Cell Injury

Now that we have discussed the causes of cell injury and the morphologic changes in necrosis, we next consider in more detail the molecular basis of cell injury, and then illustrate the important principles with a few selected examples of common types of injury.

The biochemical mechanisms linking any given injury with the resulting cellular and tissue manifestations are complex, interconnected, and tightly interwoven with many intracellular metabolic pathways. Nevertheless, several general principles are relevant to most forms of cell injury:

• The cellular response to injurious stimuli depends on the type of injury, its duration, and its severity. Thus, low doses of toxins or a brief duration of ischemia may lead to reversible cell injury, whereas larger toxin doses or longer ischemic intervals may result in irreversible injury and cell death.
• The consequences of an injurious stimulus depend on the type, status, adaptability, and genetic makeup of the injured cell. The same injury has vastly different outcomes depending on the cell type; thus, striated skeletal muscle in the leg accommodates complete ischemia for 2 to 3 hours without irreversible injury, whereas cardiac muscle dies after only 20 to 30 minutes. The nutritional (or hormonal) status can also be important; clearly, a glycogen-replete hepatocyte will tolerate ischemia much better than one that has just burned its last glucose molecule. Genetically determined diversity in metabolic pathways can contribute to differences in responses to injurious stimuli. For instance, when exposed to the same dose of a toxin, individuals who inherit variants in genes encoding cytochrome P-450 may catabolize the toxin at different rates, leading to different outcomes. Much effort is now directed toward understanding the role of genetic polymorphisms in responses to drugs and toxins. The study of such interactions is called pharmacogenomics. In fact, genetic variations influence susceptibility to many complex diseases as well as responsiveness to various therapeutic agents. Using the genetic makeup of the individual patient to guide therapy is one example of “personalized medicine.”
• Cell injury results from functional and biochemical abnormalities in one or more of several essential cellular components (Fig. 1–14). The principal targets and biochemical mechanisms of cell injury are: (1) mitochondria and their ability to generate ATP and ROS under pathologic conditions; (2) disturbance in calcium homeostasis; (3) damage to cellular (plasma and lysosomal) membranes; and (4) damage to DNA and misfolding of proteins.
• Multiple biochemical alterations may be triggered by any one injurious insult. It is therefore difficult to assign any one mechanism to a particular insult or clinical situation in which cell injury is prominent. For this reason, therapies that target individual mechanisms of cell injury may not be effective.

Figure 1–14 The principal biochemical mechanisms and sites of damage in cell injury. ATP, adenosine…

With this background, we can briefly discuss the major biochemical mechanisms of cell injury.

Depletion of ATP

ATP, the energy store of cells, is produced mainly by oxidative phosphorylation of adenosine diphosphate (ADP) during reduction of oxygen in the electron transport system of mitochondria. In addition, the glycolytic pathway can generate ATP in the absence of oxygen using glucose derived either from the circulation or from the hydrolysis of intracellular glycogen. The major causes of ATP depletion are reduced supply of oxygen and nutrients, mitochondrial damage, and the actions of some toxins (e.g., cyanide). Tissues with a greater glycolytic capacity (e.g., the liver) are able to survive loss of oxygen and decreased oxidative phosphorylation better than are tissues with limited capacity for glycolysis (e.g., the brain). High-energy phosphate in the form of ATP is required for virtually all synthetic and degradative processes within the cell, including membrane transport, protein synthesis, lipogenesis, and the deacylation-reacylation reactions necessary for phospholipid turnover. It is estimated that in total, the cells of a healthy human burn 50 to 75 kg of ATP every day!

