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 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 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

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

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

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

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

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….

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

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

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

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

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

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

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

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

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

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

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.

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…..

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) 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

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. GB.UNN.10 24

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

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

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)

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

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

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

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

? 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

? 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

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

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

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

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)

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

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

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

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

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

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:- GB.UNN.10 43

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.

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

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

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

Rancidity of butter

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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