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

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Presentation on theme: "The biochemistry of cell injury and cell death Dr Stephany Veuger."— Presentation transcript:

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

2 Overview Part A Review causes of cellular damage Review causes of cellular damage Types of cellular damage Types of cellular damage Mechanisms of cell death Mechanisms of cell death Biochemical events that lead to cell death Biochemical events that lead to cell death Part B Free radicals Free radicals Diseases associated with free radical damage Diseases associated with free radical damage

3 Learning Outcomes Understand how the basic functions of the cell are affected by injury Understand how the basic functions of the cell are affected by injury Discuss morphological and biochemical changes in response to injury Discuss morphological and biochemical changes in response to injury Be able to explain the types of cell death Be able to explain the types of cell death Describe the biochemical changes in response to ischaemia Describe the biochemical changes in response to ischaemia

4 Causes of cell injury Physical Physical Chemical Chemical Infectious Infectious Immunologic Immunologic Genetic derangement Genetic derangement Nutritional and Oxygen Imbalances Nutritional and Oxygen Imbalances Metabolic changes Metabolic changes

5 Cellular damage SUBLETHAL Damage is minimal Damage is minimal Recovery RecoveryLETHAL Continued damage Continued damage Damage is massive Damage is massive

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

7 Morphological indicators of cell injury Alterations to plasma membrane Alterations to plasma membrane Cytoskeleton damage Cytoskeleton damage Mitochondrial condensation Mitochondrial condensation Mitochondrial swelling Mitochondrial swelling Dilatation of ER Dilatation of ER Ribosome detachment Ribosome detachment Alterations to lysosomes Alterations to lysosomes

8 Morphological changes following sub-lethal injury Mitochondrial swelling (low amplitude swelling) Mitochondrial swelling (low amplitude swelling) -vacuoles distort cristae -reversible ER swelling ER swelling -loss of ribosomes High amplitude swelling High amplitude swelling -cristae destroyed - irreversible ATP-dependent processes affected

9 Morphological changes following sub-lethal injury Under the microscope, these changes are seen as; Cellular swelling Cellular swelling Pale cytoplasm Pale cytoplasm Small intracellular vacuoles Small intracellular vacuoles CLOUDY SWELLING or HYDROPIC DEGENERATION Accumulation of lipid Accumulation of lipid FATTY CHANGE

10 Fatty Change Deficiency in lipid acceptor proteins, preventing export of formed triglycerides Deficiency in lipid acceptor proteins, preventing export of formed triglycerides -carbon tetrachloride, malnutrition, hypoxis Increased mobilisation of free FA into cells Increased mobilisation of free FA into cells - diabetes mellitus and nutritional deprivation Increased conversion of fatty acids to triglycerides Increased conversion of fatty acids to triglycerides -alcohol abuse Reduced oxidation of triglycerides to acetyl-coA Reduced oxidation of triglycerides to acetyl-coA -hypoxia, toxins

11 Cell survival - Following injury, major cellular components need to be maintained to promote survival ; Cell membranes Cell membranes Mitochondria Mitochondria Cytoskeleton Cytoskeleton Cellular DNA Cellular DNA - These systems are not interdependent - Threshold – death

12 Plasma membrane Integrity following injury is ESSENTIAL Direct Direct Failure of phospholipid biosynthesis Failure of phospholipid biosynthesis Particularly vulnerable to free radical attack Particularly vulnerable to free radical attack Degradation of phospholipids by Ca2+ dependent phospholipases Degradation of phospholipids by Ca2+ dependent phospholipases

13 Morphological changes following lethal injury High amplitude swelling High amplitude swelling Morphological changes to the nucleus Morphological changes to the nucleus Appearance of membrane blebs and holes Appearance of membrane blebs and holes Dissolution of the nucleus Dissolution of the nucleus Distinct structural changes to cell leading to dissolution of cell via release of lysosomal enzymes Distinct structural changes to cell leading to dissolution of cell via release of lysosomal enzymes AUTOLYSIS AUTOLYSIS

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

15 Summary I Cell have limited capacity to adapt to change Cell have limited capacity to adapt to change Mild injury can be accommodated by cells but is evident by biochemical and morphological changes Mild injury can be accommodated by cells but is evident by biochemical and morphological changes Sub-lethal –reversible Sub-lethal –reversible Injury that is sufficient to cause morpholgical changes to the nucleus is usually lethal 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 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 Apoptosis Necrosis Necrosis Different morphological changes

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

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

19 Biochemical determinants of necrotic change ATP ATP Calcium homeostasis Calcium homeostasis pH pH Reactive Oxygen Species (ROS) Reactive Oxygen Species (ROS) Intracellular antioxidant levels Intracellular antioxidant levels

