2. Cell injxxxury (Etiology +Pathogenesis).pptx

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1. Cell injury Dr. Sidra Batool (Pharm.D ( R.Ph .), M.Phil. Pharmacology) Shahida Islam College of Pharmacy

2. Cell Physiology Cell membrane: Semi-permeable membrane with Pumps for ionic and osmotic homeostasis Nucleus: Nucleolus (Synthesis of Ribosomal RNA) Mitochondria: Oxidative Phosphorylation (main source of Atp ) Endoplasmic reticulum (ER), ribosome and Golgi apparatus: protein synthesis and transport Lysosome: endocytosis, phagocytosis, pinocytosis followed by degradation Peroxisome: catalase and oxidase enzymes, metabolism of H2O2 fatty acid

3. Cell Morphology

4. Homeostasis “ When a cell maintaining its normal structure and function, it is called as Homeostasis.” When a cell encounters physiological stresses or pathological stimuli, it undergoes three processes. It maintains its homeostasis If stimuli become excessive then it undergo adaptation. (change in structure but function is same). E.g. Atrophy ( ↓ in size of cell). Hyperplasia (↑ in no. of cell) If the adaptive capability is exceeded cell injury develop.

5. Cellular Responses to Stress and Noxious Stimuli

6. Cell Injury Cell injury is defined as the effect of a variety of stresses due to etiological agents a cell encounters resulting in changes in its internal and external environment. In general, cells of the body have an inbuilt mechanism to deal with changes in the environment to an extent. The cellular response to stress may vary and depends upon the following two variables: Host factors i.e. the type of cell and tissue involved. Factors pertaining to injurious agent i.e. extent and type of cell injury. Usual morphological changes Functional implication and disease outcomes

7. Types Cell injury is of two types:
1- Reversible injury : Within certain limits if the stimulus is removed injury is reversible that cell returns to its homeostasis. 2- Irreversible injury : With severe or powerful stress the injury is reversible and as an injury is irreversible and as a result the affected cells die.

8. Etiology/Causes of cell injury: The cells may be broadly injured by two main ways of causes: By genetic causes By acquired causes Genetic causes Development defect: error on morphogenesis Cytogenetic defect: chromosomal Abnormalities

9. Etiology/Causes of cell injury: On the other hand major acquired causes of cell injury are grouped as follows. Oxygen deprivation Physical agents Chemical agents and drugs Infectious agents Immunological reactions Genetic derangements Nutritional imbalance

10. Oxygen deprivation Hypoxia – deficiency of oxygen (↓in O2 concentration at cellular level) which interfere with aerobic respiration. It is an important cause of cell injury (Oxygen problem- altitude & hemoglobin-anemia) Ischemia – loss of blood supply (arterial flow or reduced venous drainage) Inadequate oxygenation of the blood Decreased oxygen-carrying capacity of the blood After severe blood loss (Local e.g. embolus; systemic e.g. cardiac failure) Depending on the severity of the hypoxic state, cells may adapt, undergo injury, or die.

11. Chemical agents and drugs Any chemical agent may cause cell injury even glucose or salts if sufficiently concentrated may effect osmotic environment so that injury of cell occurs. In addition, common poisons interfere with cellular metabolism. If atp level below critical levels, affected cell will die. The list of pharmaceuticals may have toxic effects on cell is enormous. Some act directly but most have their effect through metabolites (metabolism of alcohol to acetaldehyde is one example. Hypertonic concentration of salt – deranging electrolyte homeostasis Poisons – arsenic, cyanide, or mercuric salts Insecticides and Herbicides Air pollutant – carbon monoxide Occupational hazard – asbestos Alcohol

12. Physical agents Physical agents such as trauma extreme of temperature, radiation, changes in pressure, electric shock, have effect on cells. Exposure of tissue to extreme heat or cold results in direct injury that is often irreversible, resulting in a pattern of coagulation necrosis Sudden changes in pressure can cause cellular disruption ( e.g a hammer blow to thumb) Electrical currents can cause direct breakdown of cell membranes that may be irreversible.

