1. The biochemistry of cell injury and cell death
Dr Stephany Veuger

2. 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

3. Learning Outcomes
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

4. Causes of cell injury Physical Chemical Infectious Immunologic
Genetic derangement
Nutritional and Oxygen Imbalances
Metabolic changes
Although different agents may have different initial targets, the final pathways are often similar

5. Cellular damage SUBLETHAL Damage is minimal Recovery LETHAL
Continued damage
Damage is massive
Response of the cell depends on nature, duration and severity of injuru

6. Mechanisms of cell injury
Injurious agents can affect the cell at a number of levels by damaging :
Plasma membrane
Aerobic respiration and ATP production
Protein synthesis
Genetic machinery

7. Morphological indicators of cell injury
Alterations to plasma membrane
Cytoskeleton damage
Mitochondrial condensation
Mitochondrial swelling
Dilatation of ER
Ribosome detachment
Alterations to lysosomes
Cytoplasmic Blebbing early on in face of reversible cell injury on cell surface
Membrane blebs involves disruption of cytoskeleton-membrane interactions this may be Ca mediated
Mito swellin sign of irreversible damage
Transition from mito condensation to swellijg ..ca

8. Morphological changes following sub-lethal injury
Mitochondrial swelling (low amplitude swelling)
-vacuoles distort cristae
-reversible
ER swelling
-loss of ribosomes
High amplitude swelling
-cristae destroyed
-irreversible
ATP-dependent processes affected
Usually considerable delay before unjury is seen as morphological chnages
Ultrastructure of mito is clear predictor of irreversible damage

9. Morphological changes following sub-lethal injury
Under the microscope, these changes are seen as;
Cellular swelling
Pale cytoplasm
Small intracellular vacuoles
CLOUDY SWELLING or HYDROPIC DEGENERATION
Accumulation of lipid
FATTY CHANGE
Niormal cell had higher potassium but lower sodium levels maintained by pumps which requjire ATP
Significance of fqatty change?
Hepatocytes…cells with high fattunrover….

10. Fatty Change
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
4 main causes
Fattyu change occurs after metabolic disturbances eg diabetes or alcohol abuse

11. Cell survival
Following injury, major cellular components need to be maintained to promote survival ;
Cell membranes
Mitochondria
Cytoskeleton
Cellular DNA
- These systems are not interdependent
- Threshold – death
Sentence re damage to mito ….sign of irreversible damage…mito important primary or secondary targets

12. 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
Direct – immune mediated
Primary and secondary
Primary immune eg complement, vuruses bacteria
Secondary ischaemia, los of ATP

13. Morphological changes following lethal injury
High amplitude swelling
Morphological changes to the nucleus
Appearance of membrane blebs and holes
Dissolution of the nucleus
Distinct structural changes to cell leading to dissolution of cell via release of lysosomal enzymes
AUTOLYSIS
Lysosome – how is cell normall protected? – lysosomal enzymes only active in acid conditons

14. Morphological changes following lethal injury (nucleus)
PYKNOSIS
-condensation of nuclear chromatin
Loss of nucleolus
KARRYORRHEXIS
-fragmentation of the nucleus
KARYOLYSIS
-complete dissolution of nuclear material

15. Summary I Cell have limited capacity to adapt to change
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

16. Cell death -Follows irreversible cell damage
-Can be by accident or design
Apoptosis
Necrosis
Different morphological changes

17. Apoptosis Routine – repair and cell cycle (p53)
Programmed – co-ordinated- “shrinkage”
Stimuli mediated by immune system ; cytokines
Autophagy (self digestion)

18. Necrosis Massive damage to cellular systems
Uncontrolled loss of large numbers of cells
Extensive organelle and cell “swelling”
Rupture of plasma membrane and dissolution of the cell

19. Biochemical determinants of necrotic change
ATP
Calcium homeostasis pH
Reactive Oxygen Species (ROS)
Intracellular antioxidant levels
uncontrolled entry of Ca2+ into cytosol important final common pathway in many causes of cell death
Oxidative stress

20. ATP Produced by cellular respiration biosynthesis
Critical for function of many transport pumps
Critical for cell signalling processes
Cloudy swelling and fatty change
Major comoponent of injury is alteration of mem permeability due to alteraions in pumps
Cloudy sewllin due to ionic balance disruotion
Fatty vhange –low aTP leads to detachment of ribosomes and hence decreased rtein synthesis this eads to less transport of lipid out of cells.

21. Calcium Normal concentration in cytosol very low
-rapidly removed by ATP-dependent pumps
-bound to buffering proteins (calbindin, parvalbumin)
Increased intracellular calcium brought about by;
-↑permeability of Ca2+ channel
-direct membrane damage
-ATP depletion
-mitochondrial damage
Intracellular ca normally stored in mito (and ER) therefore mito dmaage…..
ATP …pumps therefore demosntrating the interdependnece…..

