Mechanisms Of Cell Injury Lecture Notes PDF

Mechanisms of Cell Injury Dr Georgios Paraschou DVM, DACVP, DipRCPath, MRCVS modified from Dr Pompei Bolfa, Dr. María José Navarrete Talloni and Dr. Allan Kessell Learning Objectives Recognise the major causes of cell injury Detail the major mechanisms of cell injury, and their outcome Plan Normal c...

Mechanisms of Cell Injury Dr Georgios Paraschou DVM, DACVP, DipRCPath, MRCVS modified from Dr Pompei Bolfa, Dr. María José Navarrete Talloni and Dr. Allan Kessell Learning Objectives Recognise the major causes of cell injury Detail the major mechanisms of cell injury, and their outcome Plan Normal cell and homeostasis Cell injury Definition Causes Mechanisms i. Depletion of ATP ii. Mitochondrial damage iii. Ca2+ influx and loss of Ca2+ homeostasis iv. Oxidative stress v. Defects in membrane permeability vi. Damage to DNA and Proteins Normal Cell Smallest section of a living animal Review your histology notes and your pathology textbook. We encourage you to watch http://www.youtube.com/w atch?v=1IqsE8CVTms Homeostasis Homeo (Gr.): for similar, like, resembling Stasis (Gr.): stay Definition: tendency to stability in the normal body states of the organism = the ability to maintain internal equilibrium by adjusting its physiological processes Homeostasis Definition Cell injury = disruption of cell homeostasis or steady state Injury may come from outside the cell, or inside Injury affects one or more of important cellular structures Response of cell to injury : adaptation : degeneration : death of cell Can be : reversible – with eventual healing : irreversible – progression from degeneration to death Morphology of cell Injury What the injury looks like is variable and depends on: - what has caused the injury - extent of injury - duration of injury - cell type affected Causes Oxygen deficiency Infectious agents common/important Immunological dysfunction Bovine liver: Theileriosis Anoxic necrosis most common Workload imbalance : atrophy, hyperplasia, hypertrophy, metaplasia Physical agents : heat, cold, crush, friction, UV radiation, electrocution Nutritional imbalances : calorie deficiency/excess, vit/mineral deficiency (and excess) Genetic derangement : especially selective breeding Toxins Aging Horse trachea- prolonged intubation Oxygen Deficiency Hypoxia: partial reduction in O2 delivery to a tissue Anoxia: no O2 delivery to a tissue What causes hypoxia/anoxia? ‒ Inadequate oxygenation of blood  Heart failure  Respiratory failure ‒ Reduced transport of O2 in blood  Anemia  Carbon monoxide toxicosis ‒ Reduction in blood supply = ischemia  Thrombosis ‒ Blockage of cell respiratory enzymes  Cyanide toxicosis 7 Feline Thromboembolism Secondary to Hypertrophic Cardiomyopathy Gross pathology Infectious Agents  Viruses ‒ Obligate intracellular “parasites”  use host cell enzyme systems ‒ Cell survival depends on method viruses leave the cell  Bacteria ‒ Toxins ‒ Overwhelming and uncontrolled replication Turkey Histomoniasis Staphylococcal Epidermitis  Fungal (mycosis) ‒ Progressive, chronic inflammatory disease  Protozoan ‒ Replicate in specific host cells  cell destruction  Metazoan parasites ‒ Inflammation, tissue distortion, utilization of host nutrients 13 Immune Dysfunction Immune system fails to respond - Congenital defects: Severe Combined Immunodeficiency (SCIDS, Arabian foals)  antigen receptors (lymphocytes) - Acquired defects - May be transient (but not always) : Results from damage to lymphoid tissue : Viral infections, Chemicals, Drugs Immune system over-responds or aberrant reaction - Autoimmune diseases - Hypersensitivity reactions : anaphylaxis, Flea allergy dermatitis, Feline asthma BAL Cat 14 Workload Imbalance: often leads to cell adaptation Some cells can compensate : - increased workload : hypertrophy, hyperplasia - decreased workload : atrophy, some forms of oncosis Some cells cannot compensate : - degeneration and possible death See previous/later Mechanisms of Cell Injury 6 Mechanisms Mechanisms of Cell Injury: Depletion of ATP ATP Produced through 2 primary metabolic pathways 1. Aerobic: The Kreb’s cycle Glucose + Oxygen  CO2 + H2O + energy (2900 kJ/mol) 2. Anaerobic: Glycolysis Glucose  Lactic acid + energy (120 kJ/mol) Both require glucose ATP is required for almost all synthetic and degradative processes within the cell. Depletion of ATP is a fundamental cause of necrotic cell injury 17 Mechanisms of Cell Injury: Depletion of ATP Depletion of 5% to 10% = BAD Na+/K+ ATPase pump failure - Cell swelling - ER swelling - Plasma membrane damage Altered cell metabolism - Anaerobic glycolysis: Depletion of glycogen stores Increased lactic acid  ↓pH  loss of enzyme function Ribosome detachment - Decreased protein synthesis 18 Mechanisms of Cell Injury: Depletion of ATP Culminates in irreversible mitochondrial and lysosomal membrane damage 19 Mechanisms of Cell Injury 6 Mechanisms These 2 mechanisms of cell injury are interrelated Mechanisms of cell injury: Mitochondrial Injury 3 major consequences 1. Formation of the mitochondrial permeability transition pore (MPTP) High-conductance channel in the mitochondrial membrane When opened, leads to loss of membrane potential failure of oxidative phosphorylation progressive depletion of ATP Cell necrosis 21 Mechanisms of cell injury: Mitochondrial Injury 2. Increased production of reactive oxygen species (ROS) See later 22 Mechanisms of cell injury: Mitochondrial Injury 3. Activation of apoptotic pathways Proteins that activate the apoptosis pathway are sequestered in the mitochondria Leakage of apoptosis activating proteins into the cytosol and leads to cell death 23 Mechanisms of Cell Injury 6 Mechanisms Mechanisms of cell injury : Loss of Ca homeostasis Accumulation of Ca2+ 3 sources: ‒Extrinsic (cell damage) ‒Intrinsic Released from SER Released from the mitochondrion Sets off a cascade of events 25 Mechanisms of cell injury : Loss of Ca homeostasis Accumulation of Ca2+= cascade of intracellular events 1. Opening of MPTP  ↓ ATP 2. Enzyme activation ‒ Phospholipases: membrane damage ‒ Proteases: membrane & cytoskeletal proteins ‒ Endonucleases: DNA & chromatin fragmentation ‒ ATPases: break down ATP, accelerates ATP depletion 26 Mechanisms of cell injury : Loss of Ca homeostasis Accumulation of Ca2+ 3 major forms of damage 1. Membrane damage 2. Nuclear damage 3. ATP depletion 27 Mechanisms of cell injury: Reactive Oxygen Species 6 Mechanisms 28 Reactive Oxygen Species = ROS Derived from oxygen normally produced during cellular respiration by mitochondria Molecules or atoms with unpaired electrons – reactive free radical cellular quenching/scavenging systems neutralise normally Excess ROS or decreased scavenging capacity = oxidative stress Mechanisms of cell injury: Reactive Oxygen Species Normal metabolic processes Reduction-oxygenation reactions For example: O2 + 2H2  2 H2O ‒ Transfer of 4 electrons in this reaction ‒ Small amounts of partially reduced intermediates are produced Superoxide anion (O2-) - 1 electron Hydrogen peroxide (H2O2) - 2 electrons Hydroxyl ions (*OH) - 1 electron 30 Pathological sources of ROS Inflammation (lecs to come) Rapid bursts of ROS produced by activated WBCs (esp. neutrophils) - generates superoxide anion (O2*-) Transition metals (ie. Iron, Copper) Frequently donate or accept free electrons Catalyze free radical formation Nitric oxide (NO) Important chemical mediator Generated by endothelial cells, macrophages, neurons, and others Can act as a free radical Can be converted into peroxynitrite anion ONOO-, or NO2, or NO3 Absorption of radiant energy H2O + ionizing radiation  *OH + H Inflammation Phagocytosis and intracellular destruction of