Cell injury
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Prepared by M.D., PhD, Marta R. Gerasymchuk

Prepared by M.D., PhD, Marta R. Gerasymchuk
Department of Pathophysiology
Ivano-Frankivsk National Medical University

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  • Hypoxia is a deficiency of oxygen, which causes cell injury by reducing aerobic oxidative respiration. Hypoxia is an extremely important and common cause of cell injury and cell death. It should be distinguished from ischemia , which is a loss of blood supply from impeded arterial flow or reduced venous drainage in a tissue. Ischemia compromises the supply not only of oxygen, but also of metabolic substrates, including glucose (normally provided by flowing blood). Therefore, ischemic tissues are injured more rapidly and severely than are hypoxic tissues. One cause of hypoxia is inadequate oxygenation of the blood due to cardiorespiratory failure. Loss of the oxygen-carrying capacity of the blood, as in anemia or carbon monoxide poisoning (producing a stable carbon monoxyhemoglobin that blocks oxygen carriage), is a less frequent cause of oxygen deprivation that results in significant injury. Depending on the severity of the hypoxic state, cells may adapt, undergo injury, or die. For example, if the femoral artery is narrowed, the skeletal muscle cells of the leg may shrink in size (atrophy). This reduction in cell mass achieves a balance between metabolic needs and t-he available oxygen supply. More severe hypoxia induces injury and cell death. Excerpted from Robbins, Cotran and Kumar, 8 th Edition. 10/10/13
  • Pathologic Effects of Free Radicals. The effects of ROS and other free radicals are wide-ranging, but three reactions are particularly relevant to cell injury (see Fig. 1-20 ):    •    Lipid peroxidation in membranes . In the presence of O 2 , free radicals may cause peroxidation of lipids within plasma and organellar membranes. Oxidative damage is initiated when the double bonds in unsaturated fatty acids of membrane lipids are attacked by O 2 -derived free radicals, particularly by   . OH. The lipid–free radical interactions yield peroxides, which are themselves unstable and reactive, and an autocatalytic chain reaction ensues (called propagation ), which can result in extensive membrane damage.    •    Oxidative modification of proteins . Free radicals promote oxidation of amino acid side chains, formation of protein-protein cross-linkages ( e.g ., disulfide bonds), and oxidation of the protein backbone. Oxidative modification of proteins may damage the active sites of enzymes, disrupt the conformation of structural proteins, and enhance proteasomal degradation of unfolded or misfolded proteins, raising havoc throughout the cell.    •    Lesions in DNA . Free radicals are capable of causing single- and double-strand breaks in DNA, cross-linking of DNA strands, and formation of adducts. Oxidative DNA damage has been implicated in cell aging (discussed later in this chapter) and in malignant transformation of cells ( Chapter 7 ). Excerpted from Robbins, Cotran, and Kumar, 8 th Edition. 10/10/13
  • ACCUMULATION OF OXYGEN-DERIVED FREE RADICALS (OXIDATIVE STRESS) Cell injury induced by free radicals, particularly reactive oxygen species, is an important mechanism of cell damage in many pathologic conditions, such as chemical and radiation injury, ischemia-reperfusion injury (induced by restoration of blood flow in ischemic tissue), cellular aging, and microbial killing by phagocytes. Free radicals are chemical species that have a single unpaired electron in an outer orbit (see Table 1-3). Energy created by this unstable configuration is released through reactions with adjacent molecules, such as inorganic or organic chemicals—proteins, lipids, carbohydrates, nucleic acids—many of which are key components of cell membranes and nuclei. Moreover, free radicals initiate autocatalytic reactions, whereby molecules with which they react are themselves converted into free radicals, thus propagating the chain of damage. Reactive oxygen species (ROS) are a type of oxygen-derived free radical whose role in cell injury is well established. ROS are produced normally in cells during mitochondrial respiration and energy generation, but they are degraded and removed by cellular defense systems. Thus, cells are able to maintain a steady state in which free radicals may be present transiently at low concentrations but do not cause damage. When the production of ROS increases or the scavenging systems are ineffective, the result is an excess of these free radicals, leading to a condition called oxidative stress . Oxidative stress has been implicated in a wide variety of pathologic processes, including cell injury, cancer, aging, and some degenerative diseases such as Alzheimer disease. ROS are also produced in large amounts by leukocytes, particularly neutrophils and macrophages, as mediators for destroying microbes, dead tissue, and other unwanted substances. Therefore, injury caused by these reactive compounds often accompanies inflammatory reactions, during which leukocytes are recruited and activated ( Chapter 2 ). 10/10/13
  • Ischemic injury is the most common clinical expression of cell injury by oxygen deprivation. The most useful models for studying ischemic injury involve complete occlusion of one of the end-arteries to an organ ( e.g ., a coronary artery) and examination of the tissue ( e.g ., cardiac muscle) in areas supplied by the artery. Complex pathologic changes occur in diverse cellular systems during ischemia. Up to a certain point, for a duration that varies among different types of cells, the injury is amenable to repair, and the affected cells can recover if oxygen and metabolic substrates are again made available by restoration of blood flow. With further extension of the ischemic duration, cell structure continues to deteriorate, owing to relentless progression of ongoing injury mechanisms. With time, the energetic machinery of the cell—the mitochondrial oxidative powerhouse and the glycolytic pathway—becomes irreparably damaged, and restoration of blood flow (reperfusion) cannot rescue the damaged cell. Even if the cellular energetic machinery were to remain intact, irreparable damage to the genome or to cellular membranes will ensure a lethal outcome regardless of reperfusion. This irreversible injury is usually manifested as necrosis , but apoptosis may also play a role. Under certain circumstances, when blood flow is restored to cells that have been previously made ischemic but have not died, injury is often paradoxically exacerbated and proceeds at an accelerated pace . As a consequence, reperfused tissues may sustain loss of cells in addition to cells that are irreversibly damaged at the end of ischemia . This is a clinically important process that contributes to net tissue damage during myocardial and cerebral infarction, as described in Chapter 12 and Chapter 28. This so-called ischemia-reperfusion injury (discussed later) is particularly significant because appropriate medical treatment can decrease the fraction of cells that may otherwise be destined to die in the "area at risk." Excerpted from Robbins, Cotran and Kumar, 8 th Edition. 10/10/13
  • Excerpted from Robbins, Cotran and Kumar, 8 th Edition. 10/10/13
  • 10/10/13
  • Insulin and numerous growth factors activate tyrosine kinases , which transmit cellular effects via other kinases, enzymes, and transport proteins. The tyrosine kinases can themselves be part of the receptor, or can attach themselves to the receptor on activation. Kinases frequently act by phosphorylating other kinases and thereby trigger a kinase cascade . Thus, the mitogen-activated protein kinase (MAP kinase) is activated by another kinase (MAP kinase kinase). This “snowball effect” results in an avalanche-like increase of the cellular signal. The p-38 kinase and the Jun kinase that regulate gene expression via transcription factors are also activated via such cascades. Other signaling molecules, such as the small G proteins (p21Ras) or transcription factors (e.g.,c-Jun, c-Fos, c-Myc, NF&B, AP-1), are important for signal transduction of growth factors and in apoptosis.