Figure 1–15 The functional and morphologic consequences of depletion of intracellular adenosine…

Significant depletion of ATP has widespread effects on many critical cellular systems (Fig. 1–15):

• The activity of plasma membrane ATP-dependent sodium pumps is reduced, resulting in intracellular accumulation of sodium and efflux of potassium. The net gain of solute is accompanied by iso-osmotic gain of water, causing cell swelling and dilation of the ER.
• There is a compensatory increase in anaerobic glycolysis in an attempt to maintain the cell’s energy sources. As a consequence, intracellular glycogen stores are rapidly depleted, and lactic acid accumulates, leading to decreased intracellular pH and decreased activity of many cellular enzymes.
• Failure of ATP-dependent Ca²⁺ pumps leads to influx of Ca²⁺, with damaging effects on numerous cellular components, described later.
• Prolonged or worsening depletion of ATP causes structural disruption of the protein synthetic apparatus, manifested as detachment of ribosomes from the rough ER (RER) and dissociation of polysomes into monosomes, with a consequent reduction in protein synthesis. Ultimately, there is irreversible damage to mitochondrial and lysosomal membranes, and the cell undergoes necrosis.

Mitochondrial Damage and Dysfunction

Figure 1–16 Role of mitochondria in cell injury and death. Mitochondria are…

Mitochondria may be viewed as “mini-factories” that produce life-sustaining energy in the form of ATP. Not surprisingly, therefore, they are also critical players in cell injury and death (Fig. 1–16). Mitochondria are sensitive to many types of injurious stimuli, including hypoxia, chemical toxins, and radiation. Mitochondrial damage may result in several biochemical abnormalities:

• Failure of oxidative phosphorylation leads to progressive depletion of ATP, culminating in necrosis of the cell, as described earlier.
• Abnormal oxidative phosphorylation also leads to the formation of reactive oxygen species, which have many deleterious effects, described below.
• Damage to mitochondria is often associated with the formation of a high-conductance channel in the mitochondrial membrane, called the mitochondrial permeability transition pore. The opening of this channel leads to the loss of mitochondrial membrane potential and pH changes, further compromising oxidative phosphorylation.
• The mitochondria also contain several proteins that, when released into the cytoplasm, tell the cell there is internal injury and activate a pathway of apoptosis, discussed later.

Influx of Calcium

Figure 1–17 Sources and consequences of increased cytosolic calcium in cell injury. ATP, adenosine…

The importance of Ca²⁺ in cell injury was established by the experimental finding that depleting extracellular Ca²⁺ delays cell death after hypoxia and exposure to some toxins. Cytosolic free calcium is normally maintained by ATP-dependent calcium transporters at concentrations as much as 10,000 times lower than the concentration of extracellular calcium or of sequestered intracellular mitochondrial and ER calcium. Ischemia and certain toxins cause an increase in cytosolic calcium concentration, initially because of release of Ca²⁺ from the intracellular stores, and later resulting from increased influx across the plasma membrane. Increased cytosolic Ca²⁺activates a number of enzymes, with potentially deleterious cellular effects (Fig. 1–17). These enzymes include phospholipases (which cause membrane damage), proteases (which break down both membrane and cytoskeletal proteins), endonucleases (which are responsible for DNA and chromatin fragmentation), and adenosine triphosphatases (ATPases) (thereby hastening ATP depletion). Increased intracellular Ca²⁺levels may also induce apoptosis, by direct activation of caspases and by increasing mitochondrial permeability.

Accumulation of Oxygen-Derived Free Radicals (Oxidative Stress)

Free radicals are chemical species with a single unpaired electron in an outer orbital. Such chemical states are extremely unstable, and free radicals readily react with inorganic and organic chemicals; when generated in cells, they avidly attack nucleic acids as well as a variety of cellular proteins and lipids. In addition, free radicals initiate reactions in which molecules that react with free radicals are themselves converted into other types of free radicals, thereby propagating the chain of damage.

Reactive oxygen species (ROS) are a type of oxygen-derived free radical whose role in cell injury is well established. Cell injury in many circumstances involves damage by free radicals; these situations include ischemia-reperfusion (discussed later on), chemical and radiation injury, toxicity from oxygen and other gases, cellular aging, microbial killing by phagocytic cells, and tissue injury caused by inflammatory cells.