20 ATP Produced by cellular respiration Produced by cellular respiration biosynthesis biosynthesis Critical for function of many transport pumps Critical for function of many transport pumps Critical for cell signalling processes Critical for cell signalling processes Cloudy swelling and fatty change Cloudy swelling and fatty change

21 Calcium Normal concentration in cytosol very low Normal concentration in cytosol very low -rapidly removed by ATP-dependent pumps -bound to buffering proteins (calbindin, parvalbumin) Increased intracellular calcium brought about by; Increased intracellular calcium brought about by; -↑permeability of Ca2+ channel -direct membrane damage -ATP depletion -mitochondrial damage

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 Membrane damage Destabilising of cytoskeleton Reduced ATP

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

24 Effect of ROS on biomolecules

25 Changes in metabolism Accumulation of materials as a result of changes in metabolism may compromise normal function of cell Accumulation of materials as a result of changes in metabolism may compromise normal function of cell Lipid (fatty change already covered) Lipid (fatty change already covered) Protein –kidneys, reversible Protein –kidneys, reversible Carbohydrate-diabetes, glycogen storage disorders Carbohydrate-diabetes, glycogen storage disorders pigments pigments

26 ISCHAEMIA Excellent example of the cellular response to a damaging stimulus ISCHAEMIA = LACK OF OXYGEN SUPPLY HYPOXIA =LACK OF OXYGEN

27 Definitions HYPOXIA HYPOXIA -decrease in oxygen in arterial blood or tissues ISCHAEMIA ISCHAEMIA -local anaemia, leading to hypoxia eg. Obstruction to blood flow to organ/tissue INFARCTION INFARCTION -sudden insufficiency of blood supply producing macroscopic areas of necrosis (eg. MI) -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 Shift from aerobic to anaerobic respiration Reduction in ATP Reduction in ATP Failure of ATP-dependent pumps (Na+/K+, ATPase and Ca2+) Failure of ATP-dependent pumps (Na+/K+, ATPase and Ca2+) Failure to maintain intracellular ionic balance Failure to maintain intracellular ionic balance Accumulation of Na+ in cytoplasm Accumulation of Na+ in cytoplasm Ingress of calcium and water and outflow of potassium ions 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 ribosomes detach Protein synthesis ceases Protein synthesis ceases Calcium – activation of several destructive enzyme systems Calcium – activation of several destructive enzyme systems Phospholipid synthesis ceases Phospholipid synthesis ceases Further disruption of membranes

30 Biochemical and morphological changes due to Ischaemia; pH (III) Anaerobic respiration results in lactic acid production Anaerobic respiration results in lactic acid production Intracellular pH decreases Intracellular pH decreases Membranes under acid attack Membranes under acid attack pH further augmented via phosphate ions produced by Ca2+ activated phosphatases pH further augmented via phosphate ions produced by Ca2+ activated phosphatases Fall in pH stimulates pyknosis Fall in pH stimulates pyknosis

31 Biochemical and morphological changes due to Ischaemia; pH (IV) Lysosomes Lysosomes Release of destructive enzymes leads to karryhrrexis and karyolyiss Release of destructive enzymes leads to karryhrrexis and karyolyiss Cell death Cell death Neighbouring cells injured Neighbouring cells injured Initial changes in ischaemia reversible but nuclear changes catastrophic for cell Initial changes in ischaemia reversible but nuclear changes catastrophic for cell

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

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

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

35 Summary II Cells die by two main pathways Cells die by two main pathways Biochemical determinants of injury and death ATP, Ca2+, pH, ROS Biochemical determinants of injury and death ATP, Ca2+, pH, ROS Ischaemia most common injury in clinical medicine 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? What are free radicals? Sources of free radicals Sources of free radicals Types of free radicals (ROS) Types of free radicals (ROS) Types of free radical damage Types of free radical damage Diseases associated with free radicals Diseases associated with free radicals Anti-oxidant mechanisms Anti-oxidant mechanisms

38 Learning Outcomes Define the terms free radical and reactive oxygen species Define the terms free radical and reactive oxygen species Characterise the major reactive oxygen species and their sources Characterise the major reactive oxygen species and their sources Discuss the negative effects of ROS on bio-molecules Discuss the negative effects of ROS on bio-molecules Describe the cellular defence mechanisms against free radicals 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 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 A radical that can move freely within cell and across membranes is a free radical Highly unstable and extremely reactive Highly unstable and extremely reactive

40 Free radicals Most molecules found in the body are not 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. 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 When this happens, a free radical chain reaction results

41 Sources of free radicals Ionising radiation Ionising radiation Chemicals Chemicals Exposure to excess oxygen Exposure to excess oxygen Cell respiration Cell respiration Inflammation Inflammation

