is a balance problem

ROS production

Normal respiration, cellular enzymes and stress

ROS removal

Antioxidants and detoxifying enzymes

Versus

Oxidant load exceeds protection

Slide 11 · Sources of intracellular oxidants

Metabolism

Electron-transfer reactions

Inflammation

Activated leukocytes

Radiation / toxins

Reactive intermediates

Excess oxidants

Risk of molecular injury

Sources of ROS in cell injury; Not every source specifically proves ferroptosis.

Slide 12 · Major reactive oxygen intermediates

Species

Biochemical significance

Superoxide (O₂•⁻)

Partially reduced oxygen; Precursor of other ROS.

Hydrogen peroxide (H₂O₂)

Nonradical ROS; Substrate for iron-driven chemistry.

Hydroxyl radical (•OH)

Highly reactive species capable of molecular injury.

H₂O₂ is a reactive oxygen species but not a free radical.

Slide 13 · Iron accelerates oxidant chemistry

Fe²⁺

Redox-active iron

H₂O₂

Available peroxide

•OH formation

Fenton chemistry

Lipid injury

Radical attack on membranes

Fenton reaction is an important general model of iron-catalyzed injury.

Slide 14 · Fenton reaction: A causal example

Ferrous iron (Fe²⁺) reacts with hydrogen peroxide.

The reaction produces ferric iron (Fe³⁺), hydroxyl radical (•OH) and hydroxide.

Hydroxyl radicals are highly reactive toward cellular molecules.

Do not equate all ferroptotic lipid oxidation with one chemical reaction.

Slide 15 · Iron binding limits free-radical chemistry

Restricted iron

Transferrin and ferritin bind iron in transport and storage.

Metal-binding proteins limit free-ion reactivity.

This reduces oxidant-generating chemistry.

Available redox-active iron

Greater catalytic capacity under permissive conditions.

Can cooperate with ROS to amplify injury.

Specific cellular pools matter more than iron abundance alone.

Slide 16 · Why iron reduction can prevent ferroptosis

Accessible iron falls

Reduced redox catalysis

Radical pressure falls

Less lipid oxidation

Membrane integrity

Better preserved

Death prevented

In susceptible experimental settings

Slide 17 · Membrane phospholipids are the substrate

Schematic phospholipid bilayer

Head groups

Water-facing

Fatty-acid tails

Membrane core

Intact barrier

Selective transport

Original editable schematic — not a micrograph

Slide 18 · Unsaturated lipid bonds are vulnerable

Double bonds in unsaturated membrane fatty acids are sites of radical attack.

The initial oxidative event creates reactive lipid intermediates.

Susceptibility depends on membrane lipid composition.

The specific phospholipid landscape is an important biological modifier.

Slide 19 · Lipid peroxidation begins with radical attack

Initiation

Radical attacks unsaturated lipid

Lipid radical

Reactive intermediate

Oxygen addition

Lipid peroxyl radical

Hydroperoxide

Oxidized phospholipid accumulates

Slide 20 · Peroxidation propagates as a chain

Oxidized lipid

Radical intermediate

Neighbor lipid

Hydrogen abstraction

New lipid radical

Reaction continues

Damage spreads

Autocatalytic propagation

Schematic chain propagation; Reaction rates and stoichiometry not quantified.

Slide 21 · Unchecked oxidation destabilizes the bilayer

Schematic phospholipid bilayer

Oxidation

Altered membrane lipids

Protein context

Normal interactions perturbed

Functional loss

Barrier weakened

Original editable schematic — not a micrograph

Slide 22 · Membrane fluidity changes

Lipid composition and organization govern membrane fluidity.

Extensive oxidation perturbs lipid packing and membrane behavior.

Impaired fluidity alters the physical environment of integral proteins.

This is one route from chemical damage to cellular dysfunction.

Slide 23 · Lipid–protein interactions are disturbed

Intact membrane

Lipids support normal organization of membrane proteins.

Protein activities depend partly on local lipid environment.

After extensive lipid oxidation

Altered bilayer may disturb protein interactions.

Transporters and receptors may no longer function normally.

Slide 24 · Ion and nutrient transport become abnormal

Lipid oxidation

Membrane physical properties change

Transport failure

Ion/nutrient homeostasis disturbed

Cell dysfunction

Metabolism and gradients destabilize

Slide 25 · Membrane signaling is not spared

Plasma membranes organize receptors and signaling proteins.

