Transcript Free radicals

The biochemistry of
cell injury and cell death
Dr Stephany Veuger
Overview
Part A
 Review causes of cellular damage
 Types of cellular damage
 Mechanisms of cell death
 Biochemical events that lead to cell death
Part B
 Free radicals
 Diseases associated with free radical damage
Learning Outcomes
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Understand how the basic functions of the cell
are affected by injury
Discuss morphological and biochemical changes
in response to injury
Be able to explain the types of cell death
Describe the biochemical changes in response to
ischaemia
Causes of cell injury
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Physical
Chemical
Infectious
Immunologic
Genetic derangement
Nutritional and Oxygen Imbalances
Metabolic changes
Cellular damage
SUBLETHAL
 Damage is minimal
 Recovery
LETHAL
 Continued damage
 Damage is massive
Mechanisms of cell injury
Injurious agents can affect the cell at a number of
levels by damaging :
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Plasma membrane
Aerobic respiration and ATP production
Protein synthesis
Genetic machinery
Morphological indicators of
cell injury
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Alterations to plasma membrane
Cytoskeleton damage
Mitochondrial condensation
Mitochondrial swelling
Dilatation of ER
Ribosome detachment
Alterations to lysosomes
Morphological changes following
sub-lethal injury
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Mitochondrial swelling (low amplitude swelling)
-vacuoles distort cristae
-reversible
ER swelling
-loss of ribosomes
High amplitude swelling
-cristae destroyed
-irreversible
ATP-dependent processes affected
Morphological changes following
sub-lethal injury
Under the microscope, these changes are seen as;
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Cellular swelling
Pale cytoplasm
Small intracellular vacuoles
CLOUDY SWELLING or HYDROPIC DEGENERATION
Accumulation of lipid
FATTY CHANGE
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Fatty Change
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Deficiency in lipid acceptor proteins, preventing
export of formed triglycerides
-carbon tetrachloride, malnutrition, hypoxis
Increased mobilisation of free FA into cells
- diabetes mellitus and nutritional deprivation
Increased conversion of fatty acids to triglycerides
-alcohol abuse
Reduced oxidation of triglycerides to acetyl-coA
-hypoxia, toxins
Cell survival
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Following injury, major cellular components
need to be maintained to promote survival ;
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Cell membranes
Mitochondria
Cytoskeleton
Cellular DNA
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- These systems are not interdependent
- Threshold – death
Plasma membrane
Integrity following injury is ESSENTIAL
 Direct
 Failure of phospholipid biosynthesis
 Particularly vulnerable to free radical attack
 Degradation of phospholipids by Ca2+
dependent phospholipases
Morphological changes following
lethal injury
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High amplitude swelling
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Morphological changes to the nucleus
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Appearance of membrane blebs and holes
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Dissolution of the nucleus
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Distinct structural changes to cell leading to dissolution of
cell via release of lysosomal enzymes
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AUTOLYSIS
Morphological changes following
lethal injury (nucleus)
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PYKNOSIS
-condensation of nuclear chromatin
Loss of nucleolus
KARRYORRHEXIS
-fragmentation of the nucleus
KARYOLYSIS
-complete dissolution of nuclear material
Summary I
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Cell have limited capacity to adapt to change
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Mild injury can be accommodated by cells but is
evident by biochemical and morphological
changes
Sub-lethal –reversible
Injury that is sufficient to cause morpholgical
changes to the nucleus is usually lethal
Dissolution of nuclear and cytoplasmic contents
is caused by the release of lysosomal enzymes
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Cell death
-Follows irreversible cell damage
-Can be by accident or design
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Apoptosis
Necrosis
Different morphological changes
Apoptosis
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Routine – repair and cell cycle (p53)
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Programmed – co-ordinated- “shrinkage”
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Stimuli mediated by immune system ; cytokines
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Autophagy (self digestion)
Necrosis
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Massive damage to cellular systems
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Uncontrolled loss of large numbers of cells
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Extensive organelle and cell “swelling”
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Rupture of plasma membrane and dissolution of
the cell
Biochemical determinants of
necrotic change
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ATP
Calcium homeostasis
pH
Reactive Oxygen Species (ROS)
Intracellular antioxidant levels
ATP
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Produced by cellular respiration
biosynthesis
Critical for function of many transport pumps
Critical for cell signalling processes
Cloudy swelling and fatty change
Calcium
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Normal concentration in cytosol very low
-rapidly removed by ATP-dependent pumps
-bound to buffering proteins (calbindin, parvalbumin)
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Increased intracellular calcium brought about by;
-↑permeability of Ca2+ channel
-direct membrane damage
-ATP depletion
-mitochondrial damage
Cytosolic free calcium is a potent
destructive agent
CALCIUM STORES
Mitochondria
ER lumen
Pumped to extracellular space
Bound to binding proteins
Released following cell injury
FREE Ca 2+
Activation of
ATPases
Activation of
phospholipases
Activation of
proteases
Reduced ATP
Membrane
damage
Destabilising of
cytoskeleton
Reative Oxygen species (ROS)
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Most important free radicals in the body are the
oxygen-derived free radicals
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Attack bio-molecules
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Lipid peroxidation - decreases membrane
fluidity and destabilises membrane receptors.
