Unit 1 - Cell Degeneration and Death - Textbook PDF

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Unit One – Cell Degeneration and Cell Death Mechanisms of Cell Injury and Cell Death General Principals 

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The cellular response to injurious stimuli depends on the type of injury, its duration and severity. o Low doses of toxins or small period of Ischemia (restricted blood supply) may lead to reversible cell injury o Larger does and longer ischemic may result in irreversible injury and cell death Consequences of injurious stimulus depend on type, status, adaptability and generic makeup of the injured cell o Ex: Striated skeletal muscle in the leg can withgo 2-3 hours of ischemia without irreversible injury. Cardiac muscle can die about 20-30 mins o Ex: Glycogen-replete hepatocyte can survive ischemia better than one that has just burned its last glucose o Pharmacogenomics – understanding the role of genetic polymorphisms in response to drugs and toxins o Precision medicine: using genetic makeup to guide therapy Cell injury usually results from functional and biochemical abnormalities in one or more of a limited number of essential cellular components o Different insults and endogenous perturbations affect different organelles and biochemical pathways o Ex: Deprivation of oxygen or nutrients (hypoxia and ischemia) impairs energy-dependent cell functions (culminating in necrosis – premature death of cells). Damage to proteins and DNA trigger apoptosis. Any initiating trigger may activate one or more mechanisms

Hypoxia and Ischemia Deficiency of oxygen leads to failure of many energy-dependent pathways and death of cells by necrosis. Most frequent causes of cell injury and necrosis in clinical medicine. Background:   

Cellular ATP is produced from adenosine diphosphate (ADP) by oxidative phosphorylation during reduction of oxygen in the electron transport chain of mitochondria. ATP is required for membrane transport, protein synthesis, lipogenesis and diacylation-reacylation reactions necessary for phospholipid turnover A healthy human burns 50-75 kg of ATP every day

Cells that are subjected to stress of hypoxia but do not die activate mechanisms that are induced by transcription factors of hypoxia inducible factor 1 (HIF-1) family 

HIF-1 stimulated synthesis of proteins that help cell survive in low oxygen o Vascular endothelial growth factor (VEGF): a protein that stimulate the growth of new vessels to attempt to increase blood flow and supply of oxygen

Other proteins: cause adaptive changes in cellular metabolism by stimulating uptake of glucose and glycolysis and reducing mitochondrial oxidative phosphorylation. (ie less oxygen needed)  Anaerobic glycolysis can make ATP without oxygen from circulation or hydrolysis of intracellular glycogen  Liver and striated muscle: are more likely to survive hypoxia because they have greater glycolytic capacity (from presence of glycogen). Brain has limited glucose stores – would not be able to survive long periods of hypoxia Warburg effect: Rapidly proliferating normal cells and cancer cells rely on aerobic glycolysis to produce (A lot of) their energy. Metabolites generated by glycolysis and TCA cycle (kreb cycle) serve as precursors for the synthesis of cellular constituents (proteins, lipids and nucleic acids) needed for cell growth. o

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Persistent/sever hypoxia and ischemia can lead to failure of ATP generation and depletion of ATP in cells. This can any effects on cellular systems: 

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Reduced activity of plasma membrane ATP-dependent sodium pumps o Accumulation of sodium o Efflux of potassium o Cell swelling: A result of gain of water due to gain of solute. Dilates the ER Increase in anaerobic glycolysis o Lactic acid accumulation - Decreased pH o Decrease activity of cellular enzymes Structural disruption of the protein synthetic apparatus as a result of prolonged depletion of ATP o Detachment of ribosomes from rough ER o Dissociation of polysomes into monosomes – reduction in protein synthesis Increased reactive oxidative species (ROS) o Reperfusion injury: Hypoxia predisposes cells to ROS-mediate damage if blood flow/oxygen delivery is re-established Irreversible damage to mitochondrial and lysosomal membranes – cell undergoes necrosis (cell death) o Membrane damage – late in cell injury – caused by diverse mechanisms o Apoptosis by mitochondrial pathway contributes as well

Ischemia-Reperfusion Injury This restoration of blood flow to ischemic but viable tissues results in increased cell injury (especially in myocardial and cerebral ischemia). Normally results in the recovery of reversibly injured cells. Mechanisms accounting for exacerbation of cell injury from ischemic tissues: 

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New damage during reoxygenation by increased ROS. o ROS is generated by injured cells with damaged mitochondria o ROS is generated by infiltrating leukocytes Inflammation may increase o Enhances influx of leukocytes and plasma proteins o Complement proteins can bind to injured tissues or antibodies

Oxidative Stress Cellular abnormalities that are induced by ROS, which belong to a group of molecules known as free radicals.  