  • DAMAGE TO DNA AND PROTEINS (See Fig. 1-20 on slide) Cells have mechanisms that repair damage to DNA, but if this damage is too severe to be corrected ( e.g ., after exposure to DNA damaging drugs, radiation, or oxidative stress), the cell initiates a suicide program that results in death by apoptosis. A similar reaction is triggered by improperly folded proteins, which may be the result of inherited mutations or external triggers such as free radicals. Because these mechanisms of cell injury typically cause apoptosis, they are discussed later in the chapter. Before concluding our discussion of the mechanisms of cell injury, it is useful to consider the possible events that determine when reversible injury becomes irreversible and progresses to cell death. The clinical relevance of this question is obvious—if we can answer it we may be able to devise strategies for preventing cell injury from having permanent deleterious consequences. However, the molecular mechanisms connecting most forms of cell injury to ultimate cell death have proved elusive, for several reasons. The “point of no return,” at which the damage becomes irreversible, is still largely undefined, and there are no reliable morphologic or biochemical correlates of irreversibility. Two phenomena consistently characterize irreversibility — the inability to reverse mitochondrial dysfunction (lack of oxidative phosphorylation and ATP generation) even after resolution of the original injury, and profound disturbances in membrane function . As mentioned earlier, injury to lysosomal membranes results in the enzymatic dissolution of the injured cell that is characteristic of necrosis. Leakage of intracellular proteins through the damaged cell membrane and ultimately into the circulation provides a means of detecting tissue-specific cellular injury and necrosis using blood serum samples. Cardiac muscle, for example, contains a specific isoform of the enzyme creatine kinase and of the contractile protein troponin ; liver (and specifically bile duct epithelium) contains an isoform of the enzyme alkaline phosphatase; and hepatocytes contain transaminases . Irreversible injury and cell death in these tissues are reflected in increased levels of such proteins in the blood, and measurement of these biomarkers is used clinically to assess damage to these tissues. 10/10/13
  • Apoptosis is a pathway of cell death that is induced by a tightly regulated suicide program in which cells destined to die activate enzymes that degrade the cells' own nuclear DNA and nuclear and cytoplasmic proteins. Apoptotic cells break up into fragments, called apoptotic bodies, which contain portions of the cytoplasm and nucleus. The plasma membrane of the apoptotic cell and bodies remains intact, but its structure is altered in such a way that these become “tasty” targets for phagocytes. The dead cell and its fragments are rapidly devoured, before the contents have leaked out, and therefore cell death by this pathway does not elicit an inflammatory reaction in the host. The process was recognized in 1972 by the distinctive morphologic appearance of membrane-bound fragments derived from cells, and named after the Greek designation for “falling off.” It was quickly appreciated that apoptosis was a unique mechanism of cell death, distinct from necrosis, which is characterized by loss of membrane integrity, enzymatic digestion of cells, leakage of cellular contents, and frequently a host reaction (see Fig. 1-8 and Table 1-2 ). However, apoptosis and necrosis sometimes coexist, and apoptosis induced by some pathologic stimuli may progress to necrosis. Excerpted from Robbins, Cotran and Kumar, 8 th Edition. 10/10/13

Cell injury Presentation Transcript

  • 1. CELL DAMAGE
  • 2. ContentContent  Components and functions of normal cell.Components and functions of normal cell.  Cellular injury. Characteristics of the concept of “injury”.Cellular injury. Characteristics of the concept of “injury”.  Mechanisms and manifestations of damage of subcellularMechanisms and manifestations of damage of subcellular structures: plasmatic membrane, mitochondria,structures: plasmatic membrane, mitochondria, endoplasmatic reticulum, lysosomes, microtubules andendoplasmatic reticulum, lysosomes, microtubules and microphilaments, nucleus and cytoplasm.microphilaments, nucleus and cytoplasm.  Principles of classification of cell injuries.Principles of classification of cell injuries.  Molecular mechanisms of cell injury.Molecular mechanisms of cell injury.  Antioxidant mechanisms of cells.Antioxidant mechanisms of cells.  Cell death.Cell death.  Mechanisms of apoptosis.Mechanisms of apoptosis.  Ageing.Ageing.
  • 3. Cellular PathologyCellular Pathology ““All organ injuries start with structuralAll organ injuries start with structural or molecular alterations in cells”or molecular alterations in cells” concept began by Virchow in 1800's.concept began by Virchow in 1800's.
  • 4. NORMAL CELL
  • 5. NORMAL CELLNORMAL CELL • at the subcellular or molecular level. • all cells share the basic organelles for the synthesis of: • transport of ions and other substances. • to understand pathology, review normal structure and function of cells. “you cannot appreciate the abnormal before you understand the normal” • Present day study of disease attempts to understand how cells react to injury. proteinsproteins lipidslipids carbohydratescarbohydrates energyenergy productionproduction
  • 6. Plasma membranePlasma membrane • phospholipid bilayerphospholipid bilayer with embedded proteins / glycoproteins / glycolipids. • semipermeable membrane with pumps for ionic / osmotic homeostasis
  • 7. 1.1. Lipid peroxidation (LPO), increased generation of free radicals 2. Activation of phospholipases and lysosomal hydrolytic enzymes 3. Membrane damage via amphiphilic, detergent substances 4. Cell swelling → membrane tension, rupture 5. Inhibition of repairof the damaged membrane compounds (blockage of denovo synthesis) 6. Immune complex influence the membrane macromolecules 7.Conformation disorders of macromolecules Damage to lipid bilayer → damage to phospholypase, lipase activities of the membrane Membrane damageMembrane damage (main causes)(main causes)
  • 8. NucleusNucleus • nuclear envelope /nuclear envelope / nuclear poresnuclear pores • chromatinchromatin (euchromatin vs(euchromatin vs heterochromatin)heterochromatin) • nucleolusnucleolus (synthesis of(synthesis of ribosomal RNA)ribosomal RNA)
  • 9. MitochondriaMitochondria • inner & outer membranes, cristaeinner & outer membranes, cristae • intermembranous and inner matrixintermembranous and inner matrix compartmentscompartments • oxidative phosphorylation (main sourceoxidative phosphorylation (main source of ATP)of ATP)
  • 10. Endoplasmic reticulum (ER),Endoplasmic reticulum (ER), Ribosomes & Golgi ApparatusRibosomes & Golgi Apparatus • Rough (RER) vs smooth (SER) endoplasmic reticulum • Ribosomes (free in cytosol or attached to RER) • Polysomes (threaded by mRNA). • Condensing vacuoles/ secretory vesicles
  • 11. LysosomeLysosome• Enzymatic (acid hydrolases) digestion of materials in the cell • Endocytosis • Phagocytosis / phagosome; • Pinocytosis / pinocytotic vesicle; • Receptor-mediated endocytosis
  • 12. PeroxisomePeroxisome• Enzymes (ex. catalase,catalase, oxidasesoxidases) ! metabolism of hydrogen peroxide & fatty acid
  • 13. Cellular functionsCellular functions 1.1. MovementMovement – muscle cells can generate forces that produce motion.– muscle cells can generate forces that produce motion. 2.2. ConductivityConductivity – is the main function of nervous cells. Conduction as a– is the main function of nervous cells. Conduction as a response to a stimulus is manifested by a wave of excitation, an electricalresponse to a stimulus is manifested by a wave of excitation, an electrical potential.potential. 3.3. Metabolic absorptionMetabolic absorption – all cells take in and use nutrients and other– all cells take in and use nutrients and other substances from their environment.substances from their environment. 4.4. SecretionSecretion – certain cells are able to synthesize new substances and– certain cells are able to synthesize new substances and secrete them.secrete them. 5.5. ExcretionExcretion – all cells are able to rid themselves of waste products– all cells are able to rid themselves of waste products resulting from the metabolic breakdown of nutrients.resulting from the metabolic breakdown of nutrients. 6.6. Respiration (oxidation)Respiration (oxidation) – cells absorb oxygen which is used to– cells absorb oxygen which is used to transform nutrients into energy in the form of ATP (in mitochondria)transform nutrients into energy in the form of ATP (in mitochondria) 7.7. ReproductionReproduction – tissue growth occurs as cells enlarge and reproduce– tissue growth occurs as cells enlarge and reproduce themselves.themselves. 8.8. CommunicationCommunication – is critical for all the other functions listed above– is critical for all the other functions listed above enabling the survival of the society of cells. Constant communicationenabling the survival of the society of cells. Constant communication allows the maintenance of a dynamic steady state.allows the maintenance of a dynamic steady state.
