What are the Causes of Cellular Injury?

CELLULAR PATHOLOGY

Etiology = what initiates a process, the cause of a disease.
Pathogenesis = what is its mechanism = pathophysiology
Morphology = how it is recognized
Functional Consequences = how it produces disease
Homeostasis = maintenance of a stable internal environment despite environmental variations.

An example of this syllogism:  hemmorhagic interventricular septum
Etiology: Occluded coronary artery, ischemia of myocardium
Pathogenesis: Free radical damage, necrosis, and weakening/rupture of septum
Morphology: Signs of MI (gross and microscopic) and hole in septum
Func conseq: CV collapse and death

Priniciples of Injury:
• Cells normally exist in a steady state
• Adaptation: achieve a new steady state, survival in the presence of the stimulus is now possible
• Examples – hypertrophy of cardiac mm cells b/c of incr work load; due to incr myofilaments and mitochondria
• Cells can reach a point where they can no longer adapt  injury
• Can be reveresible or irreversible (leads to cell death)

•The system that undergoes the primary damage depends on the injurious agent
• Damage in one system leads to damage in other systems
• Inhibition of aerobic respiration  depletion of ATP  no ptn synthesis
• Morphological changes develop more slowly than the biochemical derangements
• Microscopic evidence of cell death may not be apparent for 10-12 hrs
• Reaction to an injurious stimulus depends on its duration and severity
• Results of cell injury depend on the type of cell and its enviornment – neurons are more sensitive than skeletal mm cells

Causes of Cell Injury:

• Hypoxia – most common cause of cell death
• Both MI’s and strokes are ultimately due to hypoxia
• Results from:
1. Loss of blood supply due to blood loss, blockage of vessels, etc
2. Loss of osygen-carrying capacity due to anemia, CO poisoning
3. Intracellular blockage of oxidative phosphorylation by toxins (ex – cyanide)
• Physical Agents:
1. Direct mechanical trauma
2. Extremes of temperature
3. Radiation
4. Electrical shock
5. Changes in atm pressure

• Chemical agents
• Includes toxins, manufactured products, natural products, and therapeutic drugs
• Toxicity may depend on concentration

• Biological Agents – viruses, bacteria, parasites

• Immunologic Reactions
• Example – anaphylaxis, autoimmune diseases

• Genetic Derangements
• Point mutation of DNA – sickle cell anemia
• Inborn errors of metabolism
• Extra chromosomes – Down’s syndrome

• Nutritional Imbalances
• Deficiences and excesses of vitamins, minerals, ptn, kcal, etc

Cell injury and necrosis:
• Cellular response depends on type, duration, and severity
• Consequences of cell injury depend on the type, state, and adaptability of the injured cell
• Four intracellular systems are especially vulnerable:
1. maintenance of integrity of cell mbr
2. aerobic respiration
3. ptn synthesis
4. preservation of the intregrity of the genetic apparatus of the cell

General Biochemical Mechanisms:
• Particularly vulnerable are glycolysis, TCA cycle, and oxidative phosphorylation
• ATP depletion
• Tissues with greater glycolytic ability have an advantage when ATP levels are falling
• ATP depletion and decr ATP synthesis are common consequences of ischemic and toxic injury

• Oxygen/oxygen-derived free radicals
• Have production of reduced reactive oxygen forms, free radicals as by-products
• Cells have defense systems
• Oxidative stress: imbalance b/t free-radical generating and radical-scavenging systems

• Intracellular calcium and loss of calcium homeostatsis
• Ischemia and toxins cause early incr in cytosolic Ca concentration  activates enzymes
• Enzymes that are activated are:
• Phospholipases – damages cell mbr
• Proteases
• ATPases
• Endonucleases
• Defects in membrane permeability
• Irreversible mitochondrial damage
• Cells are dependent on mitochondria for ATP
• Can be damaged by:
• Incr of cytosolic Ca
• Oxidatve stress
• Breakdown on phospholipids t/ phospholipase A2 and sphingomyelin pathways
• Lipid breakdown products – ceramide and free fatty acids