There are different types of ROS, and they are produced by two major pathways (Fig. 1–18).

Figure 1–18 Pathways of production of reactive oxygen species. A, In all cells, superoxide (O₂^•) is…

• ROS are produced normally in small amounts in all cells during the reduction-oxidation (redox) reactionsthat occur during mitochondrial respiration and energy generation. In this process, molecular oxygen is sequentially reduced in mitochondria by the addition of four electrons to generate water. This reaction is imperfect, however, and small amounts of highly reactive but short-lived toxic intermediates are generated when oxygen is only partially reduced. These intermediates include superoxide (  ), which is converted to hydrogen peroxide (H₂O₂) spontaneously and by the action of the enzyme superoxide dismutase. H₂O₂ is more stable than  and can cross biologic membranes. In the presence of metals, such as Fe²⁺, H₂O₂ is converted to the highly reactive hydroxyl radical ^•OH by the Fenton reaction.
• ROS are produced in phagocytic leukocytes, mainly neutrophils and macrophages, as a weapon for destroying ingested microbes and other substances during inflammation and host defense (Chapter 2). The ROS are generated in the phagosomes and phagolysosomes of leukocytes by a process that is similar to mitochondrial respiration and is called the respiratory burst (or oxidative burst). In this process, a phagosome membrane enzyme catalyzes the generation of superoxide, which is converted to H₂O₂. H₂O₂ is in turn converted to a highly reactive compound hypochlorite (the major component of household bleach) by the enzyme myeloperoxidase, which is present in leukocytes. The role of ROS in inflammation is described in Chapter 2.
• Nitric oxide (NO) is another reactive free radical produced in leukocytes and other cells. It can react with  to form a highly reactive compound, peroxynitrite, which also participates in cell injury.

The damage caused by free radicals is determined by their rates of production and removal (Fig. 1–19). When the production of ROS increases or the scavenging systems are ineffective, the result is an excess of these free radicals, leading to a condition called oxidative stress.

Figure 1–19 The generation, removal, and role of reactive oxygen species (ROS) in cell injury. The…

The generation of free radicals is increased under several circumstances:

• The absorption of radiant energy (e.g., ultraviolet light, x-rays). Ionizing radiation can hydrolyze water into hydroxyl (^•OH) and hydrogen (H^•) free radicals.
• The enzymatic metabolism of exogenous chemicals (e.g., carbon tetrachloride—see later)
• Inflammation, in which free radicals are produced by leukocytes (Chapter 2)

Cells have developed many mechanisms to remove free radicals and thereby minimize injury. Free radicals are inherently unstable and decay spontaneously. There are also nonenzymatic and enzymatic systems that contribute to inactivation of free radicals (Fig. 1–19).

• The rate of decay of superoxide is significantly increased by the action of superoxide dismutases (SODs) found in many cell types.
• Glutathione (GSH) peroxidases are a family of enzymes whose major function is to protect cells from oxidative damage. The most abundant member of this family, glutathione peroxidase 1, is found in the cytoplasm of all cells. It catalyzes the breakdown of H₂O₂ by the reaction 2 GSH (glutathione) + H₂O₂ → GS-SG + 2 H₂O. The intracellular ratio of oxidized glutathione (GSSG) to reduced glutathione (GSH) is a reflection of this enzyme’s activity and thus of the cell’s ability to catabolize free radicals.
• Catalase, present in peroxisomes, catalyzes the decomposition of hydrogen peroxide (2H₂O₂ → O₂ + 2H₂O). It is one of the most active enzymes known, capable of degrading millions of molecules of H₂O₂per second.
• Endogenous or exogenous antioxidants (e.g., vitamins E, A, and C and β-carotene) may either block the formation of free radicals or scavenge them once they have formed.

Reactive oxygen species cause cell injury by three main reactions (Fig. 1–19):

• Lipid peroxidation of membranes. Double bonds in membrane polyunsaturated lipids are vulnerable to attack by oxygen-derived free radicals. The lipid–radical interactions yield peroxides, which are themselves unstable and reactive, and an autocatalytic chain reaction ensues.
• Cross-linking and other changes in proteins. Free radicals promote sulfhydryl-mediated protein cross-linking, resulting in enhanced degradation or loss of enzymatic activity. Free radical reactions may also directly cause polypeptide fragmentation.
• DNA damage. Free radical reactions with thymine in nuclear and mitochondrial DNA produce single-strand breaks. Such DNA damage has been implicated in cell death, aging, and malignant transformation of cells.

In addition to the role of ROS in cell injury and killing of microbes, low concentrations of ROS are involved in numerous signaling pathways in cells and thus in many physiologic reactions. Therefore, these molecules are produced normally but, to avoid their harmful effects, their intracellular concentrations are tightly regulated in healthy cells.

Defects in Membrane Permeability

Figure 1–20 Mechanisms of membrane damage in cell injury. Decreased O₂ and increased cytosolic…

Increased membrane permeability leading ultimately to overt membrane damage is a consistent feature of most forms of cell injury that culminate in necrosis. The plasma membrane can be damaged by ischemia, various microbial toxins, lytic complement components, and a variety of physical and chemical agents. Several biochemical mechanisms may contribute to membrane damage (Fig. 1–20):

• Decreased phospholipid synthesis. The production of phospholipids in cells may be reduced whenever there is a fall in ATP levels, leading to decreased energy-dependent enzymatic activities. The reduced phospholipid synthesis may affect all cellular membranes, including the membranes of mitochondria, thus exacerbating the loss of ATP.
• Increased phospholipid breakdown. Severe cell injury is associated with increased degradation of membrane phospholipids, probably owing to activation of endogenous phospholipases by increased levels of cytosolic Ca²⁺.
• ROS. Oxygen free radicals cause injury to cell membranes by lipid peroxidation, discussed earlier.
• Cytoskeletal abnormalities. Cytoskeletal filaments act as anchors connecting the plasma membrane to the cell interior, and serve many functions in maintaining normal cellular architecture, motility, and signaling. Activation of proteases by increased cytosolic Ca²⁺ may cause damage to elements of the cytoskeleton, leading to membrane damage.
• Lipid breakdown products. These include unesterified free fatty acids, acyl carnitine, and lysophospholipids, all of which accumulate in injured cells as a result of phospholipid degradation. These catabolic products have a detergent effect on membranes. They may also either insert into the lipid bilayer of the membrane or exchange with membrane phospholipids, causing changes in permeability and electrophysiologic alterations.

The most important sites of membrane damage during cell injury are the mitochondrial membrane, the plasma membrane, and membranes of lysosomes.

• Mitochondrial membrane damage. As discussed earlier, damage to mitochondrial membranes results in decreased production of ATP, with many deleterious effects culminating in necrosis.
• Plasma membrane damage. Plasma membrane damage leads to loss of osmotic balance and influx of fluids and ions, as well as loss of cellular contents. The cells may also leak metabolites that are vital for the reconstitution of ATP, thus further depleting energy stores.
• Injury to lysosomal membranes results in leakage of their enzymes into the cytoplasm and activation of the acid hydrolases in the acidic intracellular pH of the injured (e.g., ischemic) cell. Lysosomes contain ribonucleases (RNases), DNases, proteases, glucosidases, and other enzymes. Activation of these enzymes leads to enzymatic digestion of cell components, and the cells die by necrosis.

Damage to DNA and Proteins

Cells have mechanisms that repair damage to DNA, but if this damage is too severe to be corrected (e.g., after radiation injury or oxidative stress), the cell initiates its suicide program and dies by apoptosis. A similar reaction is triggered by the accumulation of improperly folded proteins, which may result from inherited mutations or external triggers such as free radicals. Since these mechanisms of cell injury typically cause apoptosis, they are discussed later in the chapter.