  • 14. Cell InjuryCell Injury 1. Cause1. Cause •• intrinsic,intrinsic, •• extrinsicextrinsic •• infectious,infectious, •• non infectiousnon infectious 2. The type of influence.2. The type of influence. •• directdirect •• mediatedmediated •• acuteacute •• chronicchronic →→ by the courseby the course •• reversiblereversible •• irreversibleirreversible 3. Manifestations of injury.3. Manifestations of injury. a. specifica. specific b. stereotypical (non specific)b. stereotypical (non specific) 4. In dependence on the4. In dependence on the pathogenically mechanismspathogenically mechanisms of cellsof cells damage divide on: a) violent (forced); b)damage divide on: a) violent (forced); b) cytopath(ogen)iccytopath(ogen)ic Cell injuryCell injury is defined as such a change in cell structure, metabolism,is defined as such a change in cell structure, metabolism, physico-chemical properties and function which leads to impairment of itsphysico-chemical properties and function which leads to impairment of its vital activityvital activity
  • 15. REVERSIBLE CELL INJURYREVERSIBLE CELL INJURY  It occurs whenIt occurs when environmentalenvironmental changes exceed thechanges exceed the capacity of the cell tocapacity of the cell to maintain normalmaintain normal homeostasis.homeostasis.  If the stress isIf the stress is removed in tissue orremoved in tissue or if the cell withstandif the cell withstand the assault the injurythe assault the injury is reversibleis reversible
  • 16. IRREVERSIBLE CELL INJURYIRREVERSIBLE CELL INJURY  If the stress remains the severe, the cellIf the stress remains the severe, the cell injury becomes irreversible and lead toinjury becomes irreversible and lead to cell deathcell death
  • 17. Comparative analysis of reversible and irreversible cell injuryComparative analysis of reversible and irreversible cell injury 1. Mitochondrial oxygenation ↓ 2. ATP ↓ 3. Na+ K+ pump ↓ 4. Intracellular Na + , Ca2+ ↑, extracellular K+ ↑ glycolysis ↑ lactate ↑, pH ↓ 5. H20 ↑ 6. Acute cell swelling a. nuclear chromatin shrinking b. lysosomal swelling Enlargement of endoplasmic reticulum Ribosome order ↓ Protein synthesis ↓ Impaired lipid deposition Reversible injury Inreversible injury 1. Membrane damage a. Loss of phospholipids b. Cytoskeleton injury c. Free radical ↑ d. Lysis of lipids 2. Ca2+ influx ↑ Increased Ca2+ load of mitochondria Uncoupling of oxidative phosphorilation 3. Release of cytoplasmic enzymes (LDG) 4. Release of lysosomal enzymes 5. Autophagy
  • 18. The main causes ofThe main causes of cellcell injuryinjury Internal stresses • metabolic imbalances, nutritional deficiencies ormetabolic imbalances, nutritional deficiencies or excessesexcesses • genetic abnormalitiesgenetic abnormalities • acquired derangementsacquired derangements →→ hypoxiahypoxia →→ impairment inimpairment in aerobic tissue respirationaerobic tissue respiration, ischemia, ischemia →→ decrease in blooddecrease in blood supplysupply External •• physical agents (physical agents ( mechanical injury, high and lowmechanical injury, high and low temperature, radiation, electrical shock, sudden fluctuations oftemperature, radiation, electrical shock, sudden fluctuations of the barometric pressure, acceleration, etcthe barometric pressure, acceleration, etc…)…) •• natural toxins, venomsnatural toxins, venoms •• drugs, "chemicals" (Paracelsus)drugs, "chemicals" (Paracelsus) →→ abundantabundant oxygen, increase in glucose, high doses of dietary salt, poisons,oxygen, increase in glucose, high doses of dietary salt, poisons, insecticides, carbon monoxide, asbestos, drugs, socialinsecticides, carbon monoxide, asbestos, drugs, social stimulators, e.g. alcohol, narcotics.stimulators, e.g. alcohol, narcotics.
  • 19. Cell injury signsCell injury signs a. Swelling b. Dystrophy c. Thesaurismosis d. Dysplasia e. Necrosis f. Autolysis a. Decrease in function b. Cellular 1. Increase in permeability 2. Cytoplasmic enzymes leakage to the blood c. Metabolic derangements d. Injury mediators e. Synthesis impairments f. Electrolyte balance disorders Morphological Functional
  • 20. 1. Lipid: 1) free oxidation of lipids (FOL), Increased free radical generation and lipid peroxydation → oxidative stress 2) activating of phospholipase and 3) detergent action of free fat acids. 2. Impairment in calcium homeostasis (calcium stress) 3. Electrolyte-osmotic balance disorders 4. Acidosis (intracellular, extracellular) 5. Protein disorders – enzymatic derangements 6. Nucleic acid disorders (transcription, translation, DNA repair disorders) → nucleic acid stress 7. Violation of power providing of cell. Molecular mechanisms of cell injuryMolecular mechanisms of cell injury
  • 21. FREE RADICALSFREE RADICALS A commonA common "final"final pathway"pathway" in a variety ofin a variety of forms of cell injury,forms of cell injury, including injury broughtincluding injury brought about by inflammatoryabout by inflammatory cells, is generation ofcells, is generation of free radicals, i.e.,free radicals, i.e., molecular species with amolecular species with a single unpaired electronsingle unpaired electron available in an outeravailable in an outer orbital.orbital.  Single free radicalsSingle free radicals initiate chain reactionsinitiate chain reactions which destroy largewhich destroy large numbers of organicnumbers of organic moleculesmolecules
  • 22. Formation, Function, Types of Free RadicalsFormation, Function, Types of Free Radicals Ionizing Radiation Produces Hydroxyl FRs Prod. Superoxide FRs Damaged Mitochondria High Concentration of O2Prod. Superoxide &Prod. Superoxide & Hydroxyl FRsHydroxyl FRs Prod. HydrogenProd. Hydrogen Peroxide (H2O2)Peroxide (H2O2) Oxidase Reactions 1. NADPH1. NADPH oxidase inoxidase in the PMN & monocytethe PMN & monocyte cell membranecell membrane MyeloperoxidaseMyeloperoxidase Hypochlorouse acidHypochlorouse acid 2. Xanthine oxidase2. Xanthine oxidase isis a ROS – generate FRsa ROS – generate FRs Prod. Superoxide FRsProd. Superoxide FRsDrugsDrugs (e.g.(e.g. acetaminophen)acetaminophen)Conv. toConv. to acetaminophen FRsacetaminophen FRs in the liverin the liver CarbonCarbon tetrachloridetetrachloride Conv. to CCl3 FRsConv. to CCl3 FRs in the liverin the liver MetalsMetals (e.g. iron, copper)(e.g. iron, copper) Prod. Hydroxyl FRsProd. Hydroxyl FRs (Fenton reaction)(Fenton reaction) Nitric OxideNitric Oxide FRs prod. by macrophagesFRs prod. by macrophages & endothelial cells& endothelial cells Intima of elastic &Intima of elastic & muscular arteriesmuscular arteries LDL are oxidized by FRs,LDL are oxidized by FRs, lead to atherosclerotic pl.lead to atherosclerotic pl. FRs attack aFRs attack a molecule &molecule & “steal” its“steal” its electronelectronFRs : damageFRs : damage membranes &membranes & DNADNA
  • 23. FREE-RADICALFREE-RADICAL GENERATIONGENERATION  1.1. Oxidation ofOxidation of unsaturated fatty acidsunsaturated fatty acids in membranesin membranes ("lipid("lipid peroxidation", etc.)peroxidation", etc.) * Basic biologists:* Basic biologists: These are the sameThese are the same reactions that makereactions that make unsaturated fats turnunsaturated fats turn rancid.rancid.  2. Cross-linking of2. Cross-linking of sulfhydryl groups ofsulfhydryl groups of proteins.proteins.  3. Genetic mutations3. Genetic mutations Ionizing radiation: homolytic break of covalent bonds in water, DNA and other biomolecules H 2 O O H + H io n iz in g r a d ia t io n h ν
  • 24.  1. By absorbing radiant energy (UV, x-rays; striking water, these generate a hydrogen atom and a hydroxyl radical; when hydrogen peroxide contacts ferrous iron, it is cleaved into two hydroxyl radicals (* the Fenton reaction).  2. As part of normal metabolism (for example, xanthine oxidase and the P450 systems generate superoxide; our white cells use free radicals to attack and kill invaders)  3. As part of the metabolism of drugs and poisons (the most famous being CCl3.-, from carbon tetrachloride; even O2 in high concentrations generates enough free radicals to gravely injure the lungs). Free radicals may beFree radicals may be generated in thegenerated in the following ways:following ways:
