Cell_Injury_etiology and Pathophysiology.pptx

This presentation discusses about the etiology and pathophysiology of cell injury.

1. Cell Injury Etiology and Pathophysiology Dr. Swarna Dabral Assistant Professor

2. Introduction Cells are the basic building blocks of all tissues in the human body, which come together to form organs and systems. There are two main types of cells: epithelial cells, which cover surfaces and line cavities, and mesenchymal cells, which provide support and structure. In a healthy body, these cells work well together. In 1859, a scientist named Virchow introduced the idea that diseases start at the cellular level . This means that problems within cells can lead to diseases. Since then, scientists have focused on studying how changes in the structure and function of cells cause diseases. Most diseases start with some form of cell damage . This damage then leads to cells not working properly, which can affect the tissues , organs , and s ystems they are part of.

3. Cell Injury Cell injury happens when a cell faces different types of stress or harmful conditions from various causes, leading to changes in the internal or external environment of the cell. Normally, cells have ways to handle some level of stress or changes in their environment. How a cell responds to stress depends on two main factors: Host factors and Injurious agent factors HOST FACTORS: This refers to the type of cell and the tissue it is part of . Different cells and tissues react differently to stress. Type of Cell : Nerve Cells (Neurons): These cells are very sensitive to lack of oxygen. If the brain doesn't get oxygen for even a few minutes, neurons can be damaged or die . Skin Cells: Skin cells are more resilient and can survive longer without oxygen compared to nerve cells. They can also regenerate and heal faster when injured. Type of Tissue: Heart Muscle Tissue (Cardiac Muscle): If the blood supply to the heart is blocked , the heart muscle cells can quickly become damaged, leading to a heart attack . Liver Tissue: Liver cells can handle more stress and can regenerate better than heart muscle cells . The liver can recover from damage caused by alcohol or toxins more effectively. INJURIOUS AGENT FACTORS: This refers to the type and severity of the stress or injury the cell is experiencing. Different kinds and amounts of injury can affect how the cell responds. Type of Injury: Physical Injury: A cut on the skin will affect skin cells and may cause bleeding, but skin cells can heal relatively quickly. A similar injury to the brain would be much more severe and harder to heal. Chemical Injury: Exposure to strong acids can cause severe burns to the skin and underlying tissues, whereas mild acids might only cause minor irritation . Severity of Injury: Mild Stress: Mild dehydration might cause skin cells to lose some moisture, making the skin feel dry . This can be easily fixed by drinking water and moisturizing . Severe Stress: Severe dehydration affects all cells in the body , leading to serious health problems like kidney failure, which requires immediate medical attention.

4. When cells get injured, they can respond in different ways. Here are some of the main ways cells react to injury: 1. Cellular Adaptations: When there is extra work or demand on a cell, it might change to handle the stress. These changes can be seen under a microscope. Once the stress is gone, the cell usually returns to its normal state. Eg. Hypertrophy, atrophy, hyperplasia etc.

5. 2. Reversible and Irreversible Cell Injury: If the stress or injury to the cell is mild to moderate , the cell can recover . This is called reversible cell injury. If the injury is severe and long-lasting , the cell might not be able to recover and will die. This is called irreversible cell injury. Subcellular Changes and Intracellular Accumulations: Even if a cell recovers from an injury , there can be residual signs of the injury inside the cell. These signs can include changes at the tiny (subcellular changes) level or the buildup of substances (metabolites) inside the cell (intra-cellular accumulations) . To understand diseases at the cellular level, it's important to know: The causes of cell injury (etiology). How cell injury happens and how cells adapt (pathogenesis).

6. The cells may be broadly injured by two major ways: The causes of disease comprise vast majority of common diseases afflicting mankind. Based on underlying agent, the acquired causes of cell injury can be further categorised as under: Hypoxia and ischaemia : Cells of different tissues essentially require oxygen to generate energy and perform metabolic functions . Deficiency of oxygen or hypoxia results in failure to carry out these activities by the cells. Hypoxia is the most common cause of cell injury. Hypoxia may result from the following 2 ways : a. The most common mechanism of hypoxic cell injury is by reduced supply of blood to cells due to interruption i.e. ischaemia . b. Hypoxia may also result from impaired blood supply from causes other than interruption e.g. disorders of oxygen-carrying RBCs (e.g. anaemia , carbon monoxide poisoning ), heart diseases, lung diseases and increased demand of tissues . 2. Physical agents: P hysical agents in causation of disease are as under: i ) mechanical trauma (e.g. road accidents); ii) thermal trauma (e.g. by heat and cold); iii) electricity; iv) radiation (e.g. ultraviolet and ionising ); and v) rapid changes in atmospheric pressure. 3. Chemical agents and drugs : An ever-increasing list of chemical agents and drugs may cause cell injury. Impor tant examples include the following: i ) chemical poisons such as cyanide, arsenic, mercury; ii) strong acids and alkalis; iii) environmental pollutants; iv) insecticides and pesticides; v) oxygen at high concentrations; vi) hypertonic glucose and salt; vii) social agents such as alcohol and narcotic drugs; and viii) therapeutic administration of drugs

