Mechanism of Irreversible Cell Injury

Pathologic Cell Injury and Cell Death III – Mechanism of Cell Injury


Here we are, looking at irreversible cell injury now. We discussed reversible cell injury and 1 type of cell death, namely necrosis, earlier in the posts:

Mechanism of Reversible Cell Injury

Necrosis

Now, we will discuss the different ways by which cells can actually become injured, and the mechanism by which the cells become injured.

So we can start by asking: When does reversible cell injury become irreversible cell injury?

To answer this question, there are 3 key factors that must be understood:

1) The cellular response to an injurious stimulus depends on the severity, duration and nature of the injury. To explain this, if a small amount of a toxin is injected into an individual, the cells may be injured and undergo reversible injury, but since the toxin levels are low, the cell is able to recover once the toxin is cleared away. If the levels of toxins persist at a very high level, the cells will undergo very critical injuries, and may eventually die, resulting in irreversible cell injury and cell death.

2) The consequences of a cellular injury depends on the state, type, and adaptability of the cell. This means that the state of the cell before the injury is a critical factor in the development of an injury. It therefore refers to the vulnerability of the cell. For example, wouldn’t a cell that is already under stress be more likely to be easily pushed to irreversible injury? If a cell is already under nutritional deficiencies and ischemia, for example, then it is in an atrophied state, and will much more easily be pushed to cellular injury if the increased stress of another condition is added. Furthermore, the type of cell is also important in determining the consequence of a cellular injury. Imagine both striated muscles in the leg and cardiac muscles of the heart have undergone cell injury via ischemia, while the patient can simply relax the leg and allow the muscles of the leg to rest and recover from cellular injury, you cannot relax the cardiac muscles since they keep you alive. Furthermore, there are some toxins that when applied to two groups of cells, can produce no effect in one, and fatal effects in the other. An example of this is with addition of CCl4, carbon tetrachloride. The fact that some cells may be immune is likely due to the presence of enzymes that metabolize the toxin, in this case CCl4, not present in all cells due to polymorphisms (different forms of enzyme, that may not be functional) in other cells.

3) Cell Injury results from different biochemical mechanisms acting on several essential components.

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Here is an example of how different mechanisms of cellular injury trigger several different downstream pathways of cellular injury. These will all be explored in great detail below, have no fear. The important thing to remember is which cellular components are most easily damaged. During cellular injury, usually the mitochondria, cell membrane, machinery of protein synthesis and packaging, and DNA.  However, remember that a single cause of cell injury may intact, trigger several of these biochemical downstream mechanisms. 

Now, to answer the question, there are two features that characterize irreversibility from reversible injury.

Firstly, irreversible mitochondrial damage that results in ATP depletion and lack of oxidative phosphorylation despite removal of the injurious stimulus.

Secondly, profound disturbances in membrane function, especially those that affect internal ionic concentrations, and lysosomal enzyme activity, are almost always points of no return for the cell.

Cells are little, fragile things; they can be injured very easily by a variety of causes of cell injury, such as:

  1. Hypoxia/Ischemia
  2. Excess Heat or Cold
  3. Radiation
  4. Chemicals. drugs and toxins
  5. Infectious Agents
  6. Immunologic/Autoimmune
  7. Inflammation
  8. Nutrition
  9. Genetics

These causes listed above damage the cell by one or more of the following mechanisms of cell injury:

  1. Depletion of ATP
  2. Mitochondrial Damage
  3. Influx of Ca2+ and loss of Calcium Homeostasis
  4. Accumulation of Oxygen Derived Free Radicals (Oxidative Stress)
  5. Defects in Membrane Permeability
  6. Damage to DNA and Proteins

See if you can match which injury uses which mechanism! If you can’t just yet, then let’s dive into this interesting topic and see what we find out!

Depletion of ATP

You know what ATP is. It is so important, if you type “ATP” into your Google search, it’s the very fir-, wait what? It’s second right after professional tennis? …Well, alright, ATP is almost as important as tennis, which I guess is extremely important. I mean, look what you get when you image search ATP:

atp-ultimate-molecule

“Ultimate” is right, ATP is the energy currency of our body. When our body wants a process done, it pays in ATP. For example, if it wants to activate a transcription factor, it may phosphorylate an enzyme such as protein kinase A using ATP, which then acts on the transcription factor to activate it. It is also essential for several other processes such as protein synthesis,lipogenesis and membrane transport to name a few. Thus, it is essentially the cellular cash, except the consequence of having no cellular cash or ATP, is cell death. 