  • 25. Reactive oxygen and nitrogen species ROS/RNS Free radical – each molecule or its fragment, which can exists independently and contains one or two unpaired electrons Reactive oxygen species - species, which contain one or more oxygen atoms and are much more reactive than molecular oxygen ROS/RNS Free radicals superoxide radical hydroperoxyl radical hydroxyl radical nitric oxide hydrogen peroxide
  • 26. Some characteristics of ROS ROS Symbol Half- life Properties Superoxide radical O2 •- 10-6 s poor oxidant, quite toxic & is deployed by the immune system to kill invading microorganisms. Hydroperoxyl radical HO2 • stronger oxidant than O2 •- Hydrogen peroxide H2 O2 minits oxidant, diffuses across membranes Hydroxyl radical OH• 10-9 s extremely reactive, diffuses only to very low distance Alkoxyl radical LO• 10-6 s less reactive than OH• , but more than ROO• Peroxyl radical LOO• 10-2 s weak oxidant, high diffusability Singlet oxygen 1 O2 10-6 s powerful oxidizing agent
  • 27. Cellular sources of ROS xanthine oxidase hemoglobin riboflavin catecholamines Cytochrome P450 electron transport chain lipid peroxidation NADPH oxidase (oxidative burst: phagocytes) oxidases flavoproteins myeloperoxidase (oxidative burst: phagocytes) transient metals
  • 28. Oxidative Stress and Oxygen Free RadicalsOxidative Stress and Oxygen Free Radicals • Superoxide anionSuperoxide anion (O(O22 -- ) –) – may be formed via themay be formed via the cytochrome Pcytochrome P450450 systemsystem,, found in hepatocytes,found in hepatocytes, which metabolizes manywhich metabolizes many drugs and toxins – itdrugs and toxins – it cancan be removedbe removed byby superoxidesuperoxide dismutasedismutase • Hydrogen peroxideHydrogen peroxide (H(H22OO22)) –– removedremoved byby catalasecatalase oror glutathione peroxidaseglutathione peroxidase • Hydroxyl radicalHydroxyl radical ((.. OH) –OH) – initiates lipid per-oxidationinitiates lipid per-oxidation and DNA damageand DNA damage
  • 29. Pathologic Effects of Free RadicalsPathologic Effects of Free Radicals  Hydroxyl radicalsHydroxyl radicals initiateinitiate lipidlipid peroxidationperoxidation, leading to severe damage to, leading to severe damage to membranes, especiallymembranes, especially in mitochondriain mitochondria  Hydroxyl radicalsHydroxyl radicals causecause oxidativeoxidative damage to proteinsdamage to proteins , which may damage, which may damage enzymes and structural proteinsenzymes and structural proteins  Hydroxyl radicalsHydroxyl radicals can induce single- andcan induce single- and double-stranddouble-strand breaks in DNAbreaks in DNA, cross-linking,, cross-linking, and formation of adducts. This could lead toand formation of adducts. This could lead to defective transcriptiondefective transcription,, accelerated cell agingaccelerated cell aging,, oror malignant transformationmalignant transformation of the cell to aof the cell to a cancerous phenotypecancerous phenotype
  • 30. Formation of ROS and peroxynitrous acid in phagocytic vacuole of phagocytes SOD – superoxid dismutase MPO - myeloperoxidase
  • 31. Cellular sources of ROS - examples O 2 O 2 - H 2 O 2 H 2 O + O H H 2 O e- e- + 2H+ e- + H+ e- + H+ H b ( F e 2 + ) - O 2 m e t H b ( F e 3 + ) + O 2 - F e 2 + + H 2 O 2 F e 3 + + H O + O H - Electron transport system: Autooxidation of hemoglobin: Fenton reaction:
  • 32. LH + HO L + H2O L + O2 LOO LH + LOO L + LOOH Formation of lipid (alkyl) radical initiated by ROS: Alkyl radical react with O2 to produce peroxyl radical: Peroxyl radical attacks another poly- unsturated FA to produce new alkyl radical and lipid peroxide: Oxidative damage to lipids –Oxidative damage to lipids – Lipid peroxidation (LPO)Lipid peroxidation (LPO) ROS
  • 33. Mechanisms of cell injury mediated by ROS and RNSMechanisms of cell injury mediated by ROS and RNS ROS a RNS Modification of aa, fragmentation and aggregation of proteins Lipid peroxidation DNA damage Membrane damage Loss of membrane integrity Damage to Ca2+ and other ion transport systems Inability to maintain normal ion gradients Activation/deactivation of various enzymes Altered gene expression Depletion of ATP Lipids Proteins DNA Cell injury/ Cell death aa – amino acids
  • 34. Antioxidants andAntioxidants and secondary defensesecondary defense systemssystems  Prevent ROS formation  Eliminate radicals by formation of nonradicals or less reactive radicals  Repair dameged molecules and cell structures  Expression of genes coding for antioxidant enzymes Antioxidants and secondary defense systems Enzyme antioxidants Nonenzymatic antioxidants Chelating agents Enzymes of repair and de novo synthesis of damaged molecules Water-soluble Lipid-soluble Endogenous Present in diet
  • 35. Increased production of ROS + decreased activity of antioxidantIncreased production of ROS + decreased activity of antioxidant system = oxidative stresssystem = oxidative stress Antioxidant systemAntioxidant system I. Enzymatic a. Superoxide dismutase (SOD) b. Catalase c. Glutathione peroxidase ( glutathione, GSH) d. ubiquinone II. Non enzymatic a. α-tocopherol b. Ascorbic acid c. Cysteine, mannitol, serotonin, selenium, riboflavin, retinol, carotinoids, d. Reduced glutathioneGlutathione system Vitamins Microelements (Selenium) Amino acids with SH group
  • 36. Antioxidant enzymes ENZYME TISSUE SITE ______ Superoxide dismutase Cu/Zn SOD primarily cytosol, nucleus Mn SOD mitochondria EC SOD extracellular fluid Catalase peroxisomes Glutathione peroxidase GPx cytosol, mitochondria Glutathione reductase GRed cytosol, mitochondria _________________________________________________
  • 37. Antioxidant enzymes O 2 - + O 2 - + 2 H + H 2 O 2 + O 2 SOD SOD scavenges superoxide radical:SOD scavenges superoxide radical: 2 H 2 O 2 2 H 2 O + O 2 Catalase decomposes hydrogen peroxid in peroxisomes :Catalase decomposes hydrogen peroxid in peroxisomes : Glutathione peroxidase (GPx) decomposes HGlutathione peroxidase (GPx) decomposes H22OO22 and lipid peroxides inand lipid peroxides in cytosol and mitochondria by help of GSH, NADPH andcytosol and mitochondria by help of GSH, NADPH and glutathionereductase (GRed):glutathionereductase (GRed): GPx GRed H2O + LOH GSSG NADPH LOOH 2GSH NADP+
  • 38. Nonenzymatic antioxidantsNonenzymatic antioxidants Endogenous antioxidants - Synthesized in the bodyEndogenous antioxidants - Synthesized in the body bilirubínbilirubín glutathione and other thiocompounds (thioredoxin)glutathione and other thiocompounds (thioredoxin) uric aciduric acid coenzyme Q (Ubichinon-10/Ubichinol-10)coenzyme Q (Ubichinon-10/Ubichinol-10) lipooic acidlipooic acid melatoninmelatonin sex hormonessex hormones 2-oxoacids (pyruvate, 2-oxoglutarate)2-oxoacids (pyruvate, 2-oxoglutarate) dipeptides containig His (carnosine, anserine)dipeptides containig His (carnosine, anserine) albumin (-SH groups)albumin (-SH groups) Dietary antioxidantsDietary antioxidants ascorbic acidascorbic acid vitamine Evitamine E carotenoidscarotenoids flavonoids – plant phenols (catechin, quercetin etc)flavonoids – plant phenols (catechin, quercetin etc) Synthetic antioxidantsSynthetic antioxidants N-acetylcystein (scavenger of ROS), deferoxamine (chelator),N-acetylcystein (scavenger of ROS), deferoxamine (chelator), alopurinol (inhibitor of XO), acetyl salicylic acid (feritine synthesis)alopurinol (inhibitor of XO), acetyl salicylic acid (feritine synthesis)
  • 39. • Vitamin EVitamin E (fat-soluble)(fat-soluble) 1) Prevents lipid1) Prevents lipid peroxidation in cellperoxidation in cell membranes;membranes; 2) Neutralizes2) Neutralizes oxidized LDLoxidized LDL • Vitamin CVitamin C (water-(water- soluble)soluble) 1) Neutralizes FRs1) Neutralizes FRs produced byproduced by pollutants andpollutants and cigarette smokecigarette smoke • Smokers haveSmokers have  levels of Vit.Clevels of Vit.C because they arebecause they are used up inused up in neutralizing FRsneutralizing FRs derived from cigarettederived from cigarette smoke.smoke. 2) Best neutralizer of2) Best neutralizer of hydroxyl FRs.hydroxyl FRs.