Free radical-mediated cell injury is seen in chemical and radiation injury, hypoxia, cellular aging, tissue injury caused by inflammatory cells and ischemia-reperfusion injury Free-radicals: chemical species with single unpaired electron in outer orbit. o Extremely unstable o Decay spontaneously o React with inorganic and organic molecules o When generated in cells, they attack nucleic acids and cellular proteins and lipids o Radical scavenges: nonenzymatic and enzymatic systems serving to inactivate free radicals o Initiate reactions in which molecules that react with free radicals are them-selves converted into other types of free-radicals – propagating a chain of damage

Generation and Removal of Reactive Oxygen Species The accumulation of ROS is determined by their rates of production and removal. Two major pathways:

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Produced normally in small amounts in all cells during reduction-oxidation (redox) reactions that occur during mitochondrial respiration. o Molecular oxygen is reduced in mitochondria to generate water – by addition of 4 electrons. Small amounts of highly reactive but short-lived toxic intermediates are generates when oxygen is reduced. They are:  Superoxide (O2 •)  Hydrogen peroxide (H2O2) (by enzyme superoxide dismutase (SOD))  Hydrogen peroxide is more stable and can cross biologic membranes.  Fenton reaction: Hydrogen peroxide  hydroxyl radical (•OH) in presence of metals (Fe2+)

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Produced in phagocytic leukocytes – mainly through neutrophils and macrophages (a weapon for destroying ingested microbes and other substances during inflammation and host defense)  Respiratory burst (oxidative burst): ROS are generated in phagosomes and phagolysosomes of leukocytes. Similar to mitochondrial respiration 1. Phagosome membrane enzyme catalyzes the generation of superoxide  H2O2 2. H2O2  hypochlorite (highly reactive – in bleach) by enzyme myeloperoxidase  Nitric oxide (NO) is also produced. React with O2 – to form peroxynitrite (highly reactive)

Generation of free radicals is increased by:    

Absorption of radiant energy (UV, x-rays). o Ionizing radiation can hydrolyze water into hydroxyl (•OH) and Hydrogen (H•) Enzymatic metabolism of exogenous chemicals (ex. Carbon tetrachloride) Inflammation – free radicals are produced by leukocytes Reperfusion of ischemic tissues

Mechanisms to remove free radicals minimizing their injurious effects  

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Rate of decay of superoxide – increased by superoxide dismutase (SOD) Glutathione (GSH) peroxidases is a family of enzymes who protect cells from oxidative damage o GSH peroxidase 1: found in cytoplasm. Breaks down H2O2 by 2GSH + H2O2 → GS-SG + 2H2O Catalase: One of most active enzymes known. Degrade millions of molecules of H2O2 per second by 2H2O2 → O2 + 2H2O

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Endogenous or exogenous anti-oxidants (Vitamins E, A and C and B-carotene) can block formation of scavenge them after they formed

Cell Injury Caused by Reactive Oxygen Species ROS cause cell injury by damaging multiple components of cells  

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Lipid peroxidation of membranes: Double bonds in membrane polyunsaturated lipids are attacked by oxygen-derived free radicals. Yield peroxides and autocatalytic chain reactions occurs. Crosslinking and other changes in proteins: o Free radicals promote sulfhydryl-mediated protein crosslinking resulting in loss of enzymatic activity. o Free radical can cause polypeptide fragmentation o Damaged proteins may fail to fold triggering unfolded protein response DNA damage: Free radical reactions with thymine produce single strand breaks. This can lead to apoptotic cell death, aging and malignant transformation of cells

Cell Injury Caused by Toxins Toxins such as environmental chemicals and substances produced by infectious pathogens, induce cell injury that results in necrotic cell death. Two mechanism that toxins induce in cell injury: 

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Direct-acting toxins: o Toxins combine with a critical molecular component or cellular organelle o Ex: Mercuric chloride poisoning (contaminate seafood) – Mercury binds with sulfhydryl groups of cell membrane proteins causing an inhibition of ATP and increased membrane permeability. o Ex: Anti-neoplastic chemotherapeutic agents induce cell damage by direct cytotoxic effects o Ex: Toxins made by microorganisms: target host cell molecules that are needed for essential functions. Ie protein synthesis and ion transport Latent Toxins