7. 4. Microbial agents : Injuries by microbes include infections caused by bacteria, rickettsiae , viru ses , fungi, protozoa, metazoa , and other parasites 5. Immunologic agents: Immunity is a ‘ double-edged sword ’—it protects the host against various injurious agents but it may also turn lethal and cause cell injury e.g. i ) hypersensitivity reactions; ii) anaphylactic reactions; and iii) autoimmune diseases. 6. Nutritional derangements: A deficiency or an excess of nutrients may result in nutritional imbalances. Nutritional defi ciency diseases may be due to overall defi ciency of nutrients (e.g. starvation), of protein calorie (e.g. marasmus (severe protein energy deficient) , of minerals (e.g. anaemia ), or of trace elements. Nutritional excess is a problem of affluent societies resulting in obesity, atherosclerosis, heart disease and hypertension . 7. Ageing : Cellular ageing or senescence leads to impaired ability of the cells to undergo replication and repair, and ultimately lead to cell death culminating in death of the individual.

8. 8. Psychogenic diseases : There are no specific biochemical or morphologic changes in common acquired mental diseases due to mental stress, strain, anxiety, overwork and frustration e.g. depression, schizophrenia. However, problems of drug addiction, alcoholism, and smoking r esult in various organic diseases such as liver damage, chronic bronchitis, lung cancer, peptic ulcer, hypertension, ischae mic heart disease etc. 9. Iatrogenic factors : Although as per Hippocratic oath, every physician is bound not to do or administer anything that causes harm to the patient, there are some diseases as well as deaths attributed to iatrogenic causes (owing to physician). Examples include occurrence of disease or death due to error in judgement by the physician and u ntoward effects of administered therapy (drugs, radiation). 10. Idiopathic diseases . Idiopathic means “of unknown cause”. Finally, although so much is known about the etiology of diseases, there still remain many diseases for which exact cause is undetermined. For example, most common form of hypertension (90%) is idiopathic (or essential) hypertension. Similarly, exact etiology of many cancers is still incompletely known. In a given situation, more than one of the above etiologic factors may be involved.

9. Pathogenesis of Cell Injury Injury to the normal cell by one or more of the above listed etiologic agents may result in a state of reversible or irreversible cell injury. The underlying alterations in biochemical systems of cells for reversible and irreversible cell injury by various agents are complex and varied. However, in general, irrespective of the type, following common scheme applies to most forms of cell injury by various agents : 1. Factors pertaining to etiologic agent and host : As mentioned above, factors pertaining to host cells and etiologic agent determine the outcome of cell injury: i ) Type, duration and severity of injurious agent: The extent of cellular injury depends upon type, duration and severity of the stimulus e.g. small dose of chemical toxin or short duration of ischaemia cause reversible cell injury while large dose of the same chemical agent or persistent ischaemia cause cell death. ii) Type, status and adaptability of target cell: The type of cell as regards its susceptibility to injury, its nutritional and metabolic status, and adaptation of the cell to hostile environment determine the extent of cell injury e.g. skeletal muscle can withstand hypoxic injury for long-time while cardiac muscle suffers irreversible cell injury after persistent ischaemia due to total coronary occlusion >20 minutes

10. Injury to the normal cell by one or more of the above listed etiologic agents may result in a state of reversible or irreversible cell injury. The underlying alterations in biochemical systems of cells for reversible and irreversible cell injury by various agents are complex and varied.

11. 2. Common underlying mechanisms: Irrespective of other factors, following essential intracellular biochemical phenomena underlie all forms of cell injury : i ) Mitochondrial damage causing ATP depletion ii) Cell membrane damage disturbing the metabolic and trans-membrane exchanges. iii) Release of toxic free radicals 3. Usual morphologic changes: Bio-chemical and molecular changes underlying cell injury from various agents become apparent first , and are associated with appearance of ultrastructural changes in the injured cell. However, eventually, gross and light microscopic changes in morphology of organ and cells appear . The morphologic changes of reversible cell injury (e.g. hydropic swelling) appear earlier while later morphologic alterations of cell death are seen (e.g. in myocardial infarction).