  • 40. Oxidative StressOxidative Stress • Absorption of radiant energy with sufficient energy to initiate the radiolysis of water, i.e., “ionizing” radiation, leads to the following reaction: H2O  . H + . OH
  • 41. ACCUMULATION OF OXYGEN-ACCUMULATION OF OXYGEN- DERIVED FREE RADICALSDERIVED FREE RADICALS (OXIDATIVE STRESS)(OXIDATIVE STRESS)  Cell injury induced by free radicals, particularly reactive oxygen species, is an important mechanism of cell damage in many pathologic conditions, such as chemical and radiation injury, ischemia-reperfusion injury (induced by restoration of blood flow in ischemic tissue), cellular aging, and microbial killing by phagocytes.  Free radicals are chemical species that have a single unpaired electron in an outer orbit. Energy created by this unstable configuration is released through reactions with adjacent molecules, such as inorganic or organic chemicals —proteins, lipids, carbohydrates, nucleic acids—many of which are key components of cell membranes and nuclei.  Moreover, free radicals initiate autocatalytic reactions, whereby molecules with which they react are themselves converted into free radicals, thus propagating the chain of damage.
  • 42. ACCUMULATION OF OXYGEN-DERIVEDACCUMULATION OF OXYGEN-DERIVED FREE RADICALS (OXIDATIVE STRESS)FREE RADICALS (OXIDATIVE STRESS)  Reactive oxygen species (ROS) are a type of oxygen-derived freeare a type of oxygen-derived free radical whose role in cell injury is well established.radical whose role in cell injury is well established.  ROS are produced normally in cells during mitochondrial respirationare produced normally in cells during mitochondrial respiration and energy generation, but they are degraded and removed byand energy generation, but they are degraded and removed by cellular defense systems.cellular defense systems.  Thus, cells are able to maintain a steady state in which free radicals mayThus, cells are able to maintain a steady state in which free radicals may be present transiently at low concentrations but do not cause damage.be present transiently at low concentrations but do not cause damage.  When the production ofWhen the production of ROS increases or the scavenging systems areor the scavenging systems are ineffective, the result is an excess of these free radicals, leading to aineffective, the result is an excess of these free radicals, leading to a condition calledcondition called oxidative stress..  Oxidative stress has been implicated in a wide variety of pathologichas been implicated in a wide variety of pathologic processes, including cell injury, cancer, aging, and some degenerativeprocesses, including cell injury, cancer, aging, and some degenerative diseases such asdiseases such as Alzheimer diseaseAlzheimer disease..  ROS are also produced in large amounts by leukocytes, particularlyare also produced in large amounts by leukocytes, particularly neutrophils and macrophages, as mediators for destroying microbes, deadneutrophils and macrophages, as mediators for destroying microbes, dead tissue, and other unwanted substances.tissue, and other unwanted substances.  Therefore, injury caused by these reactive compounds often accompaniesTherefore, injury caused by these reactive compounds often accompanies inflammatory reactions, during which leukocytes are recruited and activated..
  • 43. Reperfusion Injury andReperfusion Injury and Activated OxygenActivated Oxygen • Toxic oxygen species are generated, not during the ischemia itself, but during reperfusion, hence the term reperfusion injuryreperfusion injury • This has clinical relevance, since reperfusion of heart muscle is commonly achieved with per-cutaneous angioplasty. Patients more than 20 minutes post-infarction are at risk for reperfusion injury.
  • 44. Reperfusion Injury and Activated OxygenReperfusion Injury and Activated Oxygen Generation ofGeneration of oxygen free radicalsoxygen free radicals occurs fromoccurs from parenchymal and endothelial cells and from infiltratingparenchymal and endothelial cells and from infiltrating leukocytesleukocytes • Reactive oxygen species• Reactive oxygen species can further damagecan further damage mitochondrial membranes, which precludes generation ofmitochondrial membranes, which precludes generation of ATP and leads to cell deathATP and leads to cell death • Ischemic injury• Ischemic injury is associated withis associated with inflammation,inflammation, as aas a result of the production of cytokines and increasedresult of the production of cytokines and increased expression of adhesion molecules by hypoxic parenchymalexpression of adhesion molecules by hypoxic parenchymal and endothelial cellsand endothelial cells •• Recent data suggest that activation of theRecent data suggest that activation of the complement pathwaycomplement pathway may contribute tomay contribute to ischemia-ischemia- reperfusion injury.reperfusion injury. The complement systemThe complement system is involved in host defense and isis involved in host defense and is an important mechanism of immune injury. Knockout micean important mechanism of immune injury. Knockout mice lacking several complement proteins are resistant to thislacking several complement proteins are resistant to this type of injury.type of injury.
  • 45. Mechanisms of membrane damage in ischemia and reperfusion
  • 46. Cellular response to ischemia. ATP production by mitochondria relies on an adequate supply of oxygen and of energy substrates such as glucose. Mitochondrial function is therefore compromised soon after failure of blood supply, resulting in failure of production of ATP. One consequence of lack of ATP is failure of ATP-dependent membrane pumps, which normally pump sodium (and with it water) out of cells. Failure of membrane ion pumps leads to accumulation of sodium and water in the cell cytoplasm, with disruption of internal membrane systems. Failure of internal membrane pumps also allows free calcium to enter the cytosol, where it activates many destructive enzyme systems. Structural damage to internal membranes and the cytoskeleton, coupled with lack of ATP, leads to impairment of key synthetic pathways, including those of protein synthesis. Rupture of lysosomes and intracellular liberation of powerful hydrolytic enzymes, active at a low pH, brings about further cellular dissolution.
  • 47. Summary:Summary: • ReperfusionReperfusion generates free radicals fromgenerates free radicals from parenchymal, endothelial, andparenchymal, endothelial, and inflammatory cells in the injured tissue,inflammatory cells in the injured tissue, often producing more cellular injury thanoften producing more cellular injury than the initial ischemia, largely due tothe initial ischemia, largely due to membrane damagemembrane damage • Be able to identify grossly andBe able to identify grossly and microscopically: Myocardial infarction,microscopically: Myocardial infarction, renal infarction (pale infarct), gangrenerenal infarction (pale infarct), gangrene
  • 48. Activating of membraneActivating of membrane phospholipasephospholipase • The surplus activating of phospholipase A2 has an important value in pathogenesis of cell damage. This enzyme carries out breaking up of phospholipids of cell diaphragms to a) unsaturated fatty acids and b) lysophospholipids. • Unsaturated fatty acids, in particular arachidonic acid, under act of certain enzymes transform to biologically active matters - eicosanoids. • Lysophospholipids have ability to create a micelle and they are very strong detergents. High concentration of ions of Ca2+ in a cytoplasm is the basic reason of activation of phospholipase A2.
  • 49. Detergents action of surplus of free fatty acidsDetergents action of surplus of free fatty acids • Free fatty acids in large concentrations similarly as lysophospholipids damage bimolecular lipid layer of cellular membranes. • It is possible to select 4 basic mechanisms of increase of maintenance of free fatty acids in a cell: • 1) Entering of free fatty acids is increased cell in the presence of high level of lipids in blood, that is observed during activating of processes of lipolysis in fatty tissues (stress, diabetes). • 2) Formation of free fatty acids is increased in lysosomes (at atherosclerosis). • 3) Liberation of free fatty acids is increased from phospholipids of cellular membranes under act of phospholipases. • 4) The use of free fatty acids is broken by a cell as energy sources (diminishing of enzymes is a beta-oxidization and to the Krebs cycle during hypoxia).