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Not intrinsically active and must first be converted to reactive metabolites which then act on target cells by direct covalent binding to proteins and lipids Accomplished by cytochrome P-450 in smooth ER of liver/other organs. Most important mechanism of cell injury is formation of free radicals  Carbon tetrachloride (CCL4) – once used in dry cleaning 1. Converted into a toxic free radical mostly in liver 2. Free radical causes injury by membrane phospholipid peroxidation 3. In 30 mins of exposure, sufficient damage to ER membranes of hepatocytes and a decline in synthesis of enzymes and plasma proteins 4. 2hours – selling of the smooth ER and dissociation of ribosomes from RER 5. Decreased of synthesis of apoproteins that form complexes with triglycerides – triglyceride secretion. Results in accumulation of lipids in hepatocytes and the “fatty liver” 6. Mitochondrial injury follows. Diminishing ATP and cell swelling 7. Cell membranes are damaged by fatty aldehydes by lipid peroxidation in ER 8. Cell death  Acetaminophen

Endoplasmic Reticulum Stress The accumulation of misfolded proteins in a cell can stress compensatory pathways in ER and lead to cell death by apoptosis   

Normally, chaperones control proper folding targeting misfolded polypeptides for proteolysis (breakdown of proteins or peptides) Unfolded protein response: Protective cellular response when misfolded proteins accumulate. Increase in chaperones and decreased protein translation Activate of proapoptotic sensors of the BH3-only family and direct activation of caspases lead to apoptosis by mitochondrial pathway when there are too many misfolded proteins

Accumulation of misfolded proteins may be caused by abnormalities that increase the production of misfolded proteins or reduce the ability to eliminate them.       

Caused by gene mutations Aging is associated with decreased capacity to correct misfolding Infections when large amounts of microbial proteins are synthesized within cells Increased demand of secretory proteins like insulin in insulin-resistant states Changes in pH and redox state Neurodegenerative diseases Deprivation of glucose and oxygen

Protein misfolding may cause disease by creating deficiency of essential protein or inducing apoptosis 

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Misfolded proteins lose their activity as they are degraded – can contribute to a loss of functions which ensues cell injury o Cystic fibrosis: inherited mutation in membrane transport protein that prevents normal folding Cell death o Alzheimer disease o Huntington disease o Parkinson disease o May underlie type 2 diabetes

Amyloidosis: Improperly folded proteins can accumulate in extracellular tissues DNA Damage

Exposure of cells to radiation or chemotherapeutic agents, intracellular generation of ROS and mutations may induce DNA damage – trigging apoptotic death 1. Damage is sensed by intracellular sentinel proteins which transmit signals that lead to accumulation of p53 protein 2. p53 arrests the cell cycle (@ G1 phase) to allow for DNA to be repaired before replicated. If damage is too great, p53 will trigger apoptosis by stimulation BH3-only sensor proteins 3. BH3-only sensor proteins activate Bax and Bak (proapoptotic members of Bcl-2 family)  Cancer: p53 is mutated or absent and cells that would normal go through apoptosis survive. DNA damage may result in mutations or DNA rearrangement (translocations) that lead to neoplastic transformation Inflammation Inflammatory cells, including neutrophils, macrophages, lymphocytes and leukocytes, secrete products that evolved to destroy microbes but may damage host tissues.  

Elected by pathogens, necrotic cells and dysregulated immune response (autoimmune diseases and allergies) Classified under hypersensitivity

Common Events in Cell Injury From Diverse Causes Abnormalities characterize cell injury: Mitochondrial Dysfunction:   

Mitochondria are sensitive to many types of injurious stimuli including hypoxia, chemical toxins and radiation. Changes occur in necrosis and apoptosis Biochemical abnormalities: o Failure of oxidative phosphorylation  depletion of ATP  culminating in necrosis o Failure of oxidative phosphorylation  formation of ROS  harmful effects o Formation of high-conductance channel in mitochondrial membrane (membrane permeability transition pore)  loss of membrane potential and pH changes  compromising oxidative phosphorylation o Cytochrome c (protein) releases into cytoplasm’s  apoptosis

Defects in Membrane Permeability   

Mitochondrial membrane damage: Decrease production of ATP  necrosis Plasma membrane damage: Loss of osmotic balance and influc of fluids and ions. Loss of cellular contents. Cells can leak metabolites – vital for ATP Membrane of lysosomes Damage: leakage of their enzymes into cytoplasm. Activate acid hydrolases in the acidic intracellular pH of injured cell (maybe ischemic). Lead to enzymatic digestion of cell components then necrosis

Cholestatic Syndromes

Hepatic bile serves two major functions: 1. The emulsification of dietary fat in the lumen of the gut through the detergent action of bile salts 2. The elimination of bilirubin, excess cholesterol, xenobiotics and other waster Processes that interfere with excretion of bile lead to jaundice and icterus due to retention of bilirubin and cholestasis Jaundice: Occur in settings of increase bilirubin product (ex. Extravascular red cell hemolysis), hepatocyte dysfunction (ex. hepatitis) or obstruction of the flow of bile (ex. Impacted gallstone). These all disrupt equilibrium between bilirubin production and clearance. *Important table*

Steps of the metabolism of bilirubin (serves to break down hemoglobin) by the liver: 1. 2. 3. 4.