Quickly reviewing the physiology of ATP, we must remember that ATP is synthesized by 2 major methods:

  1. Oxidative Phosphorylation of ADP, done within the mitochondria by the reduction of oxygen using the electron transport chain.
  2. Glycolytic Pathway, which generates ATP without Oxygen, using Glucose derived from various methods, either body fluids or hydrolysis of glycogen.

ATP depletion and reduced ATP synthesis are associated with hypoxic and chemical (toxic) injury. 

Thus, the causes of ATP depletion would either be a reduced supply of oxygen and nutrients, mitochondrial damage and the actions of toxins (e.g. cyanide).

So when does ATP loss become life-threatening?

Generally, once ATP falls to a value of 5-10% of its original value, the ATP loss becomes extremely life-threatening. This is because several cellular systems are affected:

1) The very first effect is cellular swelling, discussed under Mechanism of Reversible Cellular Injury, linked above in the introduction. This occurs due to the inability of functioning of the Na+/K+-ATPase pump. As a result of this, Na+ accumulates within the cell and K+ moves out of the cell. This means the cell is no longer able to maintain internal ionic concentrations, and the point at which it is unable to do so is when the transition from reversible cell injury to irreversible cell injury is set in stone. Since water also follows Na+, it moves into the cell causing cellular swelling in the form of blebs and increased cell volume; and accumulates especially in the endoplasmic reticulum, causing ER swelling and vacuolar degeneration.

2) Alteration in cellular energy metabolism. Think about this: a process that disrupts either ATP synthesis or causes ATP depletion such as hypoxia, will result in a decrease of oxygen, and a consequent decrease in ATP production. If ATP synthesis decreases, but ATP must still be used, it makes sense to think that AMP concentrations are gradually increasing. Usually, ATP inhibits an enzyme known as phosphofructokinase-1, that is important in converting fructose-6-phosphate into fructose-1,6-bisphosphate, an extremely important step in glycolysis. Thus, we see that high ATP inhibits glycolysis. AMP however, reverses the inhibitory effect of ATP on phosphofructokinase-1, and thus promotes the formation of fructose-1,6-bisphosphate and promotes glycolysis. This means that there is a shift in energy source from oxidative phosphorylation to anaerobic glycolysis. This process promotes production of glucose for glycolysis by the breakdown of glycogen. Hence the body’s glycogen stores are rapidly decreased.

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Now recall that the end product of anaerobic respiration in animals is lactic acid. As this type of respiration is being promoted, glycogen stores and eventually fat stores and muscle proteins are broken down, all producing gradually higher amounts of lactic acid and inorganic phosphates. If allowed to build up, this acid eventually drops the intracellular pH to levels where cellular enzymes are not able to function with great efficiency.

3) As ATP driven pumps continue to gradually fail, eventually, a very important Ca2+ pump fails. Given that the intracellular concentration of calcium is 10^-7 moles and the extracellular concentration is 10^-3 moles (1000 times less – imagine the concentration gradient!), calcium rushes into the cell rapidly, causing several processes that are regulated by minute amounts of calcium to be completely disrupted. At this stage, the cell is beyond all viability.

4) Eventually, as ATP depletion continues, structural disruption begins to occur. This structural disruption is most pronounced in the apparatus of protein synthesis. These disruptions include detachment of ribosomes from the rough endoplasmic reticulum and dissociation of polysomes, ultimately resulting in a decrease in protein synthesis. 

5) Eventually, as ischemia and hypoxia continue, and the protein synthesis system is compromised, new proteins produced are often misfolded, and proteins that are present within the cell themselves become misfolded. This accumulation of misfolded proteins activate the ubiquitin-proteasome system, via an unfolded protein reaction. This is discussed under Waste Disposal in Cells. 

6) As the unfolded protein reaction destroys the proteins within the cell from the inside, lysosomal and mitochondrial membranes are irreversibly damaged (the consequence of which is discussed just below), and lysosomal enzymes and reactive oxygen species are unleashed within the cell, destroying the cell from the inside. Eventually, the cell undergoes necrosis; most often, coagulative necrosis, as discussed already under Necrosis, linked above.

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(courtesy Robins and Cotrans Pathologic Basis of Disease).

Mitochondrial Damage

Damage to mitochondria is one of the hallmarks of irreversible cell injury, and is usually a common feature of all injurious biochemical pathways that follow cell injury. We’ve seen it just above too! Mitochondrial damage occurs late in ATP depletion, and the cell dies shortly afterwards. Let us see why mitochondrial damage is so fatal to the cell.

