Ref Robbins 9/e p48 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 cel- lular proteins and lipids. In addition, free radicals initiate reactions in which molecules that react with free radicals are themselves conveed 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 estab- lished. 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). * ROS are produced normally in small amounts in all cells during the reduction-oxidation (redox) reactions that occur during mitochondrial respiration and energy genera- tion. In this process, molecular oxygen is sequentially reduced in mitochondria by the addition of four elec- trons to generate water. This reaction is imperfect, however, and small amounts of highly reactive but sho-lived toxic intermediates are generated when oxygen is only paially reduced. These intermediates include superoxide (O2 * ), which is conveed to hydro- gen peroxide (H2O2) spontaneously and by the action of the enzyme superoxide dismutase. H2O2 is more stable than O2 * and can cross biologic membranes. In the pres- ence of metals, such as Fe2+ , H2O2 is conveed to the highly reactive hydroxyl radical * OH by the Fenton reaction.ROS are produced in phagocytic leukocytes, mainly neutro- phils and macrophages, as a weapon for destroying ingested microbes and other substances during inflam- mation and host defense (Chapter 2). The ROS are gener- ated in the phagosomes and phagolysosomes of leukocytes by a process that is similar to mitochondrial respiration and is called the respiratory burst (or oxida- tive burst). In this process, a phagosome membrane enzyme catalyzes the generation of superoxide, which is conveed to H2O2. H2O2 is in turn conveed 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 inflam- mation is described in Chapter 2. * Nitric oxide (NO) is another reactive free radical pro- duced in leukocytes and other cells. It can react with O2 * to form a highly reactive compound, peroxynitrite, which also paicipates 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. 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 radi- cals and thereby minimize injury. Free radicals are inher- ently 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, glu- tathione 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 decom- position of hydrogen peroxide (2H2O2 - O2 + 2H2O). It is one of the most active enzymes known, capable of degrading millions of molecules of H2O2 per second. * Endogenous or exogenous antioxidants (e.g., vitamins E, A, and C and b-carotene) may either block the forma- tion 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 mem- brane polyunsaturated lipids are vulnerable to attack by oxygen-derived free radicals. The lipid-radical interac- tions 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 numer- ous signaling pathways in cells and thus in many physio- logic reactions. Therefore, these molecules are produced normally but, to avoid their harmful effects, their intracel- lular concentrations are tightly regulated in healthy cells.