• Mitochondrial Permeability Transition – in inner mitochondrial membrane; high-conductance channel
• Reversible in early stages
• Becomes permanent if stimuli persist  cell death
• Mitochondrial damage is associated with leakage of cytochrome c into the cytoplasm
• Can trigger apoptotic death p’ways in the cytosol

Hypoxic/Ischemic Injury:

• Hypoxia – glycolytic energy production can continue
• Ischemia – compromises the delivery of substrates for glycolysis, so anaerobic energy production stops
• Ischemia tends to injure tissues faster than hypoxia
• Ischemia  reversible cell injury  point of no return  irreversible cell injury
• When blood flow is restored: injury is often exacerbated  tissues lose more cells b/c of ischemia/reperfusion injury
• Let us summarize the drama of ischemic cell death? The drama varies from one tissue to another and with the type of injury, but for ischemic cell death of liver cells we can suggest the following, drawn from many sources. The reader should try to see it as a movie:

 Mitochondrial ATP production stops.
 The ATP-driven membrane ionic pumps run down.
 Sodium and water seep into the cell.
 The cell swells, and the plasma membrane is stretched.
 Glycolysis enables the cell to limp on for a while.
 The cell initiates a heat-shock (stress) response, which will probably not help if the ischemia persists.
 The pH drops.
 Calcium enters the cell.
 Calcium activates phospholipases, causing the cell membranes to lose phospholipid and producing lysophosphatides and fatty acids, both of which cause more membrane damage, initiating a vicious circle.
 Calcium activates proteases, damaging cytoskeletal structures; blebbing develops.
 Calcium activates ATPase, causing more loss of ATP.
 Calcium activates endonucleases, the nuclear chromatin forms clumps, some seeps out. Uric acid is released.
 Protein denaturation starts (calcium may be involved).
 All cell membranes are damaged.
 The ER and other organelles swell.
 The final blow is probably related to the massive inflow of calcium.

Reversible Cell Injury:
• First point of attack is the aerobic respiration – oxidative phosphorylation by the mitochondria  decr synthesis of ATP
• Depletion of ATP has many effects:


1. Decreased activity of Na/K pump in cell mbr • Na incr inside cell, K diffuses outside cell
• Influx of Ca into cell
• Net gain of solute  osmotic cell swelling and dilation of the ER
• Incr intracellular osmotic load b/c of accumulation of catabolites and net gain of solute

2. Switch to anaerobic glycolysis • Decr in glycogen stores, buildup of lactic acid and phosphates   decr pH of cell

3. Detachment of ribosomes • Detachment of ribosomes from ER and dissociation of polysomes  decr in ptn synthesis



• If ATP depletion gets worse:
• Cytoskeleton disintegrates  loss of ultrastructural features (ex – microvilli) and formation of blebs
• Myelin figures can be seen – Myelin figures are stacks of phospholipid bilayers in the shape of spheres, cylinders, and spirals that develop wherever cells are destroyed; derived from plasma and organellar membranes
• Mitochondria are swollen
• ER remains dilated
• Early chromatin clumping
• If oxygen is restored, all these changes are reversible

Irreversible Cell Injury:
• Hallmark of irreversibility:
1. Inability to reverse mitochonrial dysfunction
2. Profound problems in membrane fx – central factor in pathogenesis of irreversible cell injury
3. Nuclear changes are the light microscopist's hallmark for irreversible injury. PYKNOSIS is a shriveling and darkening of the nucleus attributed to very low pH. (RULE: If the nucleus is smaller and darker than a resting lymphocyte's, or is small and dark and shows no euchromatin-heterochromatin textures, that cell is very dead.) Other sure signs of cell death include KARYORRHEXIS, or fragmentation of the shriveled nucleus (into "NUCLEAR DUST"), and KARYOLYSIS, which simply means that nothing of the nucleus is visible any longer, except perhaps a purple haze.