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Mitochondrial damage is so dangerous to the cell, that the cell immediately activates stress signalling and triggers autophagy of the mitochondria (called mitophagy) so as to remove the dangerous, damaged mitochondria. If the mitochondria is allowed to remain within the cell, in a damaged state, intracellular dynamics are drastically altered.

We know why mitochondria are important. They are essential in oxidative phosphorylation, involving oxygen. Thus, without them, or due to damage to them, the cell isn’t efficiently able to produce ATP and will eventually die of ATP depletion, the mechanism of which is discussed above. However, there are other major consequences and methods by which a cell whose mitochondria is directly damaged, will die.

Before we go into that, let us consider what can damage the mitochondria directly. The mitochondria is damaged by:

  • Increases in cytosolic Calcium, coupled by an increase in inorganic phosphate and certain fatty acids.
    • High inorganic phosphate and fatty acids alone cannot damage the mitochondria but coupled with high Ca2+ are extremely damaging to a cell. Note that high Calcium alone can stilldamage mitochondria.
  • Reactive Oxygen Species
  • Oxygen Deprivation, either by Hypoxia or Ischemia
  • Defective Turnover of Mitochondrial Proteins

Thus, they are very vulnerable as they can be easily damaged by a variety of causes.

If the mitochondria are indeed damaged, then there are 3 major consequences:

1) Mitochondrial Permeability Transition.
Mitochondrial permeability transition is a phenomenon that is defined as an increase in the permeability of the mitochondrial membranes to freely allow entry of molecules less than 1500 Daltons in molecular weight. Usually the outer mitochondrial membrane contains porins, that, although allow movement of molecules up to 5000 Daltons, very tightly regulate the movement of molecules into the mitochondria. Because all molecules below 1500 Da cannot be regulated, there is a mitochondrial permeability transition (MPT). This transition is brought about by a high conductance channel, the mitochondrial permeability transition pore (MPTP).

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Notice how all solutes less than 1500 Da rush into and out of the cell in the above picture. The mitochondria slowly swells as the MPTP opens. The opening of the MPTP is trigerred primarily by an increase in intracellular calcium concentrations. This Ca2+ interacts with Ca2+ receptors on the matrix side of the MPTP, from within the mitochondria, and opens them (explained in the next section). This opening is done even faster in the presence of inorganic phosphates and certain fatty acids. Furthermore, the channel also opens when there is an abundance of reactive oxygen species, which will be explained later. The MPTP channel is closed in times of high NADH, ATP, ADP and high cations such as Mg2+, that can compete with Ca2+ for the MPTP receptors. 

When this occurs, since molecules can easily move in and out of the mitochondria along their concentration gradients, the mitochondrial membrane potential is lost and H+ ions and electrons are able to freely flow out of the mitochondria. This results in a loss of the action of the electron transport chain, and thus ATP production via oxidative phosphorylation is severely compromised. As ATP depletion occurs, the cell will eventually undergo necrosis.

Using this information, it is safe to assume that we can limit cellular injury by somehow stopping the opening of this pathologic MPTP. If we examine the structure of MPTP, we can appreciate that a protein named cyclophilin D is present in the structure of MPTP, that is crucial for the proper openng of the MPTP. This protein can be targeted by an immunosuppressive drug, cyclosporine. For example, in cases of ischemia, cyclosporine can act on cyclophilin D to reduce cellular damage initiated by the mitochondria.

2) Increase in Oxidative Stress
As a result of the opening of the MPTP channels, antioxidant molecules such as glutathione, which are typically stored in the mitochondria to combat reactive oxygen species, are now removed from the mitochondria and this allows reactive oxygen species to build up within the cell. Furthermore, improper oxidative phosphorylation by the compromised electron transport chain produces oxygen free radicals and reactive oxygen species. The mechanism by which these species destroy the cell is explained lower down.

3) Induction of Apoptosis
Apoptosis is a fancy way of saying “Cell Suicide.” It is when the cell realizes it is beyond any point of return, and destroys itself. As the mitochondria is being damaged, it begins to sequester between the inner and outer membrane, a number of pro-apoptotic proteins such as cytochrome c and proteins that indirectly activate apoptosis inducing enzymes known as caspases. Thus, as permeability increases, these proteins and pro-apoptotic molecules leak into the cytosol and trigger cell death by apoptosis.