12. 4. Functional implications and disease outcome: Eventually, cell injury affects cellular function adversely which has bearing on the body. Consequently, clinical features in the form of symptoms and signs would appear . Further course or prognosis will depend upon the response to treatment versus the biologic behavior of disease. The interruption of blood supply (i.e. ischaemia ) and impaired oxygen supply to the tissues (i.e. hypoxia ) are most common form of cell injury in human beings. Pathogenesis of hypoxic and ischaemic cell injury is, therefore, described in detail below followed by brief discussion on pathogenesis of chemical and physical (principally ionising radiation) agents.

13. Pathogenesis of Hypoxia and Ischemia Cells Cellular Functios (Membrane transport, protein synthesis, lipid synthesis O 2 ATP ATP Firstly, by aerobic respiration or oxidative phosphorylation (which requires oxygen ) in the mitochondria. Secondly, cells may subsequently switch over to anaerobic glycolytic oxidation to maintain constant supply of ATP (in which ATP is generated from glucose/glycogen in the absence of oxygen). Derived from 2 sources Decreased generation of cellular ATP: Damage by ischaemia from interruption versus hypoxia from other causes All living cells require continuous supply of oxygen to produce ATP which is essentially required for a variety of cellular functions (e.g. membrane trans port, protein synthesis, lipid synthesis and phospholipid metabolism). ATP in human cell is derived from 2 sources: Firstly, by aerobic respiration or oxidative phosphorylation (which requires oxygen) in the mitochondria. Secondly, cells may subsequently switch over to anaerobic glycolytic oxidation to maintain constant supply of ATP (in which ATP is genera ted from glucose/glycogen in the absence of oxygen ). Ischemia due to interruption in blood supply as well as hypoxia from other causes limit the supply of oxygen to the cells, thus causing decreased ATP generation from ADP. Ischaemic cell injury also causes accumulation of metabolic waste products in the cells. In hypoxia from other causes (RBC disorders, heart disease, lung disease), anaerobic glycolytic ATP generation continues, and thus cell injury is less severe. In ischemia from interruption of blood supply , aerobic respiration as well as glucose availability are both compromised resulting in more severe and faster effects of cell injury. However, highly specialised cells such as myo cardium , proximal tubular cells of the kidney, and neurons of the CNS are dependent solely on aerobic respi ration for ATP generation and thus these tissues suff er from ill-eff ects of ischaemia more severely and rapidly.

14. 2. Intracellular lactic acidosis: Nuclear clumping Due to low oxygen supply to the cell , aerobic respiration by mitochondria fails first. This is followed by switch to anaerobic glycolytic pathway for the requirement of energy (i.e. ATP). This results in rapid depletion of glycogen and accumulation of lactic acid lowering the intracellular pH. Early fall in intracellular pH (i.e. intracellular lactic acidosis) results in clumping of nuclear chromatin 3. Damage to plasma membrane pumps: Hydropic swelling and other membrane changes 1. Lack of ATP and Membrane Repair: ATP is a molecule that provides energy for many cell processes, including making phospholipids , which are essential for repairing cell membranes . Without enough ATP , cells can't produce enough phospholipids , leading to damage in the plasma membrane pumps that regulate important ions like sodium, potassium, and calcium. Sodium-Potassium Pump Failure: The sodium-potassium pump (Na+-K+ ATPase) usually uses ATP to move sodium out of the cell and potassium into the cell. When there's not enough ATP , this pump can't work properly . As a result, sodium builds up inside the cell , and potassium leaks out . The extra sodium inside the cell causes water to enter the cell to balance things out, leading to cell swelling (called hydropic swelling). Calcium Pump Failure: Damage to the cell membrane can also disrupt the calcium pump, which normally controls calcium levels in the cell. When this pump fails, too much calcium enters the cell, especially into the mitochondria . The excess calcium causes the mitochondria to swell and can lead to deposits of phospholipid-rich materials inside the cell.

15. Pathogenesis of Hypoxia and Ischemia Cells Cellular Functions (Membrane transport, protein synthesis, lipid synthesis O 2 ATP ATP Firstly, by aerobic respiration or oxidative phosphorylation (which requires oxygen ) in the mitochondria. Secondly, cells may subsequently switch over to anaerobic glycolytic oxidation to maintain constant supply of ATP (in which ATP is generated from glucose/glycogen in the absence of oxygen). Derived from 2 sources Healthy Cells Hypoxia Ischemia Interruption of blood supply, aerobic respiration as well as glucose availability are both compromised Severe ATP depletion and Severe Injury Accumulation of toxins in the cells 1. Decreased generation of cellular ATP Interruption of aerobic respiration but anaerobic respiration and ATP generation continues Less severe injury 2. Intracellular lactic acidosis: Nuclear clumping Low O2 Supply Aerobic respiration fail Anaerobic respiration start to generate ATP Glycogen Lactic acid pH Clumping of Nuclear Chromatin