Mitochondrial dysfunction • Incr of Ca and ATP depletion  mitochondria takes up more Ca  activates
    mitochondrial phospholipases accumulation of free fatty acids
• Causes changes such as mitochondrial permeablilty transtion
• Also causes changes in outer membrane
• Severe swelling of mitochondria
Loss of membrane phospholipids • Ca enters cells  activates phospholipases  phospholipid breakdown  incr in lipid breakdown products
• Extensive damge to plasma membranes; problems with permeabilility
• Massive influx of calcium into the cell occurs
• Continued loss of ptns, enzymes, conenzymes, and ribonucleic acids from the
     hyperpermeable membranes
Cytoskeletal problems • Activation of proteases by Ca  causes damage to cytoskeleton
• Can have detachemnt of cytoskeleton from mbr  mbr suceptible to stretching and tearing
Lipid breakdown products • Include free fatty acids, acyl carnitine, and lysophospholipids
• Act as detergents on membrane
• Insert into lipid bilayer
• Exchange with mebrane phospholipids
• Cause problems with permeability and retention of ions
Loss of intracellular amino acids • Glycine and other aa’s protect hypoxic cells from irreversible mbr damage in vitro
• Glycine helps ATP-depleted cells to resist the lethal effects of high Ca
• Loss of such aa’s that occurs in hypoxia predisposed to mbr injury
Swelling of lysosomes
• Injury to lysosomal membranes occurs  leakage of enzymes into the cytoplasm  activation of acid hydrolases
• Lysosomes contain RNAases, DNAases, proteases, phosphatases, glucosidases, and cathespins
• Have enzymatic degradation of cell components
Large, flocculent, amorphous densities develop in the mitochondrial matrix

• Early fall in pH is followed by a shift to neutral or alkaline pH
• Acidosis protects against lethal injury in many ischemia models and reperfusion
• Probably as a result in the inhibitory effect of low pH on enzymatic reactions

After cell death:
• Cell components are degraded
• Widespread leakage of cellular enzymes into x-cell space
• Dead cells become replaced by large masses composed of phospholipids in the form of myelin figures  phagocytosed or degraded into fatty acids
• Calcification of fatty acids can occur

Reperfusion:
• Cells may die after reperfusion, by both necrosis and apoptosis
• May be b/c cells are structurally intact, but are biochemically compromised and lose integrity during reperfusion
• New damaging processes are set in motion during reperfusion

Reactive oxygen species
• Incr in reactive oxygen species during reperfusion
• From parenchyma and endothelial cells; also infiltrating leukocytes
• Cellular anti-oxidant defenses may have damaged during ischemia, and therefore be more damaged during reperfusion

Mitochondrial permeablility transition • Can be further promoted by reactive oxygen species

Additional inflammation • Caused by cytokines and incr expression of adhesion molecules by hypoxic cells
• Recruits PMN’s to reperfused tissues  inflammation  injury


Free-Radical Induced Cell Injury:

4) Free Radicals = have single unpaired electron in outer orbit, thus extra –ve charge
Reactive Oxygen Species (ROS) = Superoxide (O2-), Hydrogen Peroxide H2O2, Hydroxyl ion OH-
Reactive Nitrogen Species = Peroxynitrite

How, in a living tissue, can any atom or molecule find itself limited to a single electron in an outer orbit? This accident can occur in several ways.
• Energy supplied by the environment can split the covalent bond between two atoms in such a way that one electron remains attached to either side (homolytic fission). This is what happens when tissues are irradiated: water molecules are split into free radicals (radiolysis) which account for much of the tissue damage. The energy of sunlight does something similar to the skin (photolysis).
• Even without the application of external energy, susceptible atoms can capture an electron; for instance, an electron that strays off the electron transport chain in a mitochondrion. Normally, the electron-transfer reactions are carried out by enzymes that are firmly embedded and properly ordered in lipid membranes, so that the production of free radicals is minimized; but this order can be broken. Under normal conditions, about 1 percent of the electrons that pass along the transport chain stray away and react with molecular oxygen; fortunately, the resulting free radical is captured by defensive free-radical scavengers and damage is avoided. The rate of this electron escape is directly proportional to the partial pressure of oxygen. In a person breathing 100 percent O2 the mitochondria of the pulmonary alveolar cells may produce five times the usual amount of free radicals, too much for the scavenger mechanisms to absorb.
• Several oxidative enzymes can produce free radicals, in some cases because the substrate diffuses away from the enzyme surface before it is wholly oxidized or reduced to an even electron number.