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Influx of Calcium and Loss of Calcium Homeostasis

Calcium is a very tightly regulated control molecule. It is involved in the activation of a large number of molecules, and is an extremely important second messenger in the body. As mentioned already, the intracellular concentration of Calcium is extremely low, between 10^-7 and 10^-8 moles. In contrast the extracellular concentration of calcium is around 10^-3 moles, or to be more specific, 13^-3 moles.

Since several regulatory mechanisms are activated by calcium, it is extremely important to keep the concentrations of calcium within the cell at a low level. This is done by pumping calcium that enters the cell into the endoplasmic reticulum (sarcoplasmic reticulum in muscle cells) and mitochondria, to be stored as intracellular stores.

Ishcemia and toxins can initially increases intracellular Ca2+ by an increased release of Ca2+ from intracellular stores, but later due to the implications of reversible injury – namely the interruption of the Ca2+ pump, either by loss of ATP or by disruption of the electron transport chain. This means that calcium flows from the much higher concentration outside the cell to the much lower concentration inside the cell, increasing intracellular Ca2+ and triggering a number of effects:

1) High intracellular Ca2+ is sequestered and stored primarily by the endoplasmic reticulum, and to a lesser extend, the mitochondria. As intracellular Ca2+ rapidly increases, the need for storage by the mitochondria increases. Ca2+ is sequestered by the mitochondria through a mitochondrial Ca2+ uniporter (MCU), which facilitates transport of Ca2+ one way, inside the mitochondria. Usually the Ca2+ moves back into the cell and outside the mitochondria via a separate channel, by diffusing down the concentration gradient using Na+/Ca2+ and Cl-/Ca2+ exchangers, but as the intracellular Ca2+ concentration increases, Calcium does not leave the mitochondria as easily. Thus, Ca2+ concentration within the mitochondria gradually increases, until it is high enough to open the mitochondrial permeability transition pore (MPTP), and cause the cascade that results in severe mitochondrial damage, ATP depletion, reactive oxygen species formation and apoptosis, as discussed above.

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2) Activation of a number of Ca2+ dependent molecules. 
Remember, it is usually an increase in Ca2+ in the cell in moderate amounts that activates a vast number of proteins and molecules by triggering intracellular signaling. An example of this is the phospholipase C-trigerred Ca2+ release that promotes the activity of Protein Kinase C, a phosphorylating enzyme. Similarly, Ca2+ is involved in the stimulation or activation of phospholipases (which break down membrane phospholipids), proteases (which break down proteins), endonucleases (responsible for DNA and chromatin fragmentation) and ATPase (depletes ATP even further).

3) Induction of Apoptosis
Increase in intracellular Ca2+ directly activates caspases, which trigger apoptosis directly, and also by the increase in mitochondrial permeability (by MPTP activation), which causes the buildup of pro-apoptotic proteins such as cytochrome c, that promotes apoptosis, as explained in the above section

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Accumulation of Oxygen Derived Free Radicals

Free radicals are chemicals that have a single unpaired electron in an outer orbit. Cell injury induced by free radicals, particularly oxygen derived free radicals, is a very important mechanism of cell damage in many pathologic conditions. Here is how a free radical looks, below:

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Notice that in the diagram above, the “red” free radical has 7 electrons in its outer shell. This means that there are 3 pairs and 1 “lonely” electron. This one lonely electron wants another companion, and the molecule itself wants to feel completed, with no lonely electrons. Therefore, it begins to “attack” other molecules to try and steal an electron from them, to become complete. If the free radical steals an electron from the stable molecule, we say that it oxidizes the stable molecule, while the free radical itself is reduced. To remember this, remember OIL RIG – Oxidation is Loss, Reduction is Gain (of electrons). Thus, by stealing an electron, the free radical becomes reduced, while its victim is oxidized.

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You can appreciate what happens here. As one free radical attacks other stable molecules, they, in turn, become free radicals. This is because when an electron is “stolen” from a stable molecule, it in turn has one unpaired electron, or a lonely electron, since its partner was stolen. It’s like one of those revenge movies where the “hero” sets out on a path of destruction. Well, in this case, it is indeed quite destructive, producing a cascade that critically injures the cells by attacking essential proteins, lipids and carbohydrates and nucleic acids.

Reactive Oxygen Species (ROS) are a type of oxygen derived free radical. They are essentially chemically reactive substances containing oxygen, such as oxygen ions and peroxide ions. These ROS are produced in moderate amounts naturally during normal oxygen metabolism, such as during mitochondrial respiration and energy generation, but they are quickly removed by cellular defense mechanisms. Thus, cells can in fact maintain a transient state of low concentrations of ROS without damaging the cell. In fact, ROS actually have functional roles within the body, in cellular signaling, modulation of gene expression, activation of MAP kinases (involved in insulin signaling) and reversible gene modulations. However, an increase in ROS production, or a decrease in ROS scavenging leads to an excess of these oxygen derived free radicals, resulting in a condition known as oxidative stress.