42 Ionising radiation

43 Reactive oxygen species (ROS) O2-O2-O2-O2- H2O2H2O2H2O2H2O2 OH OH RO RO RCOO 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 Not a free radical itself, but is dangerous because in the presence of a transition metal it quickly produces OH Hydroxyl radical Generated from H 2 O 2 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”

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 Attack of hydroxyl radical on DNA bases Thymine + OH● Thymine-OH● Hydroxythymine radical Thymine-OH● + OH●Thymine glycol

46 Effect of ROS on biomolecules

47 Effect on lipid Peroxidation of membrane lipids is the most important cause of serious acute damage to cells 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 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

48

49 Effect on DNA Reactive FRs such as the hydroxyl radical can react with both the deoxyribose and the bases of 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. 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 Bases can be affected by addition reactions, ultimately leading to mutation and cellular derangement Depletion of NADH pools Depletion of NADH pools

50 Effect on proteins Formation of disulphide bridges by oxidation of the thiol groups (-SH) of cysteine residues Formation of disulphide bridges by oxidation of the thiol groups (-SH) of cysteine residues Attack metal binding sites leading to degradation by proteases Attack metal binding sites leading to degradation by proteases Loss of biological activity eg enzymes Loss of biological activity eg enzymes Malondialdehyde - protein adducts or advanced lipoxidation end products (APE) Malondialdehyde - protein adducts or advanced lipoxidation end products (APE)

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

52 ROS as a protective mechanism Peroxisome has highest concentration of FRs Peroxisome has highest concentration of FRs Phagocytes use the generation of FRs in phagosome to attack and destroy bacteria Phagocytes use the generation of FRs in phagosome to attack and destroy bacteria RESPIRATORY BURST –rapid use of oxygen to generate FRs RESPIRATORY BURST –rapid use of oxygen to generate FRs Problem during e.g. MI. Designed to remove dead cells but causes local inflammation Problem during e.g. MI. Designed to remove dead cells but causes local inflammation

53 The superoxide radical O2●- Generated during electron transport chain Generated during electron transport chain Oxidase enzymes Oxidase enzymes O2 O2●- O2 O2●- oxidase

54 The hydroxyl radical OH● An extremely reactive species An extremely reactive species Reacts with great speed with whatever molecules are in its vicinity 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 Responsible for many of the effects of high level radiation in the human body Can be formed by fenton reaction Can be formed by fenton reaction

55 Promoters of free radical damage : Metal ions Iron and copper Iron and copper Encourage formation of hydroxyl radical Encourage formation of hydroxyl radical Fe 2+ + H 2 O Iron conjugated to protein and stored as ferritin/ transported as transferrin Iron conjugated to protein and stored as ferritin/ transported as transferrin Copper is transported as caeruloplasmin Copper is transported as caeruloplasmin Free ions = pro-oxidants Free ions = pro-oxidants Fe 3+ + OH● + OH

56 Free Radicals and disease Accumulation of damaged proteins, carbohydrates, lipids and nucleic acids contributes to a wide range of human diseases FR damageCell injury ApoptosisNecrosisAgeingCancers Athero- sclerosis Degenerative diseases Cell death

57 FRs and cardiovascular disease There is growing evidence that lipid peroxidation occurs in blood vessel walls 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. Contributes to the development of atherosclerosis raising the risk of stroke and myocardial infarction. Lipofushin Lipofushin

58 Free radicals in cancer FRs can severely damage DNA of cells which can lead to abnormal cells & cancer growth FRs can severely damage DNA of cells which can lead to abnormal cells & cancer growth FRs can convert certain chemicals into carcinogens FRs can convert certain chemicals into carcinogens DNA repair / apoptois DNA repair / apoptois -Hydroxyguanine -Hydroxyguanine

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

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

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 Removes hydrogen peroxide

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 Superoxide dismutase converts superoxide to hydrogen peroxide and oxygen Superoxide dismutase converts superoxide to hydrogen peroxide and oxygen O 2 ●- + O 2 ●- + 2H O 2 ●- + O 2 ●- + 2H catalase and glutathione peroxidase convert hydrogen peroxide to water and oxygen catalase and glutathione peroxidase convert hydrogen peroxide to water and oxygen 2H 2 O 2 2H 2 O 2 H 2 O 2 + O 2 O 2 + H 2 O

65 Free radical theory of ageing

66 Summary III Antioxidants Maintenance of cell integrity depends on a balance between FR activity and antioxidant status Maintenance of cell integrity depends on a balance between FR activity and antioxidant status Fat-soluble antioxidant vitamins are essential for controlling lipid peroxidation Fat-soluble antioxidant vitamins are essential for controlling lipid peroxidation Diet rich in fruit and vege may prevent disease Diet rich in fruit and vege may prevent disease


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