Uptake from circulation Intracellular storage Conjugation with glucuronic acid Biliary Excretion

Bilirubin and Bile Formation Bilirubin: End product of heme degradation.

1. 85% of daily production is derived from breakdown of senescent red cells by macrophages in the spleen, liver and bone marrow. 15% is derived from turnover of hepatic heme or hemoproteins and destruction of red cell precursors in bone marrow 2. Intracellular heme oxygenase oxidizes heme to biliverdin (step 1 in figure) which is reduced to bilirubin by biliverdin reductase 3. Bilirubin is then released and binds to serum albumin (step 2). Note: This is critical since bilirubin is insoluble in aqueous solutions at physiologic pH and is highly toxic to tissues 4. Albumin carries bilirubin to liver and is then taken up into hepatocytes (step 3) 5. The conjugated with one or two molecules of glucuronic acid by bilirubin uridine diphosphate (UDP)-glucuronyltransferase (UGT1A1) in ER (Step 4) 6. Water-soluble, nontoxic bilirubin glucuronides are excreted into the bile. Most of these are deconjugated in gut lumen by bacterial B-glucuronidases and degraded to colourless urobilinogens (steps 5). 7. Excreted by feces or 20% are reabsorbed in ileum and colon returned to liver and re-excreted into bile. Small amount is reabsorbed in urine

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2/3 of organic materials in bile are bile salts – formed by bile acids with taurine or glycine o Bile acids: major catabolic products of cholesterol. Family of water-soluble sterols with carboxylated side chains. Highly effective detergents. Primary role is to solubilize water-insoluble lipids secreted by hepatocytes into bile and solubilize dietary lipids in gut lumen.  Cholic acids  Chenodeoxycholic acid

Enterohepatic circulation: 95% are reabsorbed from gut lumen and recirculate to liver Pathophysiology of Jaundice Both unconjugated and conjugated bilirubin (bilirubin glucuronides) accumulate systemically Unconjugated bilirubin is insoluble and tightly bound to albumin and cannot be excreted in urine. Very small amounts are present as free anion in plasma. If amounts rise, this unbound fraction may diffuse into tissue (brain of infants) and produce toxic injury 

Severe hemolytic disease: unbound plasma fraction increases or when protein-binding drugs displace bilirubin from albumin. o Erythroblastosis fetalis: hemolytic disease of newborn may lead to accumulation of unconjugated bilirubin in brain which causes neurologic damage – Kernicterus

Conjugated bilirubin: water-soluble, nontoxic and loosely bound to albumin (weak). This is excreted in urine Serum bilirubin levels vary between 0.3 – 1.2 mg/dL. Jaundice becomes evident when tehse rise to 2-2.5. Severe disease is 30-40 Defects in Hepatocellular Bilirubin Metabolism Neonatal Jaundice (or physiologic jaundice of the newborn):   

Conjugation and excreting bilirubin does not fully mature until 2 weeks of age therefore every newborn develops transient and mild unconjugated hyperbilirubinemia This is worsened by breastfeeding due to action of deconjugating enzymes in breast milk Sustained jaundice in newborn is abnormal

Hereditary Hyperbilirubinemias: 

May result from inborn errors of metabolism such as: o Gilbert syndrome: Common (7%) inherited condition that manifests as fluctuating unconjugated hyperbilirubinemia. Cause by decreased hepatic levels of glucuronosyltransferase from a mutated gene UGT1A1 (note: polymorphisms in the gene may play a role in the expression of this disorder). This syndrome is not associated with any morbidity (illness).  In contrast – Crigler-Najjar Syndrome Type 1: caused by severe glucuronosyltransferase deficiency that is fatal in infancy o Dubin-Johnson syndrome: From autosomal recessive defect in transport protein responsible for hepatocellular excretion of bilirubin glucuronides across canalicular membrane. Exhibits conjugated hyperbilirubinemia. Patients are normal expected for having a dark pigmented liver (from polymerized epinephrine metabolites – NOT bilirubin)

Intracellular Accumulations Cells can accumulate abnormal amounts of various substances and this can be harmful and cause varying degrees of injury. Can be located in cytoplasm, organelles (typically lysosomes) or nucleus and can be synthesized by the affected cells or produced elsewhere. Main pathways can be

1. inadequate remo...