22. 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

23. Reative Oxygen species (ROS)
Most important free radicals in the body are the oxygen-derived free radicals
Attack bio-molecules
Lipid peroxidation - decreases membrane fluidity and destabilises membrane receptors.
Most iportant free radicals in the body are the oxygen-derived free radicals

24. Effect of ROS on biomolecules
Damaged lipid can crosslink with protein and form lipofuscin granules which are difficult to degrade and remove as part of normal cell repair. As people age these granules form liver spots on the skin and Lewy bodies in the neurones. In diabetes, the long-term consequences associated with uncontrolled hyperglycaemia are due to the cross linking and denaturation of proteins. One of the mechanisms for this is the synthesis of another ROS, methylglycol, which at high concentrations overcomes the antioxidant action of GSH.
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25. Changes in metabolism
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

26. Excellent example of the cellular response to a damaging stimulus
ISCHAEMIA
Excellent example of the cellular response to a damaging stimulus
ISCHAEMIA = LACK OF OXYGEN SUPPLY
HYPOXIA =LACK OF OXYGEN
Most common type of injury in clinical medicine
Local due to atheroscleoritic pllaques for example. On exertions, cells are starved of oxygen and convert to anaerobic resp

27. 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)

28. Biochemical and morphological changes due to Ischaemia (I)
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

29. Biochemical and morphological changes due to Ischaemia (II)
Integrity of RER relies on Na+ pump
ribosomes detach
Protein synthesis ceases
Calcium – activation of several destructive enzyme systems
Phospholipid synthesis ceases
Further disruption of membranes

30. Biochemical and morphological changes due to Ischaemia; pH (III)
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

31. Biochemical and morphological changes due to Ischaemia; pH (IV)
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

32. ? ISCHAEMIA ? ? ? ?ATP ? pH ? Protein Cell death Reduced oxidative
phosphorylation
Anaerobic respiration
Decrease in
sodium pump
?ATP
Lactic acid ?
? pH
Potassium
ribosomes detach ?
Calcium
? Protein
synthesis ? ?
lysosomes
Water
Cell death

33. ? ISCHAEMIA ? ? ? ?ATP ? pH ? Protein Cell death Reduced oxidative
phosphorylation
Anaerobic respiration
Decrease in
sodium pump
?ATP
Lactic acid ?
? pH
Potassium
ribosomes detach ?
Calcium
? Protein
synthesis ? ?
lysosomes
Water
Cell death

34. ISCHAEMIA Cell death Reduced oxidative phosphorylation
Anaerobic respiration
Decrease in
sodium pump
ATP
Lactic acid
Potassium
ribosomes detach pH
Calcium
Protein
synthesis
pyknosis
lysosomes
Water
karyorrhexis
karyolysis
Cell death

35. Summary II Cells die by two main pathways
Biochemical determinants of injury and death ATP, Ca2+, pH, ROS
Ischaemia most common injury in clinical medicine

36. The role of free radicals and anti-oxidant mechanisms
in health and disease

37. Overview 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
The mnost impoortant FR in the bosy include the oxygen derived radicals (ROS)

38. Learning Outcomes
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

39. What is a free radical?
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

40. Free radicals 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
New radicals are formed

41. Sources of free radicals
Ionising radiation
Chemicals
Exposure to excess oxygen
Cell respiration
Inflammation
Free radicals produced by cigarette smoke =

42. Ionising radiation 80 % of effect of IR on cells is due to FRs
Body 70% water splits water to produce hydroxyl radicals

43. Reactive oxygen species (ROS)
H2O2
OH•
RO•
RCOO•
HOCl
Superoxide
leakage from the electron transport chain is the main source
Hydrogen peroxide
Not a free radical itself, but is dangerous because in the presence of a transition metal it quickly produces OH•
Hydroxyl radical
Generated from H2O2 by Fenton reaction
Organic radical
Usually produced from C=C bonds
Peroxyl radical
Generated when radicals attack lipids
Hypochlorous acid
Generated on purpose as part of immune “respiratory burst”
If these radicals contain oxygen or the molecule can generate radicals then they are called reactive oxygen species (ROS) and include:-
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44. Abstraction Stripping of electrons from other atoms or molecules
R• + HB RH + B•
Propogation
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.