Sources of Free Radicals


• May be initiated w/in cell by:
• Absorption of radiant energy – UV light, x-rays
• Enzymatic metabolism of exogenous chemicals or drugs – carbon tetrachloride
• Reduction-oxidation rxns
• Can produce superoxide anion radical (O2-), hydrogen peroxide (H2O2), and hydroxyl ions (OH-)
• Rapid bursts of superoxide production occur in activated PMN’s  - uses NADPH oxidase
• Some intracellular oxidases generate superoxide radicals
• Transition metals can donate or accept free electrons
• Reduction by Fe can be enhanced by superoxide
• Sources of iron and superoxide are required for maximal oxidative damage
• Nitric Oxide can act as a free radical; can be converted to more reactive forms

•Effects of reactive species:
• Lipid peroxidation of membranes
• Double bonds in unsaturated fatty acids in mbr lipids are attacked  yields peroxides
• Autocatalytic chain propagates  extensive damage
                                                     ROS + Lipids = Lipid Peroxidases + Lipids = More Lipid Peroxidases
• Termination can occur if free radical is captured by a scavenger in the cell mbr (Vit E)
• Oxidative modification of ptns
• Free radicals promote oxidation of amino acid side chains and ptn backbone  ptn fragmentation
• Lesion in DNA
• Free radicals react with thymine to produce single-stranded breaks in DNA

• Cell defenses:
• Free radicals are unstable and decay spontaneously
• Antioxidants – block initiation or inactivate free radicals; terminate radical damage (Vitamin E, Vitamin C,  Retinoids, etc)
• Minimize levels of iron and copper by binding ions to ptns (transferrin, ferritin, etc)
             • Enzymes can act as free-radical-scavenging systems (Detoxifying Enzymes)
            • Superoxide dimutases – both in mitochondria and cytosol; 2 O2- + 2 H+  O2 + H2O2
• Catalase – in peroxisomes; 2 H2O2  2 H2O + O2
• Glutathione peroxidase – both in mitochondria and cytosol; H2O2 + 2 GSH  2 H2O + GSSG

Chemical Injury
• Chemicals can act directly by binding to critical molecular comonent or cellular organelle
• Mercuric chloride poisoning
• Cyanide – poisons mitochondrial cytocrome oxidase
• Antineoplastic drugs

• Must be converted into reactive toxic metabolites
• Conversion is done via a P450 mixed function oxidase in the SER of the liver and other organs
• Metabolites can cause damage by direct covalent bonding
• Can form free radicals  lipid preoxidation

Carbon Tetrachloride Induced Liver Injury:
• CCl4 used in dry cleaning
• Toxic effect due to conversion by P450 in the SER: CCl4   CCl3 + Cl-
• CCl3 is a highly reactive toxic free radical  lipid peroxidation  propagation along microsomal membrane
• In the hepatocyte, this can lead to attach of PUFA’s  forms an organic radical
• Organic radical can meet with O2 to form an organic lipid peroxide radical
• Rapid breakdown of structure and fx of the ER

• Can form malondialdehyde from lipid peroxide
• Can form lipid-lipid, lipid-ptn, or ptn-ptn crosslinks

• Free radicals can attach ptns  disulfide crosslinking, ptn-ptn crosslinking, and ptn scission

• Net result is damage to the integrity of the membranes
• Have dissociation of polyribosomes from the ER and dissociation of the polyribosomes  decr ptn synthesis
• Inhibits secretion of TG b/c no apoproteins
• Hepatocyte accumulates large droplets of TG in its cytosol

• Have damage to mitochondria  decr ATP synthesis
• Also have damage to plasma mbr  changes in ionic homeostatsis  cell death