microbes. Phagocytosis of a particle (e.g., a bacterium) involves binding to receptors on the leukocyte membrane, engulfment, and fusion of the phagocytic vacuoles with lysosomes. This is followed by destruction of ingested particles within the phagolysosomes by lysosomal enzymes and by reactive oxygen and nitrogen species. The microbicidal products generated from superoxide are hypochlorite (HOCl−) and hydroxyl radical (−OH), and from nitric oxide (NO) it is peroxynitrite (OONO−). During phagocytosis, granule contents may be released into extracellular tissues (not shown). MPO, Myeloperoxidase; iNOS, inducible NO synthase; ROS, reactive oxygen species. Robbins and Cotran, The Pathologic Basis of Disease 9th ed, Figure 3-8 32 Summary : Pathological sources of ROS Oxidative Stress Implicated in a variety of pathologic processes - Cell injury - Cancer - Aging - Degenerative diseases 33 Neutralisation of ROS Removal of Free Radicals : Spontaneous decay: O2* + H2O  O2 + H2O2 : Enzymes: catalase, superoxide dismutase, glutathione peroxidase ‒ Break down H2O2 and O2* ‒ Located near sites where oxidants are formed : Storage and transport proteins - transferrin, ferritin, ceruloplasmin ‒ Bind reactive metals: Fe, Cu : Antioxidants - Vitamin E, Vitamin A, glutathione ‒ Block initiation ‒ Inactivate (scavenge) 34 Summary Effects of Ros - cell injury The generation, removal, and role of reactive oxygen species (ROS) in cell injury. The production of ROS is increased by many injurious stimuli. These free radicals are removed by spontaneous decay and by specialized enzymatic systems. Excessive production or inadequate removal leads to accumulation of free radicals in cells, which may damage lipids (by peroxidation), proteins, and deoxyribonucleic acid (DNA), resulting in cell injury. Robbins and Cotran, The Pathologic Basis of Disease 9th ed, Figure 2-20 35 Pathological effects: ROS 1) Lipid peroxidation in membranes  extensive membrane damage : formation of peroxides  autocatalytic reaction (propagation) - Decreased phospholipid synthesis - Increased phospholipid breakdown - Cytoskeletal abnormalities ‒ Activation of proteases  damaged cytoskeleton ‒ Cells stretch and rupture 36 Pathological effects: ROS 2) Oxidative modification of proteins  damage active sites, change conformation, enhance degradation - generated by monamine oxidase (MOA) in outer mitochondrial membrane : Oxidation of amino acid side chains, formation of protein cross-linkages (e.g. disulfide bonds), oxidation of protein backbone : Mitochondrial membrane damage : plasma membrane damage ‒ Loss of osmotic balance  influx of fluids and ions ‒ Loss of cell contents and metabolites : injury to lysosomal membranes ‒ Leakage of enzymes into the cytoplasm: RNases, DNases, proteases, phosphatases, glucosidases ‒ Enzymatic digestion of RNA, DNA, proteins, etc. 37 Pathological effects: ROS 3) Lesions in DNA  Cell aging, malignant transformation ‒ MOA: Single or double stranded breaks, cross-linking of DNA strands, formation of adducts 38 Summary: ROS Mechanisms of cell injury: Membrane Damage 6 Mechanisms 40 Membrane Damage Numerous mechanisms for damaging the plasma membrane Reactive oxygen species Decreased phospholipid synthesis : secondary to defective mitochondrial function and/or hypoxia Decreased production of ATP  ↓ phospholipid synthesis Affects all cellular membranes (plasma membrane, mitochondria, etc) Increased phospholipid breakdown Activation of calcium-dependent phospholipases Accumulation of lipid breakdown products - detergent effect on membranes - may insert into membranes → changes in permeability and electrophysiologic alterations 41 Membrane Damage Cytoskeletal abnormalities ‒Increased cytosolic calcium →activation of proteases →damage to cytoskeleton ‒In the presence of swelling, plasma membrane can detach from the cytoskeleton  