13. Infectious agent: Injuries by Microbes includes infection caused by bacteria, virus, fungi and protozoa etc.

14. Immunologic agents: double- edge sword-protects the host against various injurious agents but it may also cause cell injury. Immune reactions indented or incidental can nevertheless results in cell and tissue injuries. E.g anaphylaxis to a foreign protein or a drug. Hypersensitivity reaction to foreign protein or drug Reactions to endogenous self-antigens – autoimmune diseases Genetic defects: Genetic defects may result in pathologic change e.g sickle cell anemia

15. AgING Healing of injured tissues does not always results in perfect restoration of structure and function. Repeated trauma can also lead to degeneration which can lead to the cell death and lastly death of organism.

16. Genetic Derangements Decreased life of red blood cell – Thalassemia, Sickle cell anemia Inborn errors of metabolism – G6PD deficiency

17. Nutritional Imbalance Protein-calorie deficiencies Vitamin deficiencies Anorexia nervosa Excesses of lipids – Obesity , Atherosclerosis Metabolic diseases – Diabetes

18. Mechanisms of Cell Injury Cell injury results from functional and biochemical abnormalities in one or more of cellular components. The most important targets of injurious stimuli are Depletion of ATP Mitochondrial Damage Influx of Intracellular Calcium and Loss of Calcium Homeostasis Accumulation of Oxygen-Derived free radical ( Oxidative stress ) Defects in Membrane Permeability Damage to DNA and proteins

19. Mechanisms of Cell Injury Depletion of ATP Na + K + ATPase ( Na -pump ) Ca 2+ Mg 2+ ATPases ( Ca -pump ) Causes Hypoxia, Ischemia, Mitochondrial damage Chemical Injury Membrane transport Protein synthesis etc. ATP

20. Mechanisms of Cell Injury Depletion of ATP

21. Depletion of ATP Mechanisms of Cell Injury

22. Hypoxia/Ischemia-Pathogenesis of reversible & Irreversible injury

23. Mitochondrial Damage Mechanisms of Cell Injury Causes Hypoxia Cytosolic Ca 2+ Oxidative stress Lipid breakdown product

24. Mitochondrial Damage There are two major consequences of mitochondrial damage Mitochondrial damage often results in the formation of a high-conductance channel in the mitochondrial membrane, called the mitochondrial permeability transition pore . The mitochondria contain several proteins that are capable of activating apoptotic pathways; these include cytochrome c and caspases. Mechanisms of Cell Injury

25. Mitochondrial Damage Mechanisms of Cell Injury

26. INFLUX OF CALCIUM AND LOSS OF CALCIUM HOMEOSTASIS Increased intracellular Ca 2+ causes cell injury by several mechanisms. The accumulation of Ca 2+ in mitochondria results in opening of the mitochondrial permeability transition pore. Increased cytosolic Ca 2+ activates a number of enzymes , with potentially deleterious cellular effects Increased intracellular Ca 2+ levels also result in the induction of apoptosis. Mechanisms of Cell Injury

27. LOSS OF CALCIUM HOMEOSTASIS Mechanisms of Cell Injury

28. ACCUMULATION OF OXYGEN-DERIVED FREE RADICALS (OXIDATIVE STRESS) What is Free Radicals? Free radicals are chemical species that have a single unpaired electron in an outermost orbit/orbital. Energy created by this unstable configuration is released through reactions with adjacent molecules, such as proteins, lipids, carbohydrates in membranes & nucleic acid. Free radicals are highly reactive and can initiate autocatalytic reactions whereby molecules with which they react are themselves converted into free radicals to propagate the chain of damage . Mechanisms of Cell Injury