  • 50. CaCa2+2+ - homeostasis disorders- homeostasis disorders 1. Increased entrance1. Increased entrance a.a. HypercalciaHypercalcia b.b. Impaired barrier function of the membranesImpaired barrier function of the membranes (increase in peroxidation processes)(increase in peroxidation processes) 22.. Impaired effluxImpaired efflux →→ Ca-accumulationCa-accumulation a.a. Ca-pump disorder, Ca-channels impairmentsCa-pump disorder, Ca-channels impairments →→ disorders in synaptic plasticitydisorders in synaptic plasticity b.b. CaCa2+2+ - Na- Na++ exchange mechanism disorderexchange mechanism disorder
  • 51. CalciumCalcium mechanismsmechanisms •Calcium functionsCalcium functions as a messenger for the release of many intracellular enzymes. •Normally,Normally, intracellular calciumintracellular calcium levels arelevels are kept extremely low compared withkept extremely low compared with extracellular levels. Theseextracellular levels. These lowlow intracellular levels are maintained byintracellular levels are maintained by energy-dependentenergy-dependent,, membrane-membrane- associated calcium/magnesiumassociated calcium/magnesium (Ca(Ca22+/Mg+/Mg22+) ATPase+) ATPase exchangeexchange systems.systems.•IschemiaIschemia and certain toxins lead to anand certain toxins lead to an increaseincrease in cytosolic calciumin cytosolic calcium becausebecause ofof increased influx acrossincreased influx across the cellthe cell membranemembrane and the release of calciumand the release of calcium stored in thestored in the mitochondria andmitochondria and endoplasmic reticulum.endoplasmic reticulum. •The increased calciumThe increased calcium level activates alevel activates a number of enzymes with potentiallynumber of enzymes with potentially damaging effects.damaging effects. •The enzymes include the phospholipasesThe enzymes include the phospholipases responsibleresponsible for damaging the cellfor damaging the cell membrane, proteases thatmembrane, proteases that damage thedamage the cytoskeleton and membrane proteins,cytoskeleton and membrane proteins, ATPases thatATPases that break down ATP andbreak down ATP and hasten its depletion, and endonucleaseshasten its depletion, and endonucleases that fragment chromatinthat fragment chromatin.
  • 52. Cytosolic free calcium is a potent destructive agent. •The increase ofThe increase of concentration ofconcentration of CaCa22+ ions causes in+ ions causes in a cytoplasm:a cytoplasm: a)a) contraction ofcontraction of fibrillar structuresfibrillar structures of cellof cell (myofibrillar);(myofibrillar); b)b) activating ofactivating of phospholipase Aphospholipase A22 c)c) violationviolation ofof connection betweenconnection between the processes ofthe processes of oxidization andoxidization and phosphorylationphosphorylation..
  • 53. Mitochondria Ca2+ Endoplasmic reticulum Ca2+ Increase of cytosolicIncrease of cytosolic CaCa2+2+ ATP-ase Phospholipase Proteinase Endonuclease Reduction of phospholipids Decrease ATP Destruction of membrane and cytoskeleton proteins Segmentation of nuclear chromatin CaCa2+2+ Pathological stimuli Pathogenetic effects of Ca stress
  • 54. Electrolyte-osmoticElectrolyte-osmotic mechanismmechanism of cellof cell damagedamage. • It connect with Na+ and K+ ions. Aligning the concentrations of ions of Na+ and K+ on either side of cell membrane (multiplying maintenance of Na+ and degree of maintenance of K+ is in a cytoplasm) in the basis can have two mechanisms: 1) strengthening of ions diffusion through a cell membrane from extracelullar concentration and electric gradient; 2) violation of mechanisms of active transport of Na+ and K+ (Na-K- pump). •The first mechanism will be realized in the conditions of violations water- electrolyte exchange (hypernatremia, hypokaliemia) and violation of barrier function of cell membrane (increase of its ionic permeability).
  • 55. •Disorders of function of Na-K- pump can be conditioned the deficit of АТP in a cell, multiplying maintenance of cholesterol in lipids bilayer of membrane (for example, at atherosclerosis), by the action of a number of specific inhibitors Na-K-ATP-elements (for example, strophanthine (ouabaine)). •A change in maintenance of ions of Na+ and K+ is caused: •a) loss of electric potential of cellular diaphragm; •b) it was swollen cells (oedema); •c) osmotic injury of cellular membranes, which is accompanied the increase of their permeability.
  • 56. Water-ion balance impairmentWater-ion balance impairment NaNa++ - K- K++ pump disorder leads to:pump disorder leads to: 1. Rest potential impairment → changes in threshold, action potential, impulse transduction 2. Swelling of the cell 3. Osmotic tension of the membrane 4. Impairment of membrane barrier function 5. Impaired electrogenesis (ECG, EEG)
  • 57.  As the cell membrane is highly permeable to water and water follows the osmotic gradient, the cell depends on osmotic equilibrium to maintain its volume.  In order to counterbalance the high intracellular concentration of proteins, amino acids, and other organic substrates, the cell lowers the cytosolic ionic concentration. This is done by Na+/K+-ATPase, which pumps Na+ out of the cell in exchange for K+.  Normally the cell membrane is only slightly permeable for Na+, but highly permeable for K+, so that K+ diffuses out again.  This K+-efflux creates an inside negative potential which drives Cl– out of the cell. In this ionic shift, which uses up adenosine 5"-triphosphate (ATP), reduction of the cytosolic concentration of Na+ and Cl– (adding up to ca. 230 mosm/L) is much greater than the rise in cytosolic K+ concentration (ca. 140mosm/L).  Reduction in intracellular Na+ concentration by Na+/K+-ATPase is necessary not only to avoid cell swelling, but also because the steep electrochemical gradient for Na+ is utilized for a series of transport processes.  The Na+/H+ exchanger eliminates one H+ for one Na+, while the 3 Na+/Ca2+ exchanger eliminates one Ca2+ for 3 Na+.  Na+-bound transport processes also allow the (secondarily) active uptake of amino acids, glucose, etc. into the cell.  Lastly, depolarization achieved by opening the Na+ channels serves to regulate the function of excitable cells, e.g. the signal processing and transmission in the nervous system and the triggering of muscle contractions.
  • 58. • As the activity of Na+-transporting carriers and channels continuously brings Na+ into the cell, survival of the cell requires the continuous activity of Na+/K+-ATPase. • This intracellular Na+ homeostasis may be disrupted if the activity of Na+/K+-ATPase is impaired by ATP deficiency (ischemia, hypoxia, hypoglycemia). • The intracellular K+ decreases as a result, extracellular K+ rises, and the cell membrane is depolarized. • As a consequence, Cl– enters the cell and the cell swells up. These events also occur when the energy supply is compromised, or when Na+ entry exceeds the maximal transport capacity of the Na+/K+-ATPase. • Numerous endogenous substances (e.g., the neurotransmitter glutamate) and exogenous poisons (e.g., oxidants) increase the entry of Na+ and/or Ca2+ via the activation of the respective channels. • The increase in intracellular Na+ concentration not only leads to cell swelling, but also, via impairment of the 3Na+/Ca2+ exchanger, to an increase in cytosolic Ca2+ concentration. • Ca2+ produces a series of cellular effects; among others it penetrates into the mitochondria and, via inhibition of mitochondrial respiration, leads to ATP deficiency.
  • 59. MechanismMechanism of acidosisof acidosis Reasons of cell acidosis:Reasons of cell acidosis: • 1) there is the surplus entering of1) there is the surplus entering of H+ ions in cell from a extracelullarH+ ions in cell from a extracelullar environment;environment; • 2) formation of sour products in a2) formation of sour products in a cell during glycolysis activatingcell during glycolysis activating (lactic acid – lactate), violations of(lactic acid – lactate), violations of Krebs cycle (carbons acids),Krebs cycle (carbons acids), hydrolitic breakinghydrolitic breaking up(disintegration) phospholipidsup(disintegration) phospholipids of cellular membranes (free fatof cellular membranes (free fat acids, phosphoric acid) andacids, phosphoric acid) and others;others; • 3) violation of fastening of free H+3) violation of fastening of free H+ ions is as a result of insufficiencyions is as a result of insufficiency of the buffer systems of cell;of the buffer systems of cell; • 4) violation of move out H+ ions4) violation of move out H+ ions from a cell. Reason of this isfrom a cell. Reason of this is disorders of Na-H-exchangedisorders of Na-H-exchange mechanism, and also in themechanism, and also in the conditions of the broken localconditions of the broken local circulation of blood in tissue.circulation of blood in tissue.