susceptible to stretching and rupture 42 Summary Membrane Damage Mechanisms of membrane damage in cell injury. Decreased O2 and increased cytosolic Ca2+ are typically seen in ischemia but may accompany other forms of cell injury. Reactive oxygen species, which are often produced on reperfusion of ischemic tissues, also cause membrane damage (not shown). Robbins and Cotran The Pathologic Basis of Disease 9th ed Figure 2-20 43 Consequences of membrane damage Mitochondrial membrane damage ‒ open the MPTP  Leakage of pro-apoptotic proteins ‒ ↓ ATP Plasma Membrane damage ‒ Loss of osmotic balance  influx of fluids and ions ‒ Loss of cell contents and metabolites Injury to lysosomal membranes ‒ Leakage of enzymes into the cytoplasm: RNases, DNases, proteases, phosphatases, glucosidases ‒ Enzymatic digestion of RNA, DNA, proteins, etc. 44 Clinical pathology correlation: DOG membrane damage Biochem Panel Chemistry panel Analytes used to determine hepatocellular injury: Alanine aminotransferase (ALT) ‒ Located in the cytoplasm of hepatocytes ‒ Converts alanine into pyruvate ‒ Pyruvate is used for  gluconeogenesis or  the Kreb’s cycle When hepatocytes undergo cellular injury, ALT is released. 1) Cell necrosis (ruptures and releases ALT) 2) Membrane blebs that contain ALT Biochem Result Normal Sodium Potassium Na :K Chloride Calcium Bicarb Urea Creatinin Cholest Phosphate T. protein Albumin Globulin Bilirubin ALP ALT GGT AST CK Glucose 145 3.7 39 113 2.8 25 6.2 115 4.6 0.92 87 34 53 260 292 395 16 115-123 mmol/l 1.75-2.5 mmol/l 16-24 mmol/l 5.4-10.7 mmol/l 70-160 umpl/l 1.9-3.9 mmol/l 0.87-2.10 mmol/l 56-80 g/l 22-35 g/l 28-48 g/l 2-10 umol/l 5-80 U/l 5-80 U/l 1 – 10 U/l 10-60 U/l 5-300 U/l 45 1.9-3.9 mmol/l Cow with Photosensitisation June 2013 Freisian dairy cow, 180 days in milk presented down for 24 hr Clinical exam : - HR = 120, RR = 40, T = 39.8 C - unpigmented areas of skin mainly the face and udder are extensively erythemic ocassional frenzied moments of kicking out with her back legs. Analyte Cow Reference RBC 8.1 5.0 - 10.0 x1012/L Haemogl 133 80 - 150 g/l PCV 0.38 0.24 - 0.46 l/l MCV 46 40.0 - 60.0 fL MCH 16 11 - 17 pg MCHC 354 300 - 360 g/l Platelets 322 100 - 600 x109/L WBC 13.5 4.0 - 12.0 x109/L Neut 5.5 0.6 - 4.0 Band 3.8 0.0 - 0.2 Lymphs 3.6 2.5 - 7.5 Monos 0.5 0.0 - 0.9 Eosinos 0.0 0.0 - 0.8 Fibrinogen 12.0 H Analyte Tot Protein Albumin Globulins A:G Ratio Creatinine CK AST GLDH Bilirubin GGT Calcium Phosphate Magnesium BOHB 3.0 - 7.0 g/L For analytes out of range: Cow 83 31 52 0.6 67 3648 451 684 83 1041 2.27 1.57 1.06 0.3 Reference 58 - 80 g/L 22 - 36 g/L 24 - 40 g/L 0.8 - 1.9 90 - 120 umol/L 50 - 400 U/L 60 - 150 U/L 0 - 20 U/L 2 - 18 umol/L 0 - 36 U/L 2.00 - 3.00 mmol/L 1.29 - 2.26 mmol/L 0.70 - 1.20 mmol/L 0.0 - 0.9 mmol/L i) name and quantitate the abnormality ii) list its likely significance, and cause. Mechanisms of cell injury: Protein and DNA damage 6 Mechanisms 49 Protein Damage Accumulation of misfolded proteins ‒ Genetic mutations ‒ Free radical damage Cells have repair mechanisms for misfolded proteins When overwhelmed  proteins accumulated in the ER →“ER stress”  initiates apoptosis Normal prion protein Scrapie prion protein 50 DNA damage: repair, apoptosis, senescence, cancer Radiation, cytotoxic anticancer drugs, hypoxia ‒ Direct damage ‒ Free radical damage Cells have repair mechanisms When overwhelmed  initiates apoptosis 51 Summary  Many causes of cell injury  Severity of injury depends on the agent, extent of injury, duration of injury and the cell type.  6 major mechanisms of cell injury:  Decreased ATP, mitochondrial damage, entry of calcium, increased ROS, membrane damage, protein misfolding, and DNA damage  All mechanisms have multiple effects on cells.  Cells have systems in place to mediate and repair damage.  Severe damage leads to cell apoptosis or necrosis 52