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This is usually resisted by cellular defense mechanisms so as long as they are active. An example of a cellular defense mechanism has already been discussed – antioxidants, such as glutathione in mitochondria.

Antioxidants are, exactly as the name says, molecules that resist oxidative stress, or a buildup of oxygen derived free radicals, ROS. Antioxidants both resist the formation of free radicals and scavenge and remove free radicals when they have been formed. It does this by acting as a reducing agent and willingly entering an oxidized state. This is because it is in fact more stable in a state where it has lost an electron. Thus, it donates its electron to the free radical, converting it to a free radical and preventing the continuation of the cascade. It, itself does not become a free radical since it remains stable.

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Oxidative Stress is a serious condition, implicated in a very wide number of diseases. Look at all the diseases it is involved in below!

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So now that we know what free radicals are, and what diseases they are involved in, we can discuss how exactly these free radicals are formed, and the exact mechanisms by which they carry out their destructive cascade.

Generation of Free Radicals

Free radicals are generated very easily by the body, by a number of ways:

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1) Reduction-Oxidation reactions that occur in normal metabolic processes.
Remember that as part of normal oxidative phosphorylation, molecular oxygen (O2) is the final electron acceptor at the end of the electron transport chain. Oxygen itself as an atom has 6 electrons in its outer shell (3 pairs of electrons), and Hydrogen only has 1 electron to donate. Usually, 2 H molecules donate their 2 electrons to one Oxygen, and 2 H molecules to the other oxygen (because O2), to produce 2 molecules of water.

This conversion is usually catalyzed by a number of oxidative enzymes throughout the cell. During this conversion, intermediate oxygen-derived free radicals are produced based on incomplete donation of electrons to the oxygen. For example if only 1 electron is donated to the oxygen, then there are 3 pairs and 1 lonely electron, and the oxygen becomes a free radical called superoxide anion (O2-)􏰄 . Superoxide anions are in fact, the main method by which cells exert oxidative defenses that destroy pathogens, phagocytosed material and fragments of necrotized cells.
If 2 electrons are donated, 1 to each oxygen atom, then  hydrogen peroxide (H2O2) is formed.
If 3 electrons are donated, 2 to one oxygen atom, and 1 to another, then while one oxygen is stable, the other oxygen become a free radical bound to only 1 hydrogen atom, a hydroxyl ion radical (•OH). This •OH radical is in fact, the most reactive radical. Note that when H2O2 is produced in excess, it is converted to •OH by reaction with the superoxide anion. This is known as the Haber-Weiss Reaction.

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2) Absorption of Radiant Energy
Radiant energy, or radiation, includes X-rays and UV rays. Ionizing radiation is simply radiation that carries enough energy to liberate and remove electrons from atoms and molecules, thereby ionizing them and generating free radicals.  Ionizing radiation of this sort can hydrolyze water into •OH and H radicals.

3) Inflammation
During the normal process of inflammation, rapid bursts of ROS are actually produced in activated leukocytes as a mechanism of self-defense. This mechanism is a mechanism of inflammation, and will be explained when I talk about Inflammation. Some intracellular oxidases such as xanthase oxidase can produce free radicals, particularly, superoxide anion, (O2-). Furthermore, oxidases in peroxisomes particularly produce large amounts of H2O2.

4) Enzymatic Metabolism
The enzymatic metabolism of drugs and exogenous substances can produce free radicals that are not technically oxygen derived, and thus not ROS, but are still extremely dangerous. eg. CCl4, metabolized can form •CCl3).

5) Transition Metals
It is possible for transition metals such as Iron and Copper to contribute to the formation of free radicals. This is because they can very easily accept or donate electrons during chemical reactions. The most important example of this is the Fenton Reaction, that occurs between Fe2+ (Fe, hence Fenton Reaction) and H2O2, that produces •OH radicals.

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There is also a photo-Fenton reaction, used in industries since Fe3+ is more easily obtainable. This reaction involves using photons of light as catalysts, but the important factor to consider is the ultimate generation of the hydroxyl radical. The Fenton reaction usually is also promoted by the effects of the superoxide anion (O2-), and thus, high amounts of superoxide anion and Fe2+ promotes the formation of free radicals.