  • 60. Conclusions of acidosis of cell:Conclusions of acidosis of cell: a)a) change conformation ofchange conformation of protein molecules withprotein molecules with violation of them function andviolation of them function and properties;properties; b)b) increase of permeability ofincrease of permeability of cellular membranes;cellular membranes; c)c) activating enzymes ofactivating enzymes of lysosomes.lysosomes. • If there is a lack of OIf there is a lack of O22, energy, energy metabolism switches tometabolism switches to anaerobic glycolysis.anaerobic glycolysis. • The formation of lactic acid,The formation of lactic acid, which dissociates into lactatewhich dissociates into lactate and H+, causes cytosolicand H+, causes cytosolic acidosis that interferes withacidosis that interferes with the functions of thethe functions of the intracellular enzymes, thusintracellular enzymes, thus resulting in the inhibition ofresulting in the inhibition of the glycolysis so that this lastthe glycolysis so that this last source of ATP dries up.source of ATP dries up. MechanismMechanism of acidosis.of acidosis.
  • 61. Proteins MechanismsProteins Mechanisms • The proteins mechanisms of cell damage contain: • 1) inhibition enzymes (reverse and irreversible); • 2) denaturation, that violation of native structure of albumins molecules as a result of conditioned the break connections of changes of the second or tertiary structures of proteins; • 3) proteolisis, that is carried out under the action of lysosomal enzymes.
  • 62. Proteins used in diagnosis of tissue damage by blood testing
  • 63. The nucleic mechanismsThe nucleic mechanisms ► It’s conditioned with violationsIt’s conditioned with violations of nucleic acids (DNA, RNA).of nucleic acids (DNA, RNA). Its disturbance of replication,Its disturbance of replication, transcription and translationtranscription and translation processes.processes. ► Thus, universal mechanismsThus, universal mechanisms of increase of permeability ofof increase of permeability of cellular membranes are:cellular membranes are: 1) activating of FOL;1) activating of FOL; 2) activating of phospholipases;2) activating of phospholipases; 3) osmotic breaking up3) osmotic breaking up membranes;membranes; 4) adsorption of albumens is on4) adsorption of albumens is on a membrane.a membrane.
  • 64. The damage of mitochondrias is accompanied: 1) oppressing the processes of the cellular breathing, 2) violation of connection between the processes of oxidization and phosphorilation.
  • 65. DAMAGE TO DNA AND PROTEINSDAMAGE TO DNA AND PROTEINS  Cells haveCells have mechanisms thatmechanisms that repair damage torepair damage to DNA, but if thisDNA, but if this damage is too severedamage is too severe to be corrected (to be corrected (e.ge.g.,., after exposure to DNAafter exposure to DNA damaging drugs,damaging drugs, radiation, or oxidativeradiation, or oxidative stress), the cellstress), the cell initiates a suicideinitiates a suicide program that results inprogram that results in death by apoptosis.death by apoptosis.  A similar reaction isA similar reaction is triggered bytriggered by improperly foldedimproperly folded proteins, which mayproteins, which may be the result ofbe the result of inherited mutations orinherited mutations or external triggers suchexternal triggers such as free radicals.as free radicals.  Because theseBecause these mechanisms of cellmechanisms of cell injury typically causeinjury typically cause apoptosis, they areapoptosis, they are discussed later in thediscussed later in the chapter.chapter.
  • 66. The clinical relevance of this question isThe clinical relevance of this question is obvious—if we can answer it we may beobvious—if we can answer it we may be able to devise strategies for preventing cellable to devise strategies for preventing cell injury from having permanent deleteriousinjury from having permanent deleterious consequences. However, the molecularconsequences. However, the molecular mechanisms connecting most forms of cellmechanisms connecting most forms of cell injury to ultimate cell death have provedinjury to ultimate cell death have proved elusive, for several reasons. The “point of noelusive, for several reasons. The “point of no return,” at which the damage becomesreturn,” at which the damage becomes irreversible, is still largely undefined, andirreversible, is still largely undefined, and there are no reliable morphologic orthere are no reliable morphologic or biochemical correlates of irreversibility.biochemical correlates of irreversibility. TwoTwo phenomenaphenomena consistently characterizeconsistently characterize irreversibilityirreversibility——the inability to reversethe inability to reverse mitochondrial dysfunctionmitochondrial dysfunction (lack of(lack of oxidative phosphorylation and ATPoxidative phosphorylation and ATP generation) even after resolution of thegeneration) even after resolution of the original injury, andoriginal injury, and profoundprofound disturbances in membrane functiondisturbances in membrane function.. AsAs mentioned earlier, injury to lysosomalmentioned earlier, injury to lysosomal membranes results in the enzymaticmembranes results in the enzymatic dissolution of the injured cell that isdissolution of the injured cell that is characteristic of necrosis.characteristic of necrosis. •Before concluding our discussion of the mechanisms of cell injury, it is useful toBefore concluding our discussion of the mechanisms of cell injury, it is useful to consider the possible events that determine when reversible injury becomesconsider the possible events that determine when reversible injury becomes irreversible and progresses to cell death. The clinical relevance of this question isirreversible and progresses to cell death. The clinical relevance of this question is obvious—if we can answer it we may be able to devise strategies for preventing cellobvious—if we can answer it we may be able to devise strategies for preventing cell injury from having permanent deleterious consequences.injury from having permanent deleterious consequences.
  • 67. DAMAGE TO DNA ANDDAMAGE TO DNA AND PROTEINSPROTEINS  Leakage of intracellular proteins through theLeakage of intracellular proteins through the damaged cell membrane and ultimately into thedamaged cell membrane and ultimately into the circulation provides a means of detecting tissue-circulation provides a means of detecting tissue- specific cellular injury and necrosis using bloodspecific cellular injury and necrosis using blood serum samples.serum samples.  Cardiac muscle, for example, contains aCardiac muscle, for example, contains a specific isoform of the enzymespecific isoform of the enzyme creatinecreatine kinasekinase and of the contractile proteinand of the contractile protein troponintroponin;;  Liver (and specifically bile duct epithelium)Liver (and specifically bile duct epithelium) contains an isoform of the enzymecontains an isoform of the enzyme alkalinealkaline phosphatasephosphatase; and hepatocytes contain; and hepatocytes contain transaminasestransaminases..  Irreversible injury and cell death in these tissuesIrreversible injury and cell death in these tissues are reflected in increased levels of such proteinsare reflected in increased levels of such proteins in the blood, and measurement of thesein the blood, and measurement of these biomarkers is used clinically to assess damagebiomarkers is used clinically to assess damage to these tissues.to these tissues.
  • 68. After damage to mitochondrial membranes there is failure of ATP production and loss of the normal membrane potential of the mitochondrion. The mitochondrial membrane pores (PTPC megachannels) open and release proteins into the cytosol, which can cause apoptosis, as described below. If many mitochondria in a cell fail, causing a catastrophic reduction in ATP production, the cell will die by a non-apoptotic route.
  • 69. Mechanisms of cell death.Mechanisms of cell death. Terminology:Terminology:  Necrosis:Necrosis: Morphologic changes seen inMorphologic changes seen in dead cells within living tissue.dead cells within living tissue.  Autolysis:Autolysis: Dissolution of dead cells by theDissolution of dead cells by the cells own digestive enzymes. (not seen)cells own digestive enzymes. (not seen)  Apoptosis:Apoptosis: Programmed cell death.Programmed cell death. Physiological, for cell regulation.Physiological, for cell regulation.
  • 70. Mechanisms of cell deathMechanisms of cell death  ““ApoptosisApoptosis is a pathway of cell death that isis a pathway of cell death that is induced by a tightly-regulated suicide program ininduced by a tightly-regulated suicide program in which cells destined to die activate enzymes thatwhich cells destined to die activate enzymes that degrade the cell’s own nuclear DNA and nucleardegrade the cell’s own nuclear DNA and nuclear and cytoplasmic proteins”and cytoplasmic proteins” – Developmental morphogenesisDevelopmental morphogenesis – RadiationRadiation – Immune system regulationImmune system regulation – Viral infectionsViral infections – CancersCancers – ToxinsToxins The process was recognized in 1972 by the distinctive morphologicThe process was recognized in 1972 by the distinctive morphologic appearance of membrane-bound fragments derived from cells, andappearance of membrane-bound fragments derived from cells, and named after the Greek designation for “falling off.”named after the Greek designation for “falling off.”