Effect of ROS on biomolecules
GB.UNN.10
Changes in metabolism
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Accumulation of materials as a result of changes
in metabolism may compromise normal
function of cell
Lipid (fatty change already covered)
Protein –kidneys, reversible
Carbohydrate-diabetes, glycogen storage
disorders
pigments
ISCHAEMIA
Excellent example of the cellular response to a
damaging stimulus
ISCHAEMIA = LACK OF OXYGEN SUPPLY
HYPOXIA =LACK OF OXYGEN
Definitions
HYPOXIA
-decrease in oxygen in arterial blood or tissues
 ISCHAEMIA
-local anaemia, leading to hypoxia eg.
Obstruction to blood flow to organ/tissue
 INFARCTION
-sudden insufficiency of blood supply producing
macroscopic areas of necrosis (eg. MI)
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Biochemical and morphological
changes due to Ischaemia (I)
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Shift from aerobic to anaerobic respiration
Reduction in ATP
Failure of ATP-dependent pumps (Na+/K+,
ATPase and Ca2+)
Failure to maintain intracellular ionic balance
Accumulation of Na+ in cytoplasm
Ingress of calcium and water and outflow of
potassium ions
Cloudy Swelling and disruption of internal
membrane systems
Biochemical and morphological
changes due to Ischaemia (II)
Integrity of RER relies on Na+ pump
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ribosomes detach
Protein synthesis ceases
Calcium – activation of several destructive
enzyme systems
Phospholipid synthesis ceases
Further disruption of membranes
Biochemical and morphological
changes due to Ischaemia; pH (III)
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Anaerobic respiration results in lactic acid
production
Intracellular pH decreases
Membranes under acid attack
pH further augmented via phosphate ions
produced by Ca2+ activated phosphatases
Fall in pH stimulates pyknosis
Biochemical and morphological
changes due to Ischaemia; pH (IV)
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Lysosomes
Release of destructive enzymes leads to
karryhrrexis and karyolyiss
Cell death
Neighbouring cells injured
Initial changes in ischaemia reversible but
nuclear changes catastrophic for cell
ISCHAEMIA
Reduced oxidative
phosphorylation
Anaerobic respiration
Decrease in
sodium pump
? Potassium
? Calcium
? Water
?ATP
Lactic acid
ribosomes detach
? Protein
synthesis
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Cell death
? pH
lysosomes
ISCHAEMIA
Reduced oxidative
phosphorylation
Anaerobic respiration
Decrease in
sodium pump
? Potassium
? Calcium
? Water
?ATP
Lactic acid
ribosomes detach
? Protein
synthesis
?
Cell death
? pH
lysosomes
ISCHAEMIA
Reduced oxidative
phosphorylation
Anaerobic respiration
Decrease in
sodium pump
ATP
Lactic acid
Potassium
ribosomes detach
pH
Calcium
Water
Protein
synthesis
pyknosis
karyorrhexis
karyolysis
Cell death
lysosomes
Summary II
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Cells die by two main pathways
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Biochemical determinants of injury and death
ATP, Ca2+, pH, ROS
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Ischaemia most common injury in clinical
medicine
The role of free radicals
and anti-oxidant mechanisms
in health and disease
Overview
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What are free radicals?
Sources of free radicals
Types of free radicals (ROS)
Types of free radical damage
Diseases associated with free radicals
Anti-oxidant mechanisms
Learning Outcomes
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Define the terms free radical and reactive oxygen species
Characterise the major reactive oxygen species and their
sources
Discuss the negative effects of ROS on bio-molecules
Describe the cellular defence mechanisms against free
radicals
What is a free radical?
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A radical is an atom or molecule with one or
more unpaired electrons
A radical that can move freely within cell and
across membranes is a free radical
Highly unstable and extremely reactive
Free radicals
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Most molecules found in the body are not
radicals.
Any reactive FR generated will often react with
such non-radicals i.e. sugars, amino acids,
phospholipids, nucleotides, polysaccharides,
proteins, nucleic acids etc.
When this happens, a free radical chain
reaction results
Sources of free radicals
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Ionising radiation
Chemicals
Exposure to excess oxygen
Cell respiration
Inflammation
Ionising radiation
Ionising radiation
energy (hV)
H 2O
.
OH +
Hydroxyl
radical
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H
Reactive oxygen species (ROS)
O2-•
Superoxide
leakage from the electron transport chain is the
main source
H2O2
Hydrogen peroxide
OH•
Hydroxyl radical
Not a free radical itself, but is dangerous because
in the presence of a transition metal it quickly
produces OH•
Generated from H2O2 by Fenton reaction
RO•
Organic radical
Usually produced from C=C bonds
RCOO•
Peroxyl radical
Generated when radicals attack lipids
HOCl
Hypochlorous acid
Generated on purpose as part of immune
“respiratory burst”
GB.UNN.10
Abstraction
Stripping of electrons from other atoms or molecules
R• + HB
Propogation
RH + B•
H abstraction on sugars such as deoxyribose yields
many products, some of which are mutagenic.