29. ACCUMULATION OF OXYGEN-DERIVED FREE RADICALS (OXIDATIVE STRESS) ROS have been identified as the likely cause of cell injury in many diseases and other damaging events. These include: Organ toxicity & other gases toxicity The inflammatory process Chemical & Radiation injury Killing of microbes by the pathogenic cell Chemical carcinogenesis Cellular Aging Mechanisms of Cell Injury

30. Mechanisms of Cell Injury Formation of free radical Free radicals are formed in three ways. Absorption of radiant energy: The absorption of radiant energy (UV light, X-rays) ionizing radiations can hydrolyze water into free radicals. H2O H+ + OH−

31. Oxidation-reduction reactions Oxidation-reduction reactions that occur during normal physiological processes, cause the production of free radicals. E.g during normal respiration O2 is reduced in mitochondria by addition of 4-electrons to generate H2O. This reaction is imperfect, however, and small amounts of highly reactive but short-lived toxic intermediates are generated when oxygen is only partially reduced. These intermediates include superoxide (O2 •), which is converted to hydrogen peroxide (H2O2) spontaneously and by the action of the enzyme superoxide dismutase. H2O2 is more stable than O2 • and can cross biologic membranes. In the presence of metals, such as Fe2+, H2O2 is converted to the highly reactive hydroxyl radical •OH by the Fenton reaction.

32. ROS-Phagocytic leukocytes The ROS are generated in the phagosomes and phagolysosomes of leukocytes by a process that is similar to mitochondrial respiration and is called the respiratory burst (or oxidative burst). In this process, a phagosome membrane enzyme catalyzes the generation of superoxide, which is converted to H2O2. H2O2 is in turn converted to a highly reactive compound hypochlorite (the major component of household bleach) by the enzyme myeloperoxidase, which is present in leukocytes. Nitric oxide (NO) is another reactive free radical produced in leukocytes and other cells. It can react with superoxide to form a highly reactive compound, peroxynitrite , which also participates in cell injury

33. ACCUMULATION OF OXYGEN-DERIVED FREE RADICALS (OXIDATIVE STRESS) ROS involves in normal cell signalling, including modulation of gene regulation , activation of mitogen-activated protein (MAP) kinases , reversible protein modifications (e.g., phosphorylation and dephosphorylation) and etc. However, when present in excess under pathologic conditions, ROS may exert profound deleterious effects on signalling pathways. ROS may proceed to cell death via apoptosis as well as necrosis. Mechanisms of Cell Injury

34. Enzymatic metabolism of chemicals Enzymatic metabolism of endogenous chemicals e.g CCl4 is metabolized by hepatic oxidase enzyme into CCl3. CCl4 CCl3.

35. Chemical injury

36. Effects of free radical Various effects of free radicals are 1- Lipid peroxidation of cell membrane it is referred to a process by which the double bond present in lipid of membrane are attacked by free radicals which results in formation of peroxidase. These peroxidase are unstable reactive and give rise chain of autocatalytic reactions, cause extensive damage of cell membrane as well as organellic membrane 2- non- peroxidative mitochondrial damage

37. 3-Injury to DNA Free radicals react with the thymine nucleotide of DNA cause single stranded break in DNA which injury to DNA which results in cell death. 4- Cross linkage of proteins.
Free radicals causes cross linkage of different amino acids which result in inactivation of enzyme

38. Termination of free radical Body get rid of free radicals in three ways:
Spontaneous decay
Enzymatic degradation Certain antioxidants neutralize free radicals (vitamin E)

39. Cellular Defences against Reactive Oxygen Species detoxifying enzymes SOD (2O 2 - + 2H + → O 2 + H 2 O 2 ). Catalase (2 H 2 O 2 → 2H 2 O + O 2 ). Glutathione peroxidase ( H 2 O 2 + 2GSH → 2H 2 O + GSSG). exogenous free-radical scavengers Vitamin E (tocopherol ) is a terminal electron acceptor. Vitamin C reacts directly with O 2 - , OH• and some products of lipid peroxidation. Retinoids , the precursors of vitamin A, function as chain-breaking antioxidants.