  • 71. General biochemical mechanismsGeneral biochemical mechanisms  Defects in plasma membrane permeability.Defects in plasma membrane permeability.  Oxygen deprivation or generation of reactiveOxygen deprivation or generation of reactive oxygen species (free radical).oxygen species (free radical).  Loss of calcium homeostasis.Loss of calcium homeostasis.  MitochondrialMitochondrial damage.damage.  Chemical injuryChemical injury  Genetic variationGenetic variation
  • 72. Receptorsª Fas, (Apo1, CD95), TNF Trigger action of cytokines and hormones Provider molecules of apoptotic signal to the cell Causes: moderate injury during the entrance of O2, different actions Conduction of intracellular apoptotic signal (Bad1) Expression of apo- ptotic genes (bax, p53, Ced-3, p21) Repression of apoptosis blockaders BCL-2, XÉ Activation of cellular cysteine – photolytic – caspases + nucleases 1) Activation of death receptors-DR, 2) Mitochondria – dependent pathway: Output of Ca, proteins, cytochrome C • Lysis of cellular proteins • Fragmentation of nucleus and cytoplasm • Apoptotic bodies • Autophagocytosis Without inflammation RIP FAXD TRADD Activated caspases A p o p t o s i s
  • 73. 1. Cell shrinkage. 2. Nuclear chromatin condensation and fragmentation 3. Apoptotic bodies formation 4. Phagocytosis of apoptotic bodies by adjacent cells or macrophages. 5. Intacted membrane. Morphologic features of ApoptosisMorphologic features of Apoptosis
  • 74. Role of apoptosis in physiology and pathologyRole of apoptosis in physiology and pathology 1. During embryogenesis ( implantation, organogenesis, growth,1. During embryogenesis ( implantation, organogenesis, growth, metamorphosis)metamorphosis) 2. In adults hormone dependent involution (2. In adults hormone dependent involution (during menstrual cycle,during menstrual cycle, menopausemenopause, atrophy of, atrophy of prostate and breastsprostate and breasts)) 3. Destruction of cells in the3. Destruction of cells in the reproducingreproducing cellular populations (epithelialcellular populations (epithelial cells of intestine )cells of intestine ) 4. Cell death in tumor (regression)4. Cell death in tumor (regression) 5. Death of neutrophils in the active inflammatory process5. Death of neutrophils in the active inflammatory process 6. Death of immune cells (B,T- lymphocytes)6. Death of immune cells (B,T- lymphocytes) 7. Death of cytotoxic T- lymphocytes7. Death of cytotoxic T- lymphocytes 8. Pathological atrophy in the8. Pathological atrophy in the parenchimatous organsparenchimatous organs 9. During the some viral infections (hepatitis)9. During the some viral infections (hepatitis) 10. Temperate action of various noxious factors10. Temperate action of various noxious factors
  • 75. Confocal 3d images of nuclei from nonapoptotic (A) and apoptotic (B) cells stained with PI A B
  • 76. Morphogenesis of cell injuryMorphogenesis of cell injury Atrophy - physiological, pathological Types - local, general, dysfunctional, due to compression, blood circulation, neurogenous Necrosis, necrobiosis (protein dystrophy) Autolysis, caryorhexis, caryolysis, plasmorrhexis, plasmolysis, demarcation zone, inflammation around the necrosis
  • 77. Morphology of necrotic cellsMorphology of necrotic cells • Cell and nuclear swelling • Vacuolization of cytoplasm • Patchy chromatin condensation • Mitochondrial swelling • Plasma membrane rupture • Dissolution of chromatin • Attraction of inflammatory cells
  • 78. Relationships between sublethal and lethal cell damage. Following sublethal damageRelationships between sublethal and lethal cell damage. Following sublethal damage a cell may recover or, with persistence of the damaging stimulus, cell death maya cell may recover or, with persistence of the damaging stimulus, cell death may result. The sequential structural changes of cell death are termed 'necrosis'.result. The sequential structural changes of cell death are termed 'necrosis'. Relationships between sublethal and lethal cell damage. Following sublethal damageRelationships between sublethal and lethal cell damage. Following sublethal damage a cell may recover or, with persistence of the damaging stimulus, cell death maya cell may recover or, with persistence of the damaging stimulus, cell death may result. The sequential structural changes of cell death are termed 'necrosis'.result. The sequential structural changes of cell death are termed 'necrosis'.
  • 79. Comparison of cell death by apoptosis and necrosisComparison of cell death by apoptosis and necrosis
  • 80. Relationships between sublethal and lethal cell damage. Normal cells that are subject to a damaging stimulus may initiate apoptosis or may become sublethally damaged. If the stimulus abates, cells may recover by resynthesis of proteins and elimination of damaged components. If a damaging stimulus continues, either cells die through apoptosis or, when critical cell damage takes place, mainly through critical lack of ATP, cells die and undergo necrosis. Massively damaging stimuli, e.g. great heat or strong acids, cause immediate coagulation of proteins and death of cells.
  • 81. Adaptive responses resulting in increased tissue mass. Increased functional demand or endocrine stimulation are what usually cause hypertrophy and hyperplasia. These new patterns of growth are stable while the causative stimulus persists, but once it is removed the tissue returns to a normal pattern of growth.
  • 82. PathologyPathology of elderlyof elderly
  • 83. Ageing:Ageing: ““Progressive time related loss ofProgressive time related loss of structural and functionalstructural and functional capacity of cells leading tocapacity of cells leading to death”death” ►Senescence, Senility, SenileSenescence, Senility, Senile changes.changes. ►Ageing of a person is intimatelyAgeing of a person is intimately related to cellular ageing.related to cellular ageing.
  • 84. Factors affecting ageing:Factors affecting ageing: • Stress • Infections • Diseases • Malnutrition • Accidents • Diminished stress response. • Diminished immune response. • Good health.
  • 85. Cellular mechanismsCellular mechanisms of ageingof ageing  Cross linking proteins &Cross linking proteins & DNA.DNA.  Accumulation of toxic by-Accumulation of toxic by- products.products.  Ageing genes.Ageing genes.  Loss of repairLoss of repair mechanism.mechanism.  Free radicale injuryFree radicale injury  Telomerase shortening.Telomerase shortening.
  • 86.  General and clinical pathophysiology / Edited by Anatoliy V. Kubyshkin – Vinnytsia: Nova Knuha Publishers – 2011. – P. 134–165.  Gozhenko A.I. General and clinical pathophysiology / A.I. Gozhenko, I.P. Gurcalova // Study guide for medical students and practitioners. Edited by prof.Zaporozan, OSMU. – Odessa. – 2005.– P. 30–41.  Robbins and Cotran Pathologic Basis of Disease 8th edition./ Kumar, Abbas, Fauto. – 2007. – Chapter 1. – P. 1–30  Essentials of Pathophysiology: Concepts of Altered Health States (Lippincott Williams & Wilkins), Trade paperback (2003) / Carol Mattson Porth, Kathryn J. Gaspard. – Сhapters 1-2. – P. 1–14, 28–35.  Copstead Lee-Ellen C. Pathophysiology / Lee-Ellen C. Copstead, Jacquelyn L. Banasik // Elsevier Inc, 4th edition. – 2010. – P. 30–84.  Corwin Elizabeth J. Handbook of Pathophysiology / Corwin Elizabeth J. – 3th edition. Copyright В. – Lippincott Williams & Wilkins – 2008. – Chapter 1. – P. 3–35.  Silbernagl S. Color Atlas of Pathophysiology / S. Silbernagl, F. Lang // Thieme. Stuttgart. New York. – 2000. – P. 2–13.  Pathological physiology / Yu.I. Bondarenko, M.R. Khara, V.V. Faifura, N. Ya. Potikha. – Ternopil: Ukrmedkniga. – 2006. – 312 p.  Pathophysiology, Concepts of Altered Health States, Carol Mattson Porth, Glenn Matfin.– New York, Milwaukee. – 2009. – P. 99–109.
  • 87. Thank you for attention!