BIOCHEMICAL AND MOLECULAR MECHANISM OF CELL INJURY

BIOCHEMICAL AND MOLECULAR MECHANISM OF CELL INJURY

Mechanism of cell injury

• The cellular response to injurious stimuli depends on –

  • Type of injury

  • Its duration

  • Severity

• The consequences of cell injury depend on the type, state, & adaptability of the injured cell

• Cell injury results from biochemical and functional abnormalities in the several cellular components affecting the following

  • Aerobic respiration involving mitochondrial oxidative phosphorylation and production of ATP

  • Integrity of cell membrane on which homeostasis and integrity of cell and organelles depend

  • Protein synthesis

  • Cytoskeleton

  • Integrity of genetic apparatus of the cell

• DEPLETION OF ATP

• ATP is normally produced in two ways in mammalian cells i.e

  • By oxidative phosphorylation of Adenosine diphosphate in a reaction that results in reduction of oxygen by electron transfer system of mitochondria

  • In glycolytic pathway which can generate ATP in the absence of oxygen using glucose derived either from body fluids or from the hydrolysis of glycogen

• ATP is required for many synthetic & degradative processes which include

  • Membrane transport

  • Protein synthesis

  • Lipogenesis

  • Deacylation – reacylation reactions necessary for phospholipid turnover

• Depletion of ATP to less than 5% to 10% of normal levels leads to

  • Decrease in the activity of plasma membrane energy-dependent sodium pump

    • This leads to intracellular accumulation of sodium and efflux of potassium

    • Iso osmotic gain of water leads to swelling of cell and dilatation of the endoplasmic reticulum

  • Altered energy metabolism 

    • Due to ischemia oxidative phosphorylation decreases and there is an increase in anaerobic glycolysis

    • As a result glycogen stores decrease and there is accumulation of Lactic acid and inorganic phosphates due to hydrolysis of phosphate esters

    • This leads to decrease in pH, resulting in decreased activity of cellular enzymes

  • Failure of Ca²⁺ pump leads to influx of calcium which has damaging effects on numerous cellular components

  • Decrease in ATP leads to detachment of ribosomes from the rough endoplasmic reticulum and dissociation of polysomes into monosomes.

    • This leads to consequent reduction in the protein synthesis

    • Further there is irreversible damage to mitochondrial and lysosomal membranes and the cell undergoes necrosis

  • Depletion of oxygen and glucose leads proteins to get misfolded which triggers unfolded protein response that leads to cell death

• MITOCHONDRIAL DAMAGE 

  • Mitochondrial damage is caused by

    • Increased cytosolic calcium

    • Oxidative stress

    • Break down of phospholipids through phospholipase A₂ and sphingomyelin pathway

    • Lipid breakdown products such as free fatty acids and ceramide

  • Mitochondrial damage results in the formation of high-conductance channel, called as “membrane permeability transition” in the mitochondrial membrane

  • These channels are reversible in early stages but later becomes permanent if the damaging stimulus persists

  • This prevents maintenance of membrane potential of mitochondria which is important for oxidative phosphorylation

  • Membrane damage causes leakage of cytochrome c into the cytosol which triggers the apoptotic death pathway

 

• INFLUX OF INTRACELLULAR CALCIUM 

  • Cytoplasmic free calcium at very low level (< 0.1µmol) when compared to extracellular levels (1.3mmol)

  • In the cell calcium is sequestered in mitochondria and endoplasmic reticulum

  • Ischemia and certain toxins causes membrane damage leading to net influx of calcium into cell and release of calcium from mitochondria and endoplasmic reticulum into the cytoplasm

  • Increased calcium activates many enzymes which cause damage like

    • ATPases – hastening ATP depletion

    • Phospholipases – causing membrane damage

    • Proteases – breakdown of both membrane and cytoskeletal proteins

    • Endonucleases– DNA and chromatin fragmentation

  • Increased intracellular calcium results in increased mitochondrial permeability and induction of apoptosis

 

• ACCUMULATION OF FREE RADICALS

  • 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 proteins, lipids, carbohydrates in membranes & nucleic acid

  • Free radicals initiate autocatalytic reactions whereby molecules with which they react are themselves converted into free radicals to propagate the chain of damage  

  • Free radicals are generated by

    • The reduction –  oxidation reactions that occurs during normal metabolic process

      • Cells generate energy by reducing molecular oxygen to water

      • During this process, small amount of partially reduced reactive oxygen forms [i,e. Superoxide anion radical (O₂^–), Hydrogen peroxide (H₂O₂), and Hydroxyl ions (OH^–)] are produced in which different numbers of electrons have been transferred from O2

    • Absorption of radiant energy (UV rays , x-rays)- ionizing radiation can hydrolyze water into Hydroxyl (OH^–) and hydrogen (H) free radicals