40. DEFECTS IN MEMBRANE PERMEABILITY Causes Reactive oxygen species Decreased phospholipid synthesis Increased phospholipid breakdown Cytoskeletal abnormalities Lytic complement components Impair oxidative phosphorylation Various chemical & physical agents

41. Consequences of Membrane Damage Mitochondrial membrane damage Plasma membrane damage Injury to lysosomal membranes Lysosomes contain RNases, DNases, proteases, phosphatases and glucosidases. Activation of these enzymes leads to enzymatic digestion of proteins, RNA, DNA, and glycogen, and the cells die by necrosis .

42. DAMAGE TO DNA AND PROTEINS 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 and damaged proteins, which may be the result of inherited mutations or external triggers such as free radicals.

43. Ubiquitin and the Ubiquitin–Proteasome System Ubiquitin (Ub) is an 76-aminoacid protein . These activities are accomplished via reversible Ub conjugation with target proteins and can be divided into proteolytic and nonproteolytic pathways . Protein degradation Autophagy Endocytosis Intracellular trafficking Regulation of histones and transcription Cell cycle control Repair of DNA damage Cellular signaling

44. Morphology of cell injury Reversible cell injury Irreversible cell injury

45. Reversible cell injury Two features of reversible cell injury can be recognized under the light microscope: Cellular swelling Cellular swelling due to imbalance of ionic and fluid homeostasis Fatty change Fatty change occurs in hypoxic injury and various forms of toxic or metabolic injury & seen mainly in cells involved in and dependent on fat metabolism, such as hepatocytes and myocardial cells.

46. Morphology of reversible cell injury Plasma membrane alterations , such as blebbing, blunting, and loss of microvilli. Mitochondrial changes , including swelling and the appearance of small amorphous density aggregates. Dilation of the ER , with detachment of polysomes; intracytoplasmic myelin figures may be present. Nuclear alterations , condensation of nuclear chromatin material.

47. Irreversible cell injury Necrosis Apoptosis Autophagy

48. Morphology of Necrosis The morphologic appearance of necrosis is the result of denaturation of intracellular proteins and enzymatic digestion . Necrotic cells are unable to maintain membrane integrity and their contents often leak out & may results inflammation in the surrounding tissue. The enzymes that digest the necrotic cell are derived from the lysosomes of the dying cells themselves and from the lysosomes of leukocytes that are called in as part of the inflammatory reaction.

49. Morphology of Necrosis-cytoplasm Increased eosinophilia in hematoxylin and eosin (H & E) stains. When enzymes have digested the cytoplasmic organelles, the cytoplasm becomes vacuolated and appears moth-eaten . Dead cells may be replaced by large, whorled phospholipid masses called myelin figures that are derived from damaged cell membranes . These phospholipid precipitates are then either phagocytosed by other cells or further degraded into fatty acids; calcification of such fatty acid residues results in the generation of calcium soaps.

50. Morphology of Necrosis-nucleus Nuclear changes appear in one of three patterns Pyknosis , characterized by nuclear shrinkage and increased basophilia . Karyorrhexis , the pyknotic nucleus undergoes fragmentation. Karyolysis , the basophilia of the chromatin fades which appears to reflect loss of DNA because of enzymatic degradation by due to endonucleases .

51. Patterns of Tissue Necrosis Coagulative Liquefactive Gangrenous Caseous Fat Fibrinoid

52. Coagulative necrosis Shortly after a cell’s death, its outline is maintained . Architecture of dead tissues is preserved for a span of at least some days. Tissues exhibit a firm texture. Injury denatures proteins and enzymes blocking proteolysis of the dead cells; Eosinophilic, anucleate cells may persist for days or weeks. Ultimately the necrotic cells are removed by phagocytosis of the cellular debris by infiltrating leukocytes.

53. Liquefactive necrosis Rate of dissolution of the necrotic cells is considerably Faster Transformation of the tissue into a liquid viscous mass . The necrotic material is frequently creamy yellow because of the presence of dead leukocytes and is called pus .