Lipid oxidation can alter lipid–protein relationships.

Consequent disruption of signaling contributes to cellular failure.

Signaling changes should not be mistaken for a ferroptosis-specific test.

Slide 26 · Loss of selective permeability is lethal

Schematic phospholipid bilayer

Gap

↕

Before injury

Selective membrane permeability

During injury

Peroxidized phospholipids

After injury

Loss of barrier function

Original editable schematic — not a micrograph

Slide 27 · Different membranes, shared vulnerability

Plasma membrane

Selective permeability supports cellular homeostasis.

Barrier failure produces necrosis-like loss of integrity.

Organelle membranes

ROS can damage membranes throughout the cell.

Mitochondrial alterations may accompany ferroptosis.

Slide 28 · Ultrastructural findings reported in ferroptosis

Prominent mitochondrial cristae loss.

Disruption or rupture of the outer mitochondrial membrane.

These abnormalities have been described ultrastructurally.

Interpretation requires experimental context and appropriate methods.

Slide 29 · Cristae loss: Conceptual ultrastructure

Fewer cristae / disrupted outer boundary in the source description

Native PowerPoint schematic; Not diagnostic electron microscopy

Slide 30 · Outer mitochondrial membrane disruption

Outer membrane

Discontinuity

Fewer cristae / disrupted outer boundary in the source description

Native PowerPoint schematic; Not diagnostic electron microscopy

Slide 31 · Morphology provides clues, not proof

What morphology contributes

Can show mitochondria with diminished cristae.

Can show outer membrane disruption.

Supports a mechanism when paired with assays.

What morphology cannot settle

Cannot determine iron dependence alone.

Cannot identify a unique biochemical cascade.

Does not replace orthogonal rescue experiments.

Slide 32 · Glutathione is a central antioxidant reserve

GSH available

Reduced glutathione

Peroxide detoxification

GSH-dependent reactions

Oxidant load contained

Membranes protected

GSH-dependent protection is explicit; Downstream molecular details are supplemental.

Slide 33 · Glutathione peroxidase removes peroxides

Glutathione peroxidase uses reduced GSH in peroxide-detoxifying reactions.

Oxidized glutathione (GSSG) is generated during this process.

Cellular GSH/GSSG balance reflects redox state.

When defenses are overwhelmed, oxidized lipids can accumulate.

Slide 34 · GPX4 protects membrane phospholipids

Phospholipid hydroperoxide

Oxidized membrane lipid

GPX4 + GSH

Peroxide-reducing system

Reduced phospholipid

Lipid damage curtailed

Slide 35 · System xᶜ⁻ supports GSH synthesis

Cystine uptake

System xᶜ⁻ antiporter

Cysteine supply

Precursor availability

GSH synthesis

Antioxidant capacity

GPX4 function

Lipid peroxide detoxification

Slide 36 · Inhibiting cystine import creates vulnerability

Cystine import blocked

Less cysteine available

GSH depleted

Peroxide control weakens

Lipid oxidation rises

Iron-dependent injury

Ferroptosis

In susceptible cells

Experimental example, not a universal response in every cell type.

Slide 37 · Direct impairment of GPX4 also sensitizes cells

GPX4 inhibited

Lower lipid peroxide reduction

Phospholipid peroxides

Accumulate

Membrane injury

Propagation and failure

Cell death

Ferroptotic phenotype

Slide 38 · A parallel antioxidant defense: FSP1

FSP1 activity

NAD(P)H-dependent pathway

Ubiquinol formed

Reduced CoQ pool

Lipid radicals quenched

Protection outside GPX4 axis

Supplementary pathway; Represented schematically without claiming precise flux or location.

Slide 39 · Multiple protections converge on lipids

Protective arm

Principal role

Iron-binding proteins

Restrict redox-active metal chemistry.

GSH–GPX4

Reduce membrane lipid hydroperoxides.

FSP1–CoQ

Radical-trapping antioxidant protection.

Vitamin E and other antioxidants

Interrupt free-radical injury under suitable conditions.

Slide 40 · Injury occurs when protection is surpassed

Pro-oxidant drive

Redox-active iron + reactive lipid intermediates

Antioxidant defenses

GSH–GPX4, FSP1 and metal sequestration

Versus

Lipid peroxidation escapes control

Slide 41 · Three mechanistic levers to investigate

Promote ferroptotic injury

Increase accessible redox-active iron.

Increase oxidizable membrane substrate.

Compromise lipid-peroxide detoxification.

Oppose ferroptotic injury

Restrict accessible iron.

Interrupt radical chain propagation.

Preserve antioxidant defense function.

Slide 42 · Lipid peroxidation is not every membrane injury

Route of injury

Primary disturbance

Ferroptotic lipid damage

Iron-dependent oxidative peroxidation.

ATP depletion

Reduced phospholipid synthesis and pump function.

Calcium-activated phospholipases

Increased phospholipid breakdown.

Cytoskeletal proteolysis

Loss of membrane anchorage and stability.

Slide 43 · Ferroptosis and passive necrosis differ

Ferroptosis

Biochemically regulated lipid oxidation.

Depends on iron and antioxidant status.

May culminate in lytic morphology.

Classical accidental necrosis

Severe injury produces membrane failure.

Multiple upstream causes are possible.

A similar end point does not imply the same mechanism.

Slide 44 · Ferroptosis versus apoptosis

Characteristic

Ferroptosis

Apoptosis

Central machinery

Iron-dependent lipid oxidation

Caspase-mediated proteolysis

Membrane fate

Eventual loss of integrity

Often intact until phagocytic removal

Characteristic morphology

Mitochondrial cristae alteration

Cell shrinkage; Apoptotic bodies

Inflammatory context

Lytic end point possible

Usually limited by rapid clearance

Slide 45 · Ferroptosis versus necroptosis

Feature

Ferroptosis

Necroptosis

Principal mechanism

Iron-dependent lipid peroxidation

RIPK1/RIPK3–MLKL signaling

Membrane disruption

Oxidative lipid damage

MLKL-mediated membrane disruption

Diagnostic inference

Requires biochemical/rescue evidence

Requires pathway-specific evidence

Shared feature

Necrosis-like cell death

Necrosis-like cell death

Slide 46 · Ferroptosis versus pyroptosis

Feature

Ferroptosis

Pyroptosis

Key trigger / context

Oxidative lipid damage

Inflammasome or cytosolic LPS pathways

Key effector

Phospholipid peroxidation

Gasdermin D pore formation

Inflammatory mediators

Not defined by IL-1β processing

Associated with IL-1β release

Outcome

Membrane failure

Osmotic disruption and lysis

Slide 47 · Do not classify cell death by lysis alone

Membrane rupture

Shared morphologic end point

Competing causes

Necrosis / necroptosis / pyroptosis

Mechanistic testing

Lipids, iron, pathway inhibitors

Interpretation

Integrate convergent evidence

Ferroptosis diagnosis is mechanistic, not merely morphologic.

Slide 48 · A ferroptosis claim needs converging evidence

Show an iron- and lipid-peroxidation–linked injury phenotype.

Demonstrate biochemical evidence consistent with oxidized membrane lipids.

Test protection by interventions with relevant mechanisms.

Exclude plausible alternative regulated cell-death routes.

Slide 49 · Oxidized lipid measurement: Use caution

Informative evidence

Biochemical assays of lipid peroxidation.

Appropriate temporal relation to loss of viability.

Orthogonal lipid readouts strengthen the inference.

Limitations

Oxidative stress also occurs in other types of injury.

Single assays have technical and biological confounders.

A positive lipid signal is not a stand-alone diagnosis.

Slide 50 · Mechanism-based rescue strengthens attribution

Induced injury

Experimental perturbation

Mechanistic rescue

Iron restriction or radical trapping

Viability improves

In matched controls

Causal inference

More convincing, still contextual

Do not over-interpret nonselective compounds as pathognomonic.

Slide 51 · GPX4 status alone is insufficient

GPX4 participates in a major suppressive defense system.

Changes in protein level or expression are not equivalent to functional activity.

Parallel ferroptosis-suppressive systems also exist.

Interpret pathway markers together with lipid injury and rescue data.

Slide 52 · Experimental inference is not clinical diagnosis

In experimental models

Perturbation and rescue can interrogate causality.

Lipid oxidation and survival can be quantified.

Genetic manipulation can test mechanism.

In clinical tissue

Routine H&E cannot establish ferroptosis.