    • Rapid bursts of Superoxide production occurs in the activated polymorphonuclear leukocytes during inflammation

    • Nitric oxide –

      • NO is produced by endothelial cells, macrophages, neurons and other cell types

      • NO can act as free radical and can also be converted into highly reactive peroxynitrite anion (ONOO^–), NO₂ and NO₃^–

    • Enzymatic metabolism of exogenous chemicals and drugs generate free radicals e.g. – CCl3 from CCl4

    • Reperfusion injury

    • Transition metals such as iron and copper donate or accept free electrons during intracellular reactions and catalyze free radical formation (Fenton reaction)

 

• • Cells have defense system to prevent free radical injury

    • Normally free radicals are neutralized by anti – oxidant enzymes and substances like

      • Superoxide dismutase – present in cytoplasm and produced by mitochondria

      • Catalase – produced by peroxisomes

      • Glutathione peroxidase – produced by mitochondria and present in cytoplasm

      • Vitamin C in cytoplasm

      • Ferritin and Ceruloplasmin present in cytoplasm – Levels of active forms of iron and copper are minimized by binding of ions to storage and transport proteins like transferrin, ferritin, ceruloplasmin there by minimizing the formation of hydroxyl ion

      • Vitamins E, A and β Carotene in the plasma membrane and cell membrane of organelles

    • An imbalance between free radical generating & radical scavenging systems results in “oxidative stress”

 

• • Fenton reaction

    • Oxygen is converted into superoxide (O₂^–) by oxidative enzymes in the endoplasmic reticulum, mitochondria, plasma membrane, peroxisomes and cytosol

    • Superoxide (O₂^–) is converted into Hydrogen peroxide by dismutation. Hydrogen peroxide is also derived from oxidases in peroxisomes

    • Hydrogen peroxide is converted into Hydroxyl by the Cu²⁺/ Fe²⁺ catalyzed Fenton reaction. This reaction is reversible and hydroxyl can be converted into hydrogen peroxide in the presence of Glutathione peroxidase

    • Intracellular free iron is in the Ferric state (Fe³⁺) and must be reduced to Ferrous form to participate in reaction

 

• • Effects of Free radicals are-

    • Lipid peroxidation of membrane

    • Oxidative modification of proteins

    • Lesions in DNA

  • Lipid peroxidation of membrane –

    • Double bonds in unsaturated fatty acids of membrane lipids are attacked by oxygen derived free radicals, particularly by OH

    • This produces peroxides which initiates the autocatalytic reaction

  • Oxidative modification of proteins

    • Free radicals promote oxidation of amino acid residue side chains, formation of protein-protein cross linkages (e.g. disulfide bonds ) & oxidation of protein back bone       protein fragmentation

    • Oxidative modification of proteins damage the active sites of enzymes, disrupt the conformation of structural proteins and enhance proteosomal degradation of unfolded and misfolded proteins

  • Lesions in DNA

    • Free radicals cause single and double stranded breaks in DNA, cross – linking of DNA strands and formation of adducts

    • Oxidative DNA damage has been implicated in cell ageing & in malignant transformation

• DEFECTS IN MEMBRANE PERMEABILITY AND MEMBRANE DAMAGE

  • Early loss of selective membrane permeability leading ultimately to overt membrane damage is a consistent feature of most forms of cell injury

  • Membrane damage may effect mitochondria, plasma membrane, & other cellular membranes

  • Mechanism of membrane damage

    • Free radicals or reactive oxygen species causes membrane damage by lipid peroxidation

    • Hypoxia causes decreased production of ATP by mitochondria which leads to decreased phospholipid synthesis in all cell membranes and energy dependent enzymatic activities

    • Increased cytosolic and mitochondrial calcium results in calcium-mediated activation of endogenous phospholipases which degrades membrane phospholipids

    • Increased cytosolic calcium activates proteases which damage cytoskeletal elements and cell membrane

  • Consequences of membrane damage

    • Plasma membrane damage

      • Loss of osmotic balance and influx of fluids and ions

      • Loss of cellular contents

    • Mitochondrial membrane damage

      • Opening of mitochondrial permeability transition pore leading to decreased ATP

      • Release of proteins that trigger apoptotic death

    • Lysosomal membrane damage

      • Leads to leakage of lysosomal enzymes into the cytoplasm

      • Activation of these enzymes leads to digestion of proteins, RNA, DNA and glycogen leading to cell death by necrosis

SUMMARY

References

• Kumar, Abbas and Fausto. Robbins and Cotrans Pathologic Basis of Disease. 8th edition

• Ramdas Nayak and Rakshatha Nayak. Exam preparatory manual for undergraduates Pathology. 3rd edition