6) Nitric Oxide (NO)
This one just spells bad news. The chemical starts out as literally having the chemical symbol, “NO”. NO, a potent vasodilator, is produced by epithelial cells, macrophages, neurons and other cell types, and can individually act as a free radical. However, the main way in which NO exerts oxidative stress is by reaction with superoxide (O2-) to form peroxynitrite anion (ONOO-).

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Yep, NO produces ONOO- (Oh noooo!). You do not want NO being a free radical in your body, especially because the peroxynitrite anion radical attacks a wide variety of molecules, including DNA and proteins. Oh no!

Let us quickly talk about the most damaging of these radicals, the Hydroxyl Radical (•OH). The •OH radical is produced, in summary, by 3 mechanisms:

1) Radiolysis (via ionizing radiation) of water into •OH and H.

2) Fenton Reaction

3) Haber-Weiss Reaction.

Removal Of Free Radicals

Before we get into the mechanism of how free radicals are removed, and the body keeps safe from free radicals, remember that free radicals are very unstable, simply because they really want to find a partner for their lonely electron. Because they are so unstable, free radicals often decay spontaneously simply due to the extremely high amount of energy they carry, The decay free radicals undergo is called dismutation. 

Dismutation is a process whereby both the oxidized and reduced form of a species are produced simultaneously. For example, the superoxide anion (O2-)  usually decays to O2 and H2O2 in the presence of water. This is a dismutation. To explain this, first refer to the diagram below:

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Essentially, one superoxide anion donates its extra electron to another superoxide anion, as depicted above. Thus, the donator superoxide is oxidised (recall OIL RIG), and the receiver superoxide is reduced, becoming a peroxide ion (O2 2-). This peroxide anion is stabilized by reacting with 2 H+ ions, forming hydrogen peroxide. This explains the dismutation reaction.

Besides the normal dismutation of free radicals, the body also has natural defenses when the number of free radicals becomes elevated and uncontrollable by simple spontaneous decay. These methods include:

1) Antioxidants
Antioxidants have been explained above, so I can now be specific regarding which antioxidants are used within the body. As mentioned above, antioxidants either resist formation of free radicals entirely, or act as free radical scavengers that remove them after they have been formed. Commonly used antioxidants within the body are:

Vitamin E (alphatocepherol): This is a terminal electron acceptor, and thus reacts with free radicals to accept their extra electron, thereby blocking free radical chain reactions. Since this is a fat soluble vitamin, it mainly exerts its effects in the lipid membranes, protecting them from a phenomenon that will be explained below known as lipid peroxidation.
Vitamin C (ascorbate): This reacts directly with O2, •OH and other products of lipid peroxidation to remove them. It also serves an important role in Vitamin E regeneration, thereby preventing lipid peroxidation indirectly. Because it is water soluble, it cannot exist in the membranes and exists instead, in the cytosol.
Retinoid (precursors to Vitamin A): These are also lipid soluble, and function in similar ways to Vitamin E, acting as chain breaking antioxidants.
Glutathione: This is a reducing agent, and thus reduces or donates an electron to the free radical to make it stable. In this way, it is converted to glutathione disulphide (GSSG), as it is oxidised itself.
NO: I know we said that NO is bad, but being a potent vasodilator, it has its very important uses. It is generated as a product of an enzyme known as Nitric Oxide Synthase (NOS). NO can increase proteasomal activity, and thereby decrease cellular intake of Fe2+ by acting on the transferrin receptor, one of the receptors that intakes Fe2+, thus reducing the production of •OH by the Fenton reaction.

2) Transition Metals
It has already been mentioned that transition metals can easily form free radicals. In normal circumstances however, transition metals that are taken up and stored by being bound to a multitude of storage and transport proteins. These include Transferrin (transfers iron in blood plasma), Ferritin (stores iron in tissues), Lactoferrin (binds iron in milk) and Ceruloplasmin (stores Copper, and Iron to a lesser extent.) These transport proteins greatly reduce the availability of transport proteins, thereby preventing free radical production.

3) Enzyme Cascade
There is a cascade of 3 important enzymes that are especially important in acting against oxygen derived free radicals.

1) Superoxide Dismutase: We have already discussed dismutation of O2- above. That is exactly the reaction that this enzyme, Superoxide Dismutase (SOD) catalyses. Thus, SOD converts O2- into H2O2, via the reaction illustrated below:

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There are different variations of the SOD enzyme, each of which uses a different metal in their active site. Manganese associated SOD (Mn-SOD) is associated with the mitochondria. Copper and Zinc associated SOD (CuZn-SOD) is associated with the cytosol.