16. Healthy Cells Na+/K+ ATPase Ca+ Pumps Ischemia/Hypoxia Lack of ATP, Impaired functioning of Na+/K+ ATPase Na+ accumulation in the cells & K+ leakes /diffuses outside the cell. To balance excess sodium, water enters the cell causing cell swelling. Impairment of Ca+ channels, exxess Ca+ in the cell (mitochondria), accumulation of phospholipid rich materials and swelling 3. Damage to plasma membrane pumps: Hydropic swelling and other membrane changes

17. Healthy Cell Protein Synthesis Disperses in the cytoplasm and converts into monosomes Impaired protein synthesis Ischemia/Hypoxia RER Swells Ribosome detaches from RER, enters cytoplasm and converts to monosomes A polyribosome (or polysome or ergosome ) is a group of ribosomes bound to an mRNA molecule like “beads” on a “thread”. It consists of a complex of an mRNA molecule and two or more ribosomes that act to translate mRNA instructions into polypeptides. monosomes are a mix of mRNAs bound by a single ribosome Polysomes Polysomes Monosomes Protein Synthesis Impaired 4. Reduced protein synthesis: Dispersed ribosomes

18. Ultrastructural evidence of reversible cell membrane damage is seen in the form of : loss of microvilli, intramembranous particles and focal projections of the cytoplasm (blebs). Myelin figures may be seen lying in the cytoplasm or present outside the cell; these are derived from membranes (plasma or organellar) enclosing water and dissociated lipoproteins between the lamellae of injured membranes. Up to this point, withdrawal of acute stress that resulted in reversible cell injury can restore the cell to normal state

19. Irreversible Cell Injury Persistence of ischemia or hypoxia results in irreversible damage to the structure and function of the cell (cell death). The stage at which this point of no return or irreversibility is reached from reversible cell injury is unclear but the sequence of events is a continuation of reversibly injured cell . Two essential phenomena always distinguish irreversible from reversible cell injury A. Inability of the cell to reverse mitochondrial dysfunction on reperfusion or reoxygenation. B. Disturbance in cell membrane function in general, and in plasma membrane in particular . In addition, there is further reduction in ATP, continued depletion of proteins, reduced intracellular pH, and leakage of lysosomal enzymes into the plasma. Th ese biochemical changes have effects on the ultrastructural components of the cell

20. 1. Calcium influx: Mitochondrial damage: As a result of continued hypoxia , a large cytosolic influx of calcium ions occurs, especially after reperfusion of irreversibly injured cell. Excess intracellular calcium collects in the mitochondria disabling its function . Morphological changes are in the form of vacuoles in the mitochondria and deposits of amorphous calcium salts in the mitochondrial matrix 2. Activated phospholipases: Membrane damage: Damage to membrane function in general, and plasma membrane in particular, is the most important event in irreversible cell injury . Increased cytosolic influx of calcium in the cell acti vates endogenous phospholipases. These, in turn, degrade membrane phospholipids progressively which are the main constituent of the lipid bilayer membrane . Besides, there is also decreased replacement- synthesis of membrane phospholipids due to reduced ATP. Other lytic enzyme which is activated is ATPase which causes further depletion of ATP. Calcium Influx/Mitochondrial damage Hypoxia Ca+ influx Collects in mitochondria and impair its function Vacuoles in the mitochondria and deposits of amorphous calcium salts in the mitochondrial matrix 2. Activated Phospholipases: Membrane damage Most important event in irreversible cell injury Hypoxia Ca+ influx Activates endogenous phospholipases Degrade membrane phospholipids ATP Synthesis of membrane phospholipids ATPase+++ ATP

21. 3. Intracellular proteases: Cytoskeletal damage: The cytoskeleton is like the cell’s internal framework, made up of microfilaments, microtubules, and intermediate filaments. It helps maintain the cell’s shape and anchors the cell membrane. When a cell is damaged, certain enzymes called proteases can become activated inside the cell. These proteases break down the cytoskeleton, causing damage to the cell's structure. Additionally, if the cell swells due to injury, this physical change can also damage the cytoskeleton, leading to irreversible injury to the cell membrane. Injury 1. +++proteases Breakdown of microtubules, intermediate filaments and microfilaments 2. Cell Swelling Irreversible injury

22. 3. Nuclear Damage The nucleus, which holds the cell’s DNA, can also be damaged during cell injury. Enzymes like endonucleases and proteases can become activated and start breaking down DNA or nucleoproteins . Irreversible nuclear damage can happen in three main ways: Pyknosis: The nucleus condenses and clumps together, becoming darker and more basophilic (staining darkly with basic dyes). Karyorrhexis: The nucleus breaks apart into small fragments that scatter throughout the cytoplasm. Karyolysis : The nucleus completely dissolves and disappears. Damaged DNA activates proapoptotic proteins leading the cell to death.