54. Caseous necrosis Caseous ” (cheeselike) is derived from the friable white appearance of the area of necrosis. Necrotic area appears as a collection of fragmented or lysed cells and amorphous granular debris enclosed within a distinctive inflammatory border .

55. Fat necrosis Focal areas of fat destruction, typically resulting from release of activated pancreatic lipases into the substance of the pancreas and the peritoneal cavity. Lipases split the triglyceride esters contained within fat cells. Free fatty can combine with calcium to produce grossly visible chalky-white areas (fat saponification).

56. Fibrinoid necrosis Usually seen in immune reactions involving blood vessels. Deposits of “immune complexes,” together with fibrin that has leaked out of vessels. Bright pink and amorphous appearance in H&E stains, called “fibrinoid” (fibrin-like) by pathologists. e.g. immunologically mediated vasculitis syndromes

57. Gangrenous necrosis Gangrene is a serious and potentially life-threatening condition that arises when a considerable mass of body tissue undergo necrosis. Dry gangrene is mainly due to arterial occlusion & it’s a type of Coagulative necrosis Wet gangrene usually develops rapidly due to blockage of venous & Wet gangrene is Coagulative necrosis progressing to Liquefactive necrosis. Gas gangrene is a bacterial infection that produces gas within tissues.

58. Dry gangrene Wet gangrene

59. Apoptosis 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.

60. Morphology of Apoptosis Cell shrinkage . The cell is smaller in size; the cytoplasm is dense and the organelles, though relatively normal, are more tightly packed. Chromatin condensation . This is the most characteristic feature of apoptosis. The chromatin aggregates peripherally, under the nuclear membrane, into dense masses of various shapes and sizes. Formation of cytoplasmic blebs and apoptotic bodies . The apoptotic cell first shows extensive surface blebbing, then undergoes fragmentation into membrane-bound apoptotic bodies composed of cytoplasm and tightly packed organelles, with or without nuclear fragments. Phagocytosis of apoptotic cells or cell bodies, usually by macrophages.

61. Feature Necrosis Apoptosis Cell size Enlarged (swelling) Reduced (shrinkage) Nucleus Pyknosis ➙ karyorrhexis ➙ karyolysis Fragmentation into nucleosome-size fragments Plasma membrane Disrupted Intact; altered structure, especially orientation of lipids Cellular contents Enzymatic digestion; may leak out of cell Intact; may be released in apoptotic bodies Adjacent inflammation Frequent No Physiologic or pathologic role Invariably pathologic (culmination of irreversible cell injury) Often physiologic, means of eliminating unwanted cells; may be pathologic after some forms of cell injury, especially DNA damage

62. Biochemical Features of Apoptosis Activation of Caspases A specific feature of apoptosis is the activation of several members of a family of cysteine proteases named caspases . The term caspase is based on two properties of this family of enzymes: the “ c ” refers to a cysteine protease (i.e., an enzyme with cysteine in its active site), and “ aspase ” refers to the unique ability of these enzymes to cleave after aspartic acid residues.

63. Continued …. DNA and Protein Breakdown Apoptotic cells exhibit a characteristic breakdown of DNA into large 50- to 300-kilobase pieces. Subsequently, there is cleavage of DNA by Ca 2+ - and Mg 2+ -dependent endonucleases into fragments whose sizes are multiples of 180 to 200 base pairs, reflecting cleavage between nucleosomal subunits.

64. Continued …. Membrane Alterations and Recognition by Phagocytes. The plasma membrane of apoptotic cells changes in ways that promote the recognition of the dead cells by phagocytes. One of these changes is the movement of some phospholipids (notably phosphatidylserine) from the inner leaflet to the outer leaflet of the membrane, where they are recognized by a number of receptors on phagocytes. These lipids are also detectable by binding of a protein called annexin V ; thus, annexin V staining is commonly used to identify apoptotic cells.