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2) Catalase: Catalase is located within peroxisomes, an organelle within the cytosol.

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Catalase is especially important for the removal of H2O2, which it does by decomposing it into water and oxygen via the reaction:

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3) Glutathione Peroxidase: 
We already mentioned the role of glutathione, as a reducing agent. When Glutathione (GSH) acts as a reducing agent, it, itself is oxidized to Glutathione Disulphate (GSSG). Glutathione peroxidase catalyses the general breakdown of free radicals H2O2 and •OH using glutathione. It reacts via two reactions:

H2O2 + 2GSH ——-> 2H2O + GSSG (Breakdown of Hydrogen Peroxide)

2 •OH + 2GSH ——-> 2H2O + GSSG (Breakdown of Hydroxyl Radical)

Notice how GSH is being converted to GSSG in the presence of radicals. Thus, the intracellular ratio of GSH:GSSG is a very important indicator of the number of free radicals within a cell. A high ratio of GSH:GSSG (high GSH) means low oxidative stress, while low GSH:GSSG (high GSSG) means high oxidative stress. 

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Pathologic Effects of Free Radicals

There are 3 major ways in which free radicals cause irreversible damage to cells:

1) Lipid Peroxidation In Membranes
Lipid peroxidation is the oxidative degradation of lipids. This is most cell damaging in the membranes. In this reaction, free radicals ‘steal’ electrons from the molecules in the membranes, causing the lipid itself to now become a lipid radical. It consists of initiation, propagation and termination. Lipid peroxidation is initiated by ROS, particularly, •OH, by reacting with polyunsaturated fatty acids within the membrane. Since ROS generate lipid radicals, which are highly unstable, these lipid radicals react with oxygen to form a peroxyl-fatty acid radical. These peroxides themselves are very unstable, and reacts with other lipids to produce a free radical and lipid peroxide. Thus, it forms a free radical chain reaction, and this step is known as propagation. Finally, termination occurs when two radicals react with each other, and cancel each other out. This is a termination reaction. Molecules that react with these radicals to terminate the reaction include Vitamin E and retinoids, as discussed before.

Typically, the end products of lipid peroxidation are reactive aldehydes, such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE). HNE in particular, is a marker of lipid peroxidation, and high concentrations of HNE indicate lipid peroxidation. If membranes, particularly membranes of the lysosome and outer cell membrane are damaged, then enzymes within the lysosome can completely destroy the cell, or ECF contents can destroy the cell and disrupt calcium homeostasis.

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2) Oxidative Modification of Proteins
We already know that free radicals want another electron, so they can be complete. They therefore go around stealing electrons from other molecules. A molecule that has an electron “stolen” has lost an electron, and thus becomes oxidized. In proteins, this is a real problem, as there are several important proteins that allow our cells to stay alive.

Thus, free radicals can promote the oxidation of amino acid side chains, oxidation of the protein backbone and the formation of new protein-protein bonds such as disulphide bonds, by donating the high energy to bond formation.

This oxidation of proteins may alter the active site, rendering the protein dysfunctional if it is an enzyme, and misfolding, raising proteasome activity and consequent protein breakdown. It also can disrupt structural proteins, resulting in a collapse of the structure of the cell, destroying the cell completely.

3) Lesions in DNA
Free radicals are capable of causing single- and double-strand breaks in DNA, cross-linking of DNA strands, and formation of adducts. This sort of disruption in DNA can cause malignancies, and is also implicated in cellular aging.

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(courtesy Robbins and Cotrans Pathologic Basis of Disease)

It is still important to note that free radicals, while mostly cause death by necrosis, can also cause cellular death by apoptosis. Taking O2- (superoxide anion) into special consideration, this free radical actually is known to activate several degradative enzymes, indicating that it may be involved in apoptosis.

Defects In Membrane Permeability

Early loss of selective membrane permeability is a common feature of necrosis, not apoptosis. Eventually, overt or wholesale damage to the membrane occurs. Also note that all cellular membranes may be damaged, including those in lysosomes, peroxisomes and the cell membrane of the cell.

Mechanism of Membrane Damage

Membranes are damaged in different ways, based on different pathologies. For example, during ischemia, membrane damage may occur due to ATP depletion and the activation of phospholipases due to disruption of Ca2+ homeostasis. The plasma membrane can also be damaged due to bacterial toxins, viral proteins, lytic complement components and a variety of physical and chemical components.

All these pathologies however, follow a similar biochemical mechanisms to disrupt the membrane:

Reactive Oxygen Species: These ROS carry out lipid peroxidation to damage the membrane, as explained above.

Decreased Phospholipid Synthesis: The production of phospholipids in cells may be disrupted due to hypoxia and ischemia, or defective mitochondrial function via damage to the mitochondria. This is because ATP depletion caused by these occurrences affects energy dependent biosynthetic pathways, including the synthesis of phospholipids. Thus, the production of membranes for a wide variety of organelles, including mitochondria themselves, is disrupted.

Increased Phospholipid Breakdown: As mentioned above, traumatic cell injury can disrupt Ca2+ homeostasis, raising intracellular Ca2+ and activating Ca2+ dependent phospholipases, that break down and degrade membranes. This sort of breakdown leads to the formation of lipid breakdown products. These lipid breakdown products include molecules such as unesterifed fatty acids, acyl carnitine, lysophospholipids, which have a detergent effect on membranes. This means that the membranes become more soluble in water, and are thus more easily disrupted. Furthermore, lipid breakdown products may also insert into the cell membrane themselves, altering membrane permeability and electrophysiologic alterations.

Cytoskeletal Abnormalities: Cytoskeletal filaments serve as anchors, connecting the plasma membrane to the cell interior.

image

Upon disruption of Ca2+ homeostasis and influx of Ca2+, Ca2+ dependent proteases are activated that destroy and break down cytoskeletal filaments. Furthermore, upon cellular swelling, the first sign of cell injury, the cytoskeletal filaments may burst or be torn from the membrane, causing cellular swelling to occur much more easily, and cell shape to be much more easily disrupted or ruptured.

image

Consequences of Membrane Damage

Answering this question is actually quite easy. Just think about some organelles whose membranes are very important, and think of what will happen without them. There are 3 that should come to your mind: The cell membrane itself, lysosomes and mitochondria.

Cell Membrane: As mentioned earlier, damage to cell membrane results in a loss of osmotic gradient, and disruption of ion channels that usually regulate internal ionic concentrations. Furthermore, damage to the cell membrane can eventually cause the loss of cell contents, as well as metabolites that usually allow ATP regeneration, resulting in energy depletion.

Lysosomes: We already know about how dangerous the lysosomal enzymes are. They are all acid hydrolyses, and contain numerous enzymes that destroy various components of the cell, such as Nucleases, proteases, lipases, phosphatases, DNases, RNases, just to name a few. Release of these enzymes can destroy the entire cell from the inside, and the cell dies by necrosis.

Mitochondrial Membrane: Mitochondrial membrane damage, as discussed above, activates the mitochondrial permeability transition pore (MPTP), which causes ATP depletion due to deceased ATP synthesis, and the trigger of apoptosis.

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Detection of Cell Injury

There is a very important technique of detecting, en masse, if a region of the body is undergoing irreversible cell injury and necrosis. Since membranes are damaged, tissue-specific proteins can leak into the extracellular fluid. For example:

Acute heart injury can be indicated by the presence of an isoenzyme located only in the heart, CPKMB (creatine kinase, MB variant), and contractile protein troponin.

Liver/Bile Duct Damage can be detected by the elevated presence of enzymes Alkaline Phosphatase (ALP) together with Gamma Glutamyltransferase (GGT). 

Damage to hepatocytes of the liver is indicated by increased concentrations of transaminases.

Damage to Proteins and DNA

Usually, cells have mechanisms to repair DNA after damage. However, if damage to DNA is too severe, by DNA damaging drugs, ROS or radiation, then the cell triggers a suicide program, apoptosis.

Similarly, if enough proteins are damaged and misfolded, then the cell also triggers apoptosis. Apoptosis will be explained in the next topic in Pathology I cover.


Finally, that took a long while to do. Hope you guys like it! I’ll do reperfusion injury and hypoxia in the next post, followed by Apoptosis. Thank you for your continued support, and I really hope this is helping at least some of you out there.

VIDEOS:

Dr. Rabiul Haque  (0:00 – 36:18)

Robbins Pathology AudioBook  (30:32)

QUESTION:

1) Absorption of radiant energy can cause radiolysis of water and produce dangerous radicals. Which of the following enzymes protect and prevent the dangerous action of radicals produced by radiolysis of water? 

A. Phospholipase

B. Glutathione Peroxidase

C. Endonuclease

D. Lactate Dehydrogenase

E. Superoxide Dismutase

ANSWER: B

One thought on “Mechanism of Irreversible Cell Injury

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