23. 5. Lysosomal hydrolytic enzymes: Lysosomal damage, cell death and phagocytosis Lysosomes are small structures inside cells that contain powerful enzymes, like hydrolases, proteases, and DNases , which can break down different cell components. Normally, these enzymes are safely contained within the lysosomes. However, when a cell is injured, the membranes of the lysosomes can become damaged. This damage allows the enzymes to leak out into the cell, where they become activated due to the lack of oxygen and the acidic environment. Once activated, these enzymes start digesting the cell’s own components, leading to cell death. As the cell dies, it is replaced by structures called myelin figures. These are masses of phospholipids (fatty substances) that were part of the cell’s membranes. These myelin figures can be engulfed and digested by immune cells called macrophages, or they may form calcium deposits, known as calcium soaps. Lack of oxygen and acidic environment activates these enzymes Cytoplasm Injury/Hypoxia Damages membrane of lysosomes Leaks these enzymes Digest cell components and causes cell death Dead cell structures ger replaced with masses of phospholipids (fatty substances) called myelin figures myelin figures form calcium(deposits) soaps Myelin figures get Engulfed by phagocytes

24. Enzyme Markers in the Blood: The enzymes that leak out of the damaged cell can enter the bloodstream. Doctors can measure the levels of these enzymes in the blood to determine if cell death has occurred and to diagnose certain conditions. For example, after a heart attack (myocardial infarction), specific enzymes like SGOT (serum glutamic oxaloacetic transaminase) , LDH (lactic dehydrogenase) , CK-MB (creatine kinase-MB isoenzyme) , and cardiac troponins ( cTn ) increase in the blood, indicating damage to heart muscle cells. Ischemic-Reperfusion Injury: Cell damage from oxygen deprivation usually develops slowly, over minutes to hours. However, when blood supply is restored (reperfusion), the sudden return of oxygen can cause further injury. This is known as ischemic-reperfusion injury , where the reintroduced oxygen leads to the formation of harmful molecules called free radicals or reactive oxygen species (ROS) , which can further damage the cells. The enzymes that leak out of the damaged cell enter the bloodstream Doctors measure the levels of these enzymes in the blood to determine if cell death has occurred and to diagnose certain conditions.

25. Short term ischemia period Restoration of blood flow Outcome depends on how long they were deprived of O 2 Reversible cell injury Long term ischemia period Severe injury of cells, cannot recover, even if blood flow is restored. Irreversible Injury buildup of too much Na+ and Ca+ inside the cells because of ongoing damage to the cell membrane. Long term ischaemia restoring blood flow (reperfusion) can help the cells recover Intermediate ischemia period Cells are injured but viable But restoration of blood causes more harm This is called as reperfusion injury, common in conditions like (myocardial ischemia) or strokes (cerebral ischemia). Ischemia-Reperfusion Injury and Free Radical-Mediated Cell Injury

26. Ischemia-Reperfusion Injury and Free Radical-Mediated Cell Injury When a tissue or organ in the body is deprived of oxygen due to a lack of blood flow (ischemia), the cells within it can become injured. If blood flow is restored after a period of ischemia, the outcome for the cells depends on how long they were without oxygen. Let's break this down: 1. From Ischemia to Reversible Injury: Short Ischemia Period: If the cells were without oxygen for only a short time, restoring blood flow (reperfusion) can help the cells recover. The cells can return to their normal structure and function, which is called reversible cell injury . 2. From Ischemia to Irreversible Injury: Long Ischemia Period: If the cells were deprived of oxygen for too long, they might suffer irreversible damage even before blood flow is restored. In this case, the cells are so badly injured that they cannot recover, even if blood flow is restored. The cell death in this situation is not due to the formation of harmful molecules called free radicals but rather due to the buildup of too much sodium and calcium inside the cells because of ongoing damage to the cell membrane. 3. From Ischemia to Reperfusion Injury: Intermediate Ischemia Period: Sometimes, the cells are injured but still viable (alive) when blood flow is restored. However, instead of helping the cells recover, the return of blood flow can actually cause more harm and lead to cell death. This paradoxical worsening of cell injury is known as ischemia-reperfusion injury . This kind of injury is common in conditions like heart attacks (myocardial ischemia) or strokes (cerebral ischemia).