H abstraction on unsaturated membrane lipids is
one of the most important aspects of damage to
cells by FRs.
Addition
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Attack of hydroxyl radical on DNA bases
Thymine + OH●
Thymine-OH●
Hydroxythymine radical
Thymine-OH● + OH●
Thymine glycol
Effect of ROS on biomolecules
Effect on lipid
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Peroxidation of membrane lipids is the most
important cause of serious acute damage to cells
Malondialdehyde = marker for oxidative stress
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chain reaction of lipid peroxidation
- H abstraction from a polyunsaturated fatty
acid in a membrane or lipoprotein
- Introduction of a polar group –OOH into
hydrophobic region
- Attack of one reactive FR can oxidise
multiple fatty acid side chains to lipid
peroxides
Effect on DNA
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Reactive FRs such as the hydroxyl radical can
react with both the deoxyribose and the bases of
DNA
The sugar component will be affected by H
abstraction, resulting in many products, many
of which are mutagenic.
Bases can be affected by addition reactions,
ultimately leading to mutation and cellular
derangement
Depletion of NADH pools
Effect on proteins
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Formation of disulphide bridges by oxidation of
the thiol groups (-SH) of cysteine residues
Attack metal binding sites leading to degradation
by proteases
Loss of biological activity eg enzymes
Malondialdehyde - protein adducts or advanced
lipoxidation end products (APE)
Effect on carbohydrates
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Hydroxyl radical - H abstraction
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Depolymerisation of hyaluronic acid -Synovial
fluid viscosity
ROS as a protective mechanism
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Peroxisome has highest concentration of FRs
Phagocytes use the generation of FRs in
phagosome to attack and destroy bacteria
RESPIRATORY BURST –rapid use of oxygen
to generate FRs
Problem during e.g. MI. Designed to remove
dead cells but causes local inflammation
The superoxide radical
O2●
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Generated during electron transport chain
Oxidase enzymes
O2
oxidase
O2●-
The hydroxyl radical
OH●
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An extremely reactive species
Reacts with great speed with whatever molecules
are in its vicinity
Responsible for many of the effects of high level
radiation in the human body
Can be formed by fenton reaction
Promoters of free radical damage :
Metal ions
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Iron and copper
Encourage formation of hydroxyl radical
Fe2+ + H2O
Fe3+ + OH● + OH
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Iron conjugated to protein and stored as
ferritin/ transported as transferrin
Copper is transported as caeruloplasmin
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Free ions = pro-oxidants
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Free Radicals and disease
Accumulation of damaged proteins, carbohydrates, lipids and
nucleic acids contributes to a wide range of human diseases
FR damage
Apoptosis
Necrosis
Cell death
Cell injury
Ageing
Cancers
Atherosclerosis
Degenerative
diseases
FRs and cardiovascular disease
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There is growing evidence that lipid
peroxidation occurs in blood vessel walls
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Contributes to the development of
atherosclerosis raising the risk of stroke and
myocardial infarction.
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Lipofushin
Free radicals in cancer
FRs can severely damage DNA of cells which
can lead to abnormal cells & cancer growth
 FRs can convert certain chemicals into
carcinogens
 DNA repair / apoptois
-Hydroxyguanine
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Summary I
Free radicals
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Extremely reactive chemical species with an unpaired electron
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Produced in cells as metabolic by-products
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Produced by phagocytic cells as part of inflammatory defences
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Produced by the action of toxic compounds
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Cause cell injury
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Caused by cell injury
Summary II
Free radicals
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Free radicals can cause oxidative damage to cells
components
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The most dangerous free radical is the hydroxyl ion
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Damage by free radicals is believed to contribute to
the pathogenesis of many chronic diseases
Antioxidants
Defence systems
1) Directly – blocking formation or scavenging
2) Binding metals that catalyse ROS formation
3) Enzyme activity
Intracellular antioxidants
Glutathione peroxidase Removes hydrogen peroxide
Selenium dependent
Cytosol and mitochondria
Glutathione
Scavenger of hydroxyl radical
Superoxide dismutase
Catalase
Catalyses conversion of
superoxide to hydrogen
peroxide
Removes hydrogen peroxide
Dietary antioxidants
Vitamin E (α-tocopherol) Inhibits lipid peroxidation
Vitamin C (ascorbic acid) Inhibits pro-oxidants
Vitamin A (β-carotene)
Lipid soluble radical scavenger
Zinc
Component of
dismutase
Component of
dismutase
Component of
dismutase
Component of
peroxidase
Manganese
Copper
Selenium
superoxide
superoxide
superoxide
glutathione
Antioxidant enzymes
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Superoxide dismutase converts superoxide to hydrogen
peroxide and oxygen
O2●- + O2●- + 2H
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H2O2 + O2
catalase and glutathione peroxidase convert hydrogen
peroxide to water and oxygen
2H2O2
O2 + H2O
Free radical theory of ageing
Summary III
Antioxidants
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Maintenance of cell integrity depends on a
balance between FR activity and antioxidant
status
Fat-soluble antioxidant vitamins are essential for
controlling lipid peroxidation
Diet rich in fruit and vege may prevent disease