Acetominophen:
• Detoxified in the liver via sulfation and glucuronidation
• Small amount are converted via P450 to highly toxic metabolite  detoxified by interactions w/ GSH
• Large doses of drug ingested  GSH depleted  covalent binding of toxic metabolites  incr drug toxicity  liver cell necrosis
• Hepatotoxicity correlates with lipid peroxidation, can be reduced by administration of antibiotics

Apoptosis:
• Occurs in:
• Tissue remodeling during development
• Negative selection of B and T cells
• In response to loss of hormonal stimulation – postmenopausal, etc
• In cancer cells, spontaneously and in response to radiation or chemo
• In viral infections
• During atrophy of tissues
• Death of neutrophils during an acute inflammatory response
• Death induced by Tc cells
• Cell deletion in proliferating cell populations – intestinal crypts
• Pathologic atrophy after duct obstruction – pancreas, parotid, etc
• In response to injurious stimuli

• Morphology:
• Cell shrinkage; surface convolutions
• Chromatin condensation
• Formation of cytoplasmic blebls and apoptotic bodies
• Phagocytosis of apoptotic cells by adjacent paraenchymal cells and macrophages

• Biochemical Features
• Ptn cleavage – involves the activation of a family of cysteine proteases named caspases
• Caspases cleave nuclear scaffold and cytoskeletal ptns and triggers endonucleases
• Extensive ptn cross-linking by transglutaminase activation
• Breakdown of DNA into large pieces  interneucleosomal cleavage into smaller pieces by Ca and Mg endonucleases
• Express phosphatidylserine and/or thrombospondin in outer layers of cell mbr for recognition by macrophages

• Initiators of Apoptosis:
• Variety of signals
• Lack of growth factor or hormone
• Positive ligand-receptor interaction
• Specific injurious agents

• Signaling Pathways
• Transmembrane signals – can be negative or positive
  • FAS ligand on cytotoxic T cells – can bind to FAS receptors on target cells and trigger apoptosis
• Important in clonal selection; may play a role in killing virus-infected cells
• Ligand binding of plasma mbr receptors of the TNF superfamily

• Intracellular perturbation
• Ionizing radiation  DNA damage  apoptosis
• p53 protein
• Altered in cancers, loss of fx is important for malignant transformation
• c-myc protein
• Fx’s in cell cycle
• Overexpressed  apoptosis only in absence of an exogenous growth factor

• Certain viral proteins and bacterial toxins
• gp120 protein
• Can induce apoptosis of CD4+ T cells; imp in HIV infection

• Modulators of Apoptosis
• Specific ptns that connect death signals to execution
• bcl-2 protein – in outer mitochondrial membrane, ER, and nuclear mbr
• Altered in malignant tumors, plays a role in preventing apotosis

• bcl-x, bax, bak, and other related molecules
• Regulate apoptosis t/ dimerization reactions with other bcl-2 family members

• APAF-1
• Binds to cytochrome c and promotes apoptosis and activiting caspase 3
• Apoptotic signals results in mitochondrial permeability transitions  mitoch swelling
• Also cause incr permeability of outer mitoch mbr  release cytochrome c into cytosol
• Cytochrome c release precedes the morphologic changes of apoptosis

• Effectors of Apoptosis
• Caspases play a key role – cleave a variety of substrates
• Caspase 3  activates a cytoplasmic endonuclease  cleaves DNA b/t nucleosomes
• Gives rise to DNA ladder seen on gels of DNA from apoptotic cells
• Present new cell surface molecules – i.e. thrombospondin (facilitates phagocytosis)
• Transglutaminase activity  cross-linkage of cytoplasmic ptns
• Induced during apoptosis, may lead to incr in eosinophilia of cytoplasm

Induction of SER:
• Protracted use of barbituates  hypertrophy of the SER of hepatocytes
• Enzyme modification of barbituates uses the P-450 mixed function oxidase system
• P-450 mixed oxidase system:
• Incr solubility of a variety of compounds
• Facilatate their secretion
• Many compounds renedered more injurious by the P450 system; forms many reactive oxygen species