27. Reperfusion injury often occurs due to the excessive generation of free radicals—unstable molecules that can damage cells. Here’s how it happens: Calcium Overload: During reperfusion, too much calcium may enter the cells, leading to further cell damage and dysfunction. Excessive Generation of Free Radicals: When blood flow returns, it brings oxygen with it. The sudden influx of oxygen can lead to the production of harmful free radicals like superoxide, hydrogen peroxide (H2O2), and hydroxyl radicals. These molecules are highly reactive and can damage cell membranes, proteins, and DNA. Inflammatory Reaction: The presence of free radicals and cell damage triggers an inflammatory response, which can further injure the already weakened cells. Cell dysfunction Restoration of blood flow 1. Calcium Overload 3. Triggers inflammatory responses Severe injury weak cells Long term ischaemia 2. ROS Generation Cells Membrane, Proteins and DNA damage Excess blood brings more O 2 Too much Ca+ enters the cell production of harmful free radicals like superoxide, H2O2, and hydroxyl radicals. These are Highly reactive

28. 1. Calcium Overload Upon restoration of blood supply, the ischaemic cell is further bathed by the blood fluid that has more calcium ions at a time when the ATP stores of the cell are low . This results in further calcium overload on the already injured cells , triggering lipid peroxidation of the membrane causing further membrane damage. 2. Generation of Free Radicals While oxygen is essential for life, certain forms of oxygen, known as free radicals or reactive oxygen species (ROS) , can be harmful to cells. Free radicals are unstable molecules that have an unpaired electron in their outer orbit. Because of this, they are highly reactive and can easily damage cellular structures like proteins, lipids, and DNA. This causes Ca+ overload in injured cells Restoration of blood flow Long term ischaemia Lipid Peroxidation and Membrane Damage Ischemic cell bathed by blood fluid with more Ca+ Ions But ATP in cells are low

29. 2. Free Radicals and Their Role: Free radicals are unstable molecules formed during normal bodily processes or in response to harmful conditions. They have unpaired electrons, which makes them highly reactive. The body naturally forms and destroys these molecules, but if not properly managed, free radicals can cause significant damage to cells. Destruction of Free Radicals: Free radicals can be neutralized by specific enzymes in the body, such as: Superoxide Dismutase (SOD) Catalase Glutathione Peroxidase (GSH) These enzymes help break down free radicals, reducing their potential for harm. Oxidative Stress: If free radicals aren't broken down, they can cause oxidative stress, where they bind to and damage important molecules in cells. The hydroxyl radical is the most reactive and damaging of all the free radicals. Reactive Oxygen Species (ROS) is a term that refers to various types of oxygen-containing free radicals that can be harmful to cells. How Are Free Radicals Generated? Normal Cell Metabolism: In the cell, energy is produced through a process called oxidative phosphorylation, which occurs in the mitochondria (the cell’s powerhouses). During this process, oxygen (O2) is used to generate ATP , the energy currency of the cell. Redox Reaction: Oxygen combines with hydrogen atoms to eventually form water (H2O) in a series of steps. Each step involves the transfer of electrons, which is a part of the redox reaction . Normally, this process is tightly controlled, and oxygen is safely reduced to water without causing harm . Free Radicals neutralized by: SOD, Catalase, GSH Normal Bodily Reactions Or Injury ROS: Bind and damage cells If free radicals not broken down Results in Oxidative Stress Body forms and destroys these molecules, but if not managed causes significant harm to the cells Unstable, Highly reactive molecules with unpaired electron in there outer shell They breakdown free radicals and reduce there harm

30. Flowchart: Excessive Generation of Free Radicals Start The process begins with Normal Cell Metabolism . Oxygen Utilization in Mitochondria Oxygen (O₂) is used in the Mitochondria for energy production through Oxidative Phosphorylation . Redox Reaction In this process, Oxygen (O₂) combines with Hydrogen (H) atoms to eventually form Water (H₂O) . The process involves Four Steps of electron transfer, with each step potentially forming an intermediate molecule. Formation of Free Radicals Incomplete Electron Transfer can lead to the formation of the following reactive oxygen species (ROS): Superoxide Oxygen (O₂⁻) : Formed when one electron is transferred to oxygen. Hydrogen Peroxide (H₂O₂) : Formed when two electrons are transferred to oxygen. Hydroxyl Radical (OH⁻) : Formed when three electrons are transferred to oxygen. Cytochrome Oxidase Catalysis The enzyme Cytochrome Oxidase in the mitochondrial inner membrane catalyzes the reaction from O₂ to H₂O . Free Radical Accumulation If the production of free radicals exceeds the cell's antioxidant defenses, they Accumulate within the cell. Cell Damage Excessive free radicals can cause Cell Damage by attacking cellular components like: Proteins Lipids DNA Outcome If left unchecked, this can lead to Cell Injury or even Cell Death . Each step involves transfer of electrons (redox reactions) Energy is produced in the Mitochondria Via Oxidative Phosphorylation If the quantity of ROS exceeds the antioxidant system: causes cell damage Incomplete electron transfer causes formation of ROS In this process, O 2 is used to generate ATP O 2 combines with H to form H2O in series of step

31. Formation of Free Radicals During the process of converting oxygen to water , intermediate forms of oxygen can be created if the electron transfer is incomplete . These are the free radicals or reactive oxygen species (ROS ): Superoxide Oxygen (O₂⁻): This is formed when oxygen gains just one electron . It is the first step in the creation of reactive oxygen species. Hydrogen Peroxide (H₂O₂): When two electrons are transferred, hydrogen peroxide is formed. Although it is not a free radical itself, it can easily convert into harmful radicals in the presence of metal ions. Hydroxyl Radical (OH⁻): This is the most reactive and damaging type of free radical. It is formed when three electrons are transferred to oxygen.

