FREE RADICAL INDUCED CELL INJURY

DEFINITION:
THESE ARE EXTREMELY REACTIVE
CHEMICAL SPECIES THAT HAVE A
SINGLE UNPAIRED ELECTRON IN THE
OUTER ORBITAL.

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PRODUCTION OF FREE RADICALS

• Absorption of radiation energy.

• Endogenous oxidative reactions.
• Enzymatic metabolism of exogenous
chemicals or drugs.

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 Mechanisms of action
• By reacting with inorganic or organic chemicals.
• By initiating autocatalytic reactions.

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TYPES OF FREE RADICALS
• THREE MAIN TYPES:
• Oxygen centered Free Radicals.
• Carbon centered Free Radicals.
• Nitrogen centered Free Radicals.

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OXYGEN CENTERED FREE RADICALS

These are partially reduced toxic

intermediate oxygen species produced
intracellularly by the activity of
oxidative enzymes.

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Intracellular Sites of Production
• Cystole
• Mitochondria
• Lysosomes
• Plasma Membrane.
OXIDATIVE ENZYMES INVOLVED
• Xanthine oxidase
• Cytochrome P-450 6
OXYGEN CENTERED FREE RADICALS
• Superoxide:
_
O2 Oxidase
O2
• Hydrogen peroxide:
O2 + O2 + 2H+ SD
H 2 O2 + O 2
• Hydroxide Radicals
 Hydrolysis of water by Ionizing Radiations:
_
+
H2O H + OH
 Interaction with Transitional Metals:
(FENTION REACTION)
_ _
++ +++
Fe + H2 O Fe + OH + OH 7
Most of the Iron is in Ferric form
and it is reduced to Ferrous form
by Superoxide (Autocatalysis).
Sources of Iron and Superoxide
are req for maximum oxidative cell
damage.

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CARBON CENTERED FREE RADICALS

Carbon trichloride (CCl3) produced by enzyme

induced conversion of carbon tetrachloride.
_ _
CCl4 + e CCL3 + Cl

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NITROGEN CENTERED FREE RADICALS

• Nitric Oxide (NO) produced by invading

Leukocytes.
• Peroxynitrite anion.

. _ _
+
NO + O2 ONOO + H

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EFFECTS OF FREE RADICALS
1. Lipid peroxidation of membranes:
• Attacks double bonds existing within
unsaturated fatty acids.
• Peroxides are produced.
• Reactive species initiating
autocatalytic reactions.

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2. Lesions in DNA:
• Reacts with thymine in DNA
• Causes single strand breaks.
• Implicated in cell killing and malignant
transformation.

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3. Oxidative Modifications of Proteins

• Reacts with labile amino acids, methionine,

cystein, histidine and lysine.
• Formation of sulfhydryl mediated cross linkages.
• Fragmentation of polypeptide chains.
• Enzymes containing cross linkages are acted
upon by proteases & gets degraded leading to
havoc within the cells.

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 INACTIVATION OF FREE RADICALS
1. SPONTANEOUS DECAY
Example:
Superoxide into oxygen and H2O2.
2. NONENZYMATIC INACTIVATION by endogenous
or exogenous antioxidants.
Examples:
 Vit E.
 Sulfhydryl containing compounds like cystein and
glutathione.
 Serum proteins.
• Albumin.
• Ceruloplasmin.
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• Transferrin.
3. ENZYMATIC INACTIVATION OF FREE RADICALS.
( i ) Superoxide dismutase:
Inactivates superoxide by converting it to H 2O2.
( ii ) Catalase:
Inactivates H2O2 by converting it into water.
( iii ) Glutathione peroxidase:
Inactivates hydroxyl ion and hydrogen peroxide by
.
releasing hydrogen which then combines with OH or
H2O2 to form water.

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TYPES OF CELL INJURY EMPLOYING FREE RADICALS

• Chemical Injury.
• Radiation Injury.
• Oxygen toxicity.
• Cellular aging.
• Microbial killing.
• Inflammatory damage.
• Tumour destruction.
Final common pathway in most types of
cell injury.
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CHEMICAL INJURY

CELL INJURY INDUCED

BY THE CHEMICAL
AGENTS & DRUGS

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 TYPES OF CHEMICALS
TWO TYPES OF CHEMICALS
• WATER SOLUBLE:
Mercuric chloride
Cyanide
Anticancer chemotherapeutic Agents
• LIPID SOLUBLE:
Carbon tetrachloride
Acetaminophen (paracetamol)
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 MECHANISMS

1. Direct Cytotoxic effect

2. Indirect Mechanism by
forming reactive toxic
metabolites.
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DIRECT CYTOTOXIC EFFECT
• Water Soluble Chemicals
• By combining directly with some critical
molecular component.
• Vulnerable Cells.
• Examples:
Hg Cl 3 Poisoning
Cyanide Poisoning
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 HgCl3 POISONING

• Binds to the sulphydryl group of

membrane proteins.
• Increased membrane permeability
• Inhibition of ATPase dependent transport.