65. Mechanism of Apoptosis Apoptosis reflects several different pathways that lead to similar end points. Extrinsic pathway apoptosis Intrinsic pathway apoptosis (Mitochondrial pathway) Perforin/ granzyme pathway apoptosis Inflammatory and infectious apoptosis

66. Extrinsic pathway apoptosis Receptor–ligand interactions at the cell membrane trigger extrinsic apoptosis. Ligands are FasL & TNF- α that’s binds to their receptors Fas & TNFR (death receptors) respectively.

67. Intrinsic pathway apoptosis The mitochondrial pathway is the major mechanism of apoptosis in all mammalian cells

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69. Bcl-2 Family Protein Anti-apoptotic Proteins Pro-apoptotic Proteins Bcl-2, Bcl-xL, Mcl-1 Prevent the leakage of mitochondrial protein Stimulate their Production Growth Factors & survival signals BH3-only Proteins( sensor Proteins ) Bim, Bid, Bad, Noxa, Puma Forms oligomers, inserted into mitochondrial membrane & forms channel Bax, Bak ( effector protein ) Sensor proteins Binds with and block their function & also inhibit their synthesis

70. Liberated from their dimers & Polymerize to form MOM pores, releasing Mitochondrial proteins

71. Perforin/granzyme pathway apoptosis

72. Cellular Responses to Stress and Noxious Stimuli

73. Intracellular accumulations One of the manifestations of metabolic derangements in cells is the intracellular accumulation of abnormal amounts of various substances. Many processes result in abnormal intracellular accumulations, but most accumulations are caused by four types of abnormalities.

74. A normal endogenous substance is produced at a normal or increased rate, but the rate of metabolism is inadequate to remove it . Examples of this type of process are fatty change in the liver and reabsorption protein droplets in the tubules of the kidneys.

75. An abnormal endogenous substance, typically the product of a mutated gene, accumulates because of defects in protein folding and transport and an inability to degrade the abnormal protein efficiently . Examples include the accumulation o f mutated α1-antitrypsin in liver cells and various mutated proteins in degenerative disorders of the central nervous system

76. A normal endogenous substance accumulates because of defects, usually inherited, in enzymes that are required for the metabolism of the substance . Examples include diseases caused by genetic defects in enzymes involved in the metabolism of lipid and carbohydrates, resulting in intracellular deposition of these substances, largely in lysosomes.

77. An abnormal exogenous substance is deposited and accumulates because the cell has neither the enzymatic machinery to degrade the substance nor the ability to transport it to other sites. Accumulations of carbon particles and nonmetabolizable chemicals such as silica are examples of this type of alteration.

78. Pathologic Calcification Pathologic calcification is the abnormal tissue deposition of calcium salts, together with smaller amounts of iron, magnesium, and other mineral salts. There are two forms of pathologic calcification; DYSTROPHIC CALCIFICATION METASTATIC CALCIFICATION

79. DYSTROPHIC CALCIFICATION When the deposition occurs locally in dying tissues (coagulative, caseous, or liquefactive type necrosis, and in foci of enzymatic necrosis of fat). It occurs despite normal serum levels of calcium and in the absence of derangements in calcium metabolism. Examples are atheromas of advanced atherosclerosis & develops in aging or damaged heart valves . Whatever the site of deposition, the calcium salts appear macroscopically as fine, white granules or clumps , often felt as gritty deposits. Sometimes a tuberculous lymph node is virtually converted to stone.

80. METASTATIC CALCIFICATION Metastatic calcification may occur in normal tissues whenever there is hyperkalaemia. Causes Increased secretion of parathyroid hormone (PTH) with subsequent bone resorption Destruction of bone tissue , secondary to primary tumors of bone marrow Vitamin D–related disorders , including vitamin D intoxication and idiopathic hypercalcemia of infancy (Williams syndrome), characterized by abnormal sensitivity to vitamin D Renal failure , which causes retention of phosphate.