45. Addition Attack of hydroxyl radical on DNA bases Thymine + OH●
Hydroxythymine radical
Measure of oxidative damage in DNA
Thymine-OH● + OH●
Thymine glycol

46. Effect of ROS on biomolecules
Oxidative damage to DNA
Peroxidation of membrane
Carb damage eg synovial fluid
Proteins target them for degradation -enzymes
Most notable targets are DNA and cell membrane
Damaged lipid can crosslink with protein and form lipofuscin granules which are difficult to degrade and remove as part of normal cell repair. As people age these granules form liver spots on the skin and Lewy bodies in the neurones. In diabetes, the long-term consequences associated with uncontrolled hyperglycaemia are due to the cross linking and denaturation of proteins. One of the mechanisms for this is the synthesis of another ROS, methylglycol, which at high concentrations overcomes the antioxidant action of GSH. 46

47. Effect on lipid
Peroxidation of membrane lipids is the most important cause of serious acute damage to cells
Malondialdehyde = marker for oxidative stress
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
Membrane becomes leaky
Effects on fluidity flexibility and transport
Complete breakdown of the integrity of the membrane
Influenced by pH and leads to atherosclerosis
Peroxyl radical

48. Rancidity of butter

49. Effect on DNA
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
Malondialdehyde forms DNA adducts which are mutagenic

50. Effect on proteins
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)
FRs mainly affect proteins by causing them to be cross linked defective enzymes etc
Cross linking may also involve methionine, histidine and lysine residues

51. Effect on carbohydrates
Hydroxyl radical - H abstraction
Depolymerisation of hyaluronic acid -Synovial fluid viscosity

52. ROS as a protective mechanism
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
Tumor destruction by macrophages
Can go wrong RA Ibowel disease where excessive phagocytic activation occurs

53. The superoxide radical O2●-
Generated during electron transport chain
Oxidase enzymes
O O2●-
oxidase
In mitochondria
Including xanthine oxidase
Def in this enzyme condition chronic granulomatous –characterised by recurrent infection
P450 system

54. The hydroxyl radical OH●
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
Most dangerous radical in the body
Produces products that cannot be regenerated by the body
Gamma rays split water to procue it as shown earlier

55. Promoters of free radical damage : Metal ions
Iron and copper
Encourage formation of hydroxyl radical
Fe2+ + H2O
Iron conjugated to protein and stored as ferritin/ transported as transferrin
Copper is transported as caeruloplasmin
Free ions = pro-oxidants
Fe3+ + OH● + OH
Human body has a complex system of metal ion transport and storage

56. Free Radicals and disease
Accumulation of damaged proteins, carbohydrates, lipids and
nucleic acids contributes to a wide range of human diseases
FR damage
Cell injury
Apoptosis
Necrosis
Ageing
Cancers
Athero-
sclerosis
Degenerative
diseases
Evdence that FRs contribute to aetiology of a variety of disease statres
Including rA and the neuro degen eg parkinsons and alz
Cell death

57. FRs and cardiovascular disease
There is growing evidence that lipid peroxidation occurs in blood vessel walls
Contributes to the development of atherosclerosis raising the risk of stroke and myocardial infarction.
Lipofushin
Undegradeable lipoprotein complex
Brown atrophy of organs in old people

58. 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

59. Summary I Free radicals
Extremely reactive chemical species with an unpaired electron
Produced in cells as metabolic by-products
Produced by phagocytic cells as part of inflammatory defences
Produced by the action of toxic compounds
Cause cell injury
Caused by cell injury

60. Summary II Free radicals
Free radicals can cause oxidative damage to cells components
The most dangerous free radical is the hydroxyl ion
Damage by free radicals is believed to contribute to the pathogenesis of many chronic diseases

61. Antioxidants Defence systems
1) Directly – blocking formation or scavenging
2) Binding metals that catalyse ROS formation
3) Enzyme activity
Every cel in the body is attacked by FRs 10,000 times a day
Systems designed to mop up FRs
Because FRs are produced routinely s byproducts of metabolism…mechansism to cope with …
Intrinsic enzymes limit availability
Vitamins scavenge FRs

62. Intracellular antioxidants
Glutathione peroxidase
Removes hydrogen peroxide
Selenium dependent
Cytosol and mitochondria
Glutathione
Scavenger of hydroxyl radical
Superoxide dismutase
Catalyses conversion of superoxide to hydrogen peroxide
Catalase

63. 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 superoxide dismutase
Manganese
Copper
Selenium
Component of glutathione peroxidase

64. Antioxidant enzymes H2O2 + O2 O2 + H2O
Superoxide dismutase converts superoxide to hydrogen peroxide and oxygen
O2●- + O2●- + 2H
catalase and glutathione peroxidase convert hydrogen peroxide to water and oxygen
2H2O2
H2O2 + O2
Dismutase is an enzyme that catalyses a reaction where two of the same substrates have a different fate
Hydrogen peroxide, although n ot a radical itself, is still extremeky toxic to the cell and can react with superoidxe to form hydroxyl radical
Catalase is found in peroxisomes
O2 + H2O

65. Free radical theory of ageing
Harman in xx
Dorian grey mouse resistance to ox radical generatirs life increased by 30 %
Oxiadative damage to collagen etc –lines and wrinkels

66. Summary III Antioxidants
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