32. 1. Superoxide Dismutase: How is it formed? Mitochondrial Electron Transport: During the process in which mitochondria (the cell’s power plants) produce energy, oxygen can sometimes turn into superoxide anion. Enzymatic Reactions: Certain enzymes, like xanthine oxidase and cytochrome P450, can also produce superoxide anion in both the mitochondria and the cytosol (the liquid part inside cells). 2. Hydrogen Peroxide: What is it? Hydrogen peroxide is another type of ROS, but it’s less reactive compared to superoxide anion and hydroxyl radical. How is it formed? From Superoxide Anion : Superoxide anion is converted into hydrogen peroxide by an enzyme called superoxide dismutase (SOD). How is it broken down? Into Water: Hydrogen peroxide can be turned into water by two enzymes: Catalase: Found in peroxisomes (small structures in cells that break down harmful substances). Glutathione Peroxidase (GSH): Found in both the cytosol and mitochondria, helps neutralize hydrogen peroxide into water . 3. Hydroxyl Radical: What is it? The hydroxyl radical is one of the most reactive and dangerous types of ROS. How is it formed? Radiolysis of Water: This is when water molecules are broken down by radiation, leading to the formation of hydroxyl radicals. Fenton Reaction: This is a chemical reaction where hydrogen peroxide reacts with ferrous ions (Fe²⁺, a form of iron) to produce hydroxyl radicals. Here’s how it happens: Iron Reaction: Normally, iron in cells is in the ferric form (Fe³⁺). The Fenton reaction reduces it to the ferrous form (Fe²⁺), which then reacts with hydrogen peroxide to produce hydroxyl radicals. H2O Catalase/GSH Most dangerous Radiolysis: H2O broken down by radiation

33. Other free radicals In addition to superoxide, H2O2 and hydroxyl radicals generated during conversion of O2 to H2O reaction, a few other free radicals active in the body are as follows: i ) Nitric oxide (NO) and peroxynitrite (ONOO): NO is a chemical mediator formed by various body cells (endothelial cells, neurons, macrophages etc ), and is also a free radical. NO can combine with superoxide and forms ONOO which is a highly reactive free radical. ii) Halide reagent (chlorine or chloride) r eleased in the leucocytes reacts with superoxide and forms hypochlorous acid ( HOCl ) which is a cytotoxic free radical. I ii) Exogenous sources of free radicals include some environmental agents such as tobacco and industrial pollutants.

34. How Free Radicals Cause Cell Damage: Lipid Peroxidation: What Happens? Free radicals attack the polyunsaturated fatty acids (PUFAs) in cell membranes , creating harmful molecules called lipid hydroperoxides. Result: This process, known as lipid peroxidation, spreads damage across the cell membrane, harming other parts of the cell. Oxidation of Proteins: What Happens? Free radicals can damage proteins in cells by causing changes in amino acids (the building blocks of proteins) and breaking protein chains. Result: This leads to protein degradation, weakening the cell's structure and function. DNA Damage: What Happens? Free radicals can break the strands of DNA in both the cell nucleus and mitochondria. Result: This causes cell injury and can potentially lead to the development of cancer. Cytoskeletal Damage: What Happens? Free radicals can also disrupt the cytoskeleton, the cell’s internal framework. Result: This interference can reduce ATP production (the cell's energy source), leading to cell death. ROS Attack Polyunsaturated fatty acids in cell membrane Forms lipid hydroperoxides---Cell membrane damage Changes amino acids, degrades proteins Breaks DNA stands in cell and mitochondria Dec. ATP , and cytoskeleton damage

35. 3. Subsequent Inflammatory Reaction Inflammatory Reaction After Ischemia-Reperfusion: What Happens During Ischemia-Reperfusion? When blood supply returns to a tissue after a period of reduced or no blood flow (ischemia), an inflammatory response often follows. White blood cells called neutrophils quickly enter the tissue and consume oxygen at a rapid rate, a process known as an oxygen burst . This sudden use of oxygen leads to the release of a large amount of free radicals, which are harmful molecules that can damage cells. Role of ATP Precursors: During ischemia, the precursors of ATP (the cell’s energy currency), like ADP and pyruvate , accumulate in the tissue. When blood flow is restored, these precursors contribute to the increased production of free radicals, adding to the cell damage. Stress Proteins in Cell Injury: What Are Stress Proteins? Stress proteins are special proteins that cells produce in response to stressful conditions, such as exposure to toxins, drugs, or lack of oxygen (ischemia). These proteins help protect the cell by managing and transporting other molecules within the cell. Blood to ischemic tissues Inflammation ATP Precursors (ADP and pyruvate) WBC (neutrophils) quickly enters the tissues Consumes O2 at rapid rate (oxygen burst) Large amount of free radicals Ischemic tissues Contributes Stress proteins Help to protect cells Release in response to stress HSP and Ubiquitin