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 CYANIDE POISONING

• Directly poisons mitochondrial enzymes

• Leads to decreased ATP production.

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 2. INDIRECT MECHANISM
•Employed by Lipid soluble drugs and chemicals
•Biologically inactive.
•Converted to reactive toxic metabolism.
•Enzyme P450
•ER of liver cells and other organs.
EXAMPLES:
CCl4 Poisoning.
Paracetamol Toxicity.
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CARBON TETRACHLORIDE POISONING
• CCl4 is widely used in dry cleaning.
• Metabolised in the liver.
CCl4 in liver cells
P450 mixed function oxidase in SER
CCl3 (Toxic free radical)

Auto-oxidation of membrane phospholipids

Membrane damage to RER Release of products of lipid peroxidation

Detachment of Ribosomes Damage to plasma membrane

Lipid acceptor protein synthesis Permeability to Na, H2O + Ca

Fatty liver Cell swelling

Massive influx of Ca
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Inactivation of mitochondria, cell enzymes and denaturation of proteins
 PARACETAMOL TOXICITY

• Small amounts are converted by P-450 to a highly

toxic metabolite.
• Inactivated by interaction with reduced Glutathion.
• Depletion of reduced glutathion with large doses
• Accumulation of toxic metabolite
• Massive liver cell damage 3-5 days after intake.
• Administration of antioxidants.

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 MORPHOLOGICAL PATTERNS OF CELL INJURY

REVERSIBLE CELL INJURY

• Generalized swelling of the cell.
• Formation of cellular blebs.
• Aggegation of intramembranous particles.
• Appearance of lipid vacuoles within the cytoplasm.
• Swelling of ER and mitochondria.
• Dispersion of ribosomes.
• Appearance of small densities within the organelles.
• Clumping of nuclear chromatin.
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MORPHOLOGICAL PATTERNS OF CELL INJURY

IRREVERSIBLE CELL INJURY

Two patterns:
Necrosis
Apoptosis

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 DIFFERENCES B/W REVERSIBLE &
IRREVERSIBLE INJURY
Features REVERSIBLE IRREVERSIBLE
1. Stimuli Mild Severe
2. Pathologic Reversible Irreversible
Changes
3. Mediating Factor AMP Mediated Calcium Mediated

4. Integrity of cell Maintained, no rupture Lost, ruptured

membrane
5. Mitochondrial No swelling, small Swollen, large
Changes densities densities
6. Nuclear changes Clumping of nuclear Pyknosis,
chromatin Karyolysis,
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Karyorrhexis
 NECROSIS – CELL DEATH

Necrosis can be defined as a

morphologic pattern which appears as
the final end result of irreversible
cell injury and includes the sum of the
morphological changes that follow
cellular death in living tissues or organs.
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 WHY NECROSIS OCCURS?
• Denaturation of proteins.
• Enzymatic digestion of organelles
and other cytosolic compounds.

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 NECROSIS – MORPHOLOGICAL CHANGES

Appearance of Necrotic Cells

• Appears pink and glassy and may be vacuolated.
• Cell membranes are fragmented.
• Nuclear changes include:
 Pyknosis – Small, dense nucleus.
 Karyolysis – Faint dissolved nucleus.
 Karyorrhexis – breaking up of nucleus.

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NECROSIS – MORPHOLOGICAL CHANGES

Accompanying Morphological
Changes
• INFLAMMATION- Attraction of neutrophils by
necrotic cells
• DYSTROPHIC CALCIFICATION- Attraction of
calcium by necrotic cells.

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TYPES OF NECROSIS
Four different patterns.
1. Coagulation Necrosis.
2. Liquefaction Necrosis.
3. Caseous Necrosis.
4. Fat Necrosis
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COAGULATION NECROSIS
• Occurs due to denaturation of
cytoplasmic proteins.
• Preservation of cellular
framework.
• Occurs in heart, liver, kidney.
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LIQUEFACTION NECROSIS
• Enzymatic digestion of cellular
organelles predominates.
• Loss of cellular outlines.
• Occurs in brain and localized
bacterial infection.
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 CASEOUS NECROSIS
• Both denaturation and enzymatic
digestion operate.
• The necrotic area appears cheesy.
• Characteristic of TB lesions
anywhere in the body.

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 FAT NECROSIS
• Activation of lipases.
• Necrotic fat shows shadowy outlines
of cells and basophilic stippling as a
result of Ca-deposition.
• Occurs in adipose tissue.
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 GANGERENOUS NECROSIS
• A clinical term used in surgical practice.
• Affects mostly lower limb due to loss of
its blood supply.
• Initially there is only coagulation
necrosis.
• Later on due to the superimposed
bacterial infection it is modified by
liquefaction necrosis. 38
 CONSEQUENCES OF NECROSIS

INFLAMMATION NO INFLAMMATION

Inflammatory cells Attraction of Calcium salts

Phagocytosis Dystrophic Calcification

Prompt removal of necrotic debris

Necrotic debris converted into Calcified material

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 APOPTOSIS
Greek word meaning falling off
DEFINITION:
It is a form of cell death designated to
eliminate unwanted host cells through
activation of a coordinated, internally
programmed series of events effected
by a dedicated sets of gene products.
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EXAMPLES OF APOPTOSIS
1. During Embryogenesis.
2. Hormone Dependent Involution.
3. Cell deletion in proliferating cell
population.
4. Cell death in regressive tumours.
5. Death of Neutrophils in Acute
Inflammation.
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EXAMPLES OF APOPTOSIS (Contd.)
6. Deletion of Auto-reactive T cells in the
developing thymus.
7. Cell death induced by cytotoxic T cells
8. Pathologic atrophy in parenchymal
organs after duct obstruction.
9. Cell Injury in certain viral diseases.
10. Cell death produced by low doses of
variety of injuries stimuli.
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MORPHOLOGIC FEATURES OF APOPTOSIS

• Cell Shrinkage.
• Chromatin condensation and fragmentation.
• Formation of cytoplasmic blebs
• Apoptic bodies.
• Absence of Inflammation.

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BIOCHEMICAL FEATURES OF APOPTOSIS
• Protein cleavage.
 Cystein proteases.
 Caspases.
• Protein cross-linking.
 Transglutaminase.
• DNA breakdown.
• Plasma membrane alterations.
 Flipping off phosphatidylserine from the inner to the
outer layer of plasma membrane.
 Expression of thrombospondin
 Lead to early phagocytosis of apoptotic cells by
phagocytes.
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 MECHANISMS
• Apoptosis is the end point of an energy
dependent cascade of molecular events
initiated by certain stimuli.
• Consists of four separable but
overlapping components.
Signaling pathways.
Control and Integration.
Execution Phase.
Removal of dead cells by phagocytosis.
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 SIGNALING PATHWAYS
• Apoptotic Stimuli.
Transmitted across the plasma membrane.
Acts on intracellular regulator molecules to initiate
apoptosis.
• Survival Stimuli.
Growth Factor & certain hormones
Suppression of pre-existing death programs
• Absence or withdrawal of Survival Stimuli.
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CONTROL & INTEGRATION STAGE

• Performed by specific proteins.

• Connect death signals to the execution
programme.
• Two broad schemes:
Direct transmission of apoptic signals to
cytosol.
Indirect transmission of apoptic signals.

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CONTROL & INTEGRATION STAGE (Contd.)

DIRECT TRANSMISSTON
Two Modes:
• Cytokines mediated
• Cytotoxic T-lymphocytes mediated

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CONTROL & INTEGRATION STAGE (Contd.)

DIRECT TRANSMISSION (Contd.)

Cytokines Mediated Direct Transmission
• Two Cytokines:
 FAS
 TNF
• Combine with specific receptors on the outer
side of cells.
• Cytokines-receptor combination binds to the
specific adapter proteins on the inner side of
plasma membranes.
• This binding leads to the activation of
caspases causing apoptosis.
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CONTROL & INTEGRATION STAGE (Contd.)

DIRECT TRANSMISSION (Contd.)

CTL Mediated Direct Transmission
• CTL recognize foreign antigens present
on the surface of the infected host cell.
• Formation of two substances.
Perforin
Granzyme - B
• Both these activate caspases causing
apoptosis.
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