No single universally accepted clinical diagnostic stain.

An association does not prove an individual lesion was ferroptotic.

Slide 53 · Cancer: Vulnerability and therapeutic hypothesis

Cancer among pathologies linked with ferroptosis.

Some cancer models exhibit sensitivity to GPX4-related perturbation.

Inducing ferroptosis is an investigated antitumor strategy.

Preclinical susceptibility must not be presented as an established clinical indication.

Slide 54 · Neurodegeneration: Proposed injury mechanism

Ferroptosis with neurodegenerative disorders.

Neural tissue may be affected by altered iron and oxidative lipid injury.

Experimental studies motivate ferroptosis-inhibition strategies.

These links do not establish one uniform mechanism in every patient or disease.

Slide 55 · Stroke: Ischemic injury and oxidative context

Stroke is explicitly identified among ferroptosis-linked conditions.

Oxidative stress also occurs in ischemia-reperfusion injury.

Multiple distinct cell-death pathways may coexist in injured brain.

A clinical neuroprotective benefit from ferroptosis targeting cannot be assumed.

Slide 56 · Disease association ≠ validated therapy

What can be stated

Ferroptosis is biologically connected with selected pathologies.

Drug-target ideas follow from preclinical mechanisms.

The field has plausible therapeutic directions.

What should not be asserted

A pathology association proves causation in each patient.

A specific drug is clinically effective without trial data.

A single molecular marker validates patient-level diagnosis.

Slide 57 · Case exercise: A cell-culture death phenotype

A cell-culture perturbation reduces cystine uptake. Reduced glutathione falls, lipid oxidation rises, and viability declines. Iron reduction and a radical-trapping agent improve survival.

Discussion questions

Which observations support ferroptosis?

What alternative mechanisms and controls require examination?

Slide 58 · Case interpretation: Triangulate the mechanism

Cystine restriction

Weakens GSH protection

Lipid oxidation

Matches central lesion

Iron-sensitive rescue

Supports iron dependence

Convergent evidence

Ferroptosis is plausible

Rescue is evidence of mechanism, not absolute proof; Controls and specificity remain required.

Slide 59 · Case exercise: Lytic cell death in infection

Infected immune cells activate inflammatory caspases, cleave gasdermin D and release IL-1β through membrane pores. The cells ultimately rupture.

Discussion questions

Which form of regulated cell death is more directly supported?

Why is membrane rupture alone insufficient to infer ferroptosis?

Slide 60 · Case interpretation: Mechanism determines the label

Inflammatory caspases

Canonical/noncanonical pathways

Gasdermin D

Membrane pore formation

IL-1β release

Inflammatory output

Pyroptosis

Not iron/lipid-defined death

Slide 61 · An integrated causal framework

Iron + oxidants

Reactive chemistry

Defenses overwhelmed

GSH–GPX4 inadequate

Lipid oxidation

Peroxidation propagates

Membrane failure

Regulated lytic cell death

Simplified logic: Antioxidant failure is permissive and can occur upstream or alongside oxidation.

Slide 62 · Ferroptosis: Five defensible conclusions

Its defining lesion is iron-dependent membrane lipid peroxidation.

The lethal transition reflects oxidant burden exceeding antioxidant defenses.

It can culminate in necrosis-like plasma-membrane failure.

Mitochondrial cristae loss and outer-membrane disruption are reported ultrastructural findings.

Disease links are biologically important but not equivalent to clinical diagnostic or treatment standards.

Slide 63 · Source references

Primary textbook and contextual sections

Robbins, Cotran & Kumar: Pathologic Basis of Disease, 11th edition (2026), Chapter 2.

Ferroptosis and related key concepts in Chapter 2.

Related sections: Membrane injury, oxidative stress, free radical generation and removal, and cell-death pathways.

Slide 64 · Supplementary mechanistic references

Dixon SJ et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012;149:1060–1072. doi:10.1016/j.cell.2012.03.042.

Yang WS et al. Regulation of ferroptotic cancer cell death by GPX4. Cell. 2014;156:317–331. doi:10.1016/j.cell.2013.12.010.

Jiang X, Stockwell BR, Conrad M. Ferroptosis: mechanisms, biology and role in disease. Nat Rev Mol Cell Biol. 2021;22:266–282. doi:10.1038/s41580-020-00324-8.