36. Types of Stress Proteins: Heat Shock Proteins (HSPs): Normal Role: HSPs are found in most cells and usually act as molecular chaperones . They help with: Protein Folding: Ensuring that newly made proteins fold into the correct shape. Protein Transport: Moving proteins to the right places inside the cell. Disaggregation: Breaking down protein complexes that have clumped together. Response to Stress: When a cell is stressed (e.g., by toxins or ischemia), the levels of HSPs increase both inside the cell and in the bloodstream. Protective Role: HSPs can reduce tissue damage, such as limiting the extent of tissue death in heart attacks (myocardial infarction). They are also involved in managing protein aggregation in diseases like amyloidosis. Ubiquitin: What is Ubiquitin? Ubiquitin is another stress protein that is present in all human cells. Functions: Degradation: Ubiquitin helps mark damaged or unnecessary proteins for destruction. Synthesis: It also assists in the synthesis of new proteins. Role in Diseases: Ubiquitin is especially important in neurodegenerative diseases (e.g., Alzheimer's, Parkinson's) where it helps in managing the build-up of abnormal proteins in the brain. HSP These are carrier proteins Like HSPs, ubiquitin too directs intracellular molecules for either degradation or for synthesis. Act As Molecular chaperons (house keeping) they direct and guide metabolic molecules to the sites of metabolic activity In stress conditions thee levels elevate, in cells and they leak in plasma That is why they are called stress proteins Ubiquitin Have protective roles

37. CHEMICAL INJURY-PATHOGENESIS Chemical Injury: How Chemicals Harm Cells Two Ways Chemicals Can Cause Cell Injury: Direct Cytotoxic Effects: Some chemicals can directly damage cells without needing to be changed by the body. These chemicals typically harm the cells that are involved in processing or eliminating them. Example: Mercuric Chloride Poisoning: This chemical mainly harms cells in the digestive tract (where it is absorbed) and the kidneys (where it is excreted). Cyanide Poisoning: Cyanide directly harms cells by blocking an important enzyme in mitochondria (cytochrome oxidase), preventing the cell from producing energy (ATP). Conversion to Reactive Toxic Metabolites: Some chemicals need to be transformed by the body into a more harmful form before they can cause damage. The target cells (the ones that get damaged) might be different from the cells that actually transform the chemical. Example: Carbon Tetrachloride (CCl4): Used in the dry-cleaning industry in the past, CCl4 is converted by liver enzymes into a very harmful free radical (CCl3), which causes severe damage to liver cells. Acetaminophen: This common pain reliever can cause liver damage when it’s metabolized into toxic forms , especially in large doses.

38. 4. PHYSICAL INJURY PATHOGENESIS Physical Injury: How Physical Forces Harm Cells Types of Physical Injury: 1. Mechanical Force: Injuries caused by physical impact, such as a car accident or a fall, are significant not only medically but also legally. Severe physical trauma can lead to shock, a life-threatening condition where the body cannot maintain blood flow and oxygen delivery to vital organs. 2. Changes in Atmospheric Pressure: Rapid changes in pressure, such as those experienced by divers (decompression sickness), can cause significant injury. 3. Radiation Injury: How Radiation Harms Cells: Radiation (like X-rays) can cause injury by directly creating harmful molecules called hydroxyl radicals when it interacts with water in the body . These radicals are highly reactive and can damage cell membranes and DNA. Effects on Different Types of Cells: Proliferating Cells (cells that are actively dividing): Radiation can stop these cells from replicating their DNA, leading to cell death through a process called apoptosis (programmed cell death). This commonly affects cells like those in the skin or lining of the intestines. Non-Proliferating Cells (cells that are not actively dividing): These cells experience damage to their membranes from radiation, which leads to cell death through necrosis (uncontrolled cell death), as seen in neurons (nerve cells). Mechanical Force: Trauma and shock, body cannot maintain blood flow and O2 supply to vital organs X-Ray reacts with H2O in body to form OH-. This OH- damages DNA and cell membrane Proliferating cells are stopped from replicating there DNA, by apoptosis Non-Proliferating cells experience damage to cell membranes causing necrosis, Radiation Injury: