Free Radicals Clinical Biochemistry Dr.

Charles Saladino Part I Introduction The topic of free radicals is potentially of great importance, as it applies to the endogenous generation of mutations and damage to the DNA and to other key biomolecules and cell structures. Conversely, the topic of antioxidants explores the manner in which a variety of exogenous and endogenous molecules create, to a certain degree, an umbrella of protection against these free radicals. Both will be explored in Part I of this lecture. In strictly chemical terms, a free radical is an atom or molecule having one (or more) unpaired electron(s) looking for a partner of opposite spin. Surely, this makes the free radical a highly reactive species, potentially causing substantial changes in the structure and the function of the molecules with which it collides. What needs to be emphasized here is that this process of free radical production is naturally-occurring in living cells, generated in large part from the very large number of chemical reactions that require oxygen, including the electron transport system itself. Thus, free radicals of oxygen are a major source of biomolecular damage within the cell, and they are also involved in exogenously- produced damage, such as that which occurs in cigarette smoking (to be discussed later). Free Radical Generation As stressed above, the generation of free radicals is a natural chemical phenomenon occurring at the electron level. Such is the case in the formation of the superoxide radical, because chemical reduction (the gain of electrons) of oxygen is a normal cellular process. For example, this is quite prevalent in oxidase-catalyzed reactions, such as commonly takes place in both the microsomal oxidase system and cytochrome P450 systems, as well as in the cytochromes of the electron transport system in the mitochondria, where molecular oxygen is reduced to water. This type of radical is formed when O2 is reduced by a single electron, generating an O2-, known as the superoxide radical. You might also see this same thing written as an O2. or even O2.-. If there is reduction of O2 by a second electron, O2-2, known as a peroxy anion will result. However, within the aqueous environment, two protons can be immediately added to the peroxy anion to form H2O2 (hydrogen peroxide, a potentially cytotoxic substance itself). This conversion of the superoxide radical to hydrogen peroxide can be catalyzed by the naturally and fairly-abundant antioxidant enzyme, superoxide dismutase (SOD). Further, the hydrogen peroxide can be broken down to water and 1/2O2 in the presence of the enzyme catalase and/or glutathione peroxidase (Note: this enzyme is unusual in that the element selenium replaces the sulfur atom in a cysteine side chain.) All three enzymes are naturally occurring in the cell, and their presence is considered cytoprotective against some of the superoxide radicals and toxic hydrogen peroxide, respectively, created during the normal course of cellular metabolism.

The enzyme superoxide dismutase has an interesting configuration, in that is contains both a copper and a zinc atom. Although the Zn atom is important for the active site structure, the Cu functions to actually draw the superoxide radical into the active site, where it can be converted to hydrogen peroxide. Another significant point is that if hydrogen peroxide is not quickly reduced, then it can sometimes break down into a harmless hydroxyl ion, but also into another dangerous free radical the hydroxyl radical (OH.). As we shall see later, a variation on this reaction can occur in the lung with cigarette smoke, where Fe interacts with hydrogen peroxide to form dangerous hydroxyl radicals. This will be designated the Fenton reaction. Now you might ask, where are all these extra electrons coming from that induce free radical production? Well I think you can well appreciate that in the cytoplasm electrons are incredibly abundant and can easily go astray. However, perhaps this idea is easier to grasp if you think about the electron transport system (ETS), which is something more tangible. You can only guess at the enormous number of electrons flowing down all the ETS systems in the inner mitochondrial membranes, not to mention all the mitochondria. Simply put, electrons can leak out of the ETS very easily, instead of finding their Fe+++ targets in the cytochromes or the multi-rings structures of the FMN or CoQ. Thus, a significant percentage of the electrons leak out of the ETS and easily the very abundant oxygen that is present in the mitochondrial matrix waiting to be reduced and converted into water. Although I have been discussing variations of oxy radicals, we know that many organic molecules, such as polypeptide segments and unsaturated lipids, and even the antioxidant vitamin E, can also become free radicals. Again, these are naturally occurring processes. The importance of such free radicals is not insignificant. For example, two polypeptide chains, each containing a free radical, can cross link together and deactivate the function of those polypeptides (or enzymes etc.). These polypeptide radicals, with the unpaired electron for example at a carbon atom, could also cross link to DNA by nucleophilic attack. In fact, we know that with age, the melting curve of cow thymus nucleoprotein is higher than that of nucleoprotein from younger calf thymus, and DNA synthesis is also slower in the older tissue. It appears that proteinDNA cross linkage plays a role here, making it more difficult for the DNA to unwind, as a prerequisite for DNA replication, or for that matter, transcription. One group of molecules that is subject to free radical formation is the unsaturated fatty acids, which undergo auto-oxidation (spontaneous self-oxidation). This often occurs in the presence of a metal that catalyses the extraction of a single hydrogen atom from the carbon not involved in the double bond. This is shown in Figure 1A, where, by example, arachidonic acid undergoes such a reaction, with the formation (in line two) of several different possible free radicals, depending upon where the hydrogen extraction occurred. In the accompanying figures, anywhere you see a dot next to a carbon or oxygen, you have a free radical. Notice a few lines down in Figure 1A that once the fatty acid free radical interaction with oxygen, a peroxy free radical is formed (O-O.). Notice in 1B the cyclization of the oxygens to form hydroperoxyperoxides and various isomerization products. These are potentially cytotoxic, if they are not metabolized in some manner. In Figure 1C, we see the formation of dangerous, toxic alkoxy and epoxy radicals. These are potentially cytotoxic and carcinogenic, again depending upon the metabolism to follow.

If you look at Figure 2, one sees the likely mechanism behind smoked-induced lung cancer, mediated by free radicals. Note the exogenous Fe++ and semiquinone-induced superoxide radical formation, as well as the natural, endogenous superoxide radical formation. The endogenous radicals are produced within macrophages engaging in an oxygen burst during the metabolism used to help clear particulate matter from the lung epithelial surface. Notice the interaction of the Fe++ from the smoke with the hydrogen peroxide to produce hydroxyl radicals by way of the Fenton Reaction. Figure 3 illustrates some DNA damage from the cigarette smoke, where Fe++ is attacking the DNA sugar-phosphate backbone and is causing the opening up of the deoxyribose ring. Also, various types of DNA base damage can occur, especially from the hydroxyl radicals. Long story short: If this process is prolonged, then the DNA can be sufficiently mutated and damaged to induce cell metaplasia. If the irritant smoke-derived free radicals continue bombarding the metaplastic cells over time, the cells can transform into dysplastic cells, which transform into a full malignancy and invasion of the basement membrane underlying the bronchiolar epithelium. Although I am using the free radical damage generated form cigarette smoke, this is only a prototype model. What I mean is that these types of reactions are occurring all the time in our cells, besides that generated from exogenous challenges like smoking and radiation. Oh yes, with regard to radiation, let me say that radiation damages biomolecules by at least two mechanisms. One is direct interaction of the high energy ionizing radiation striking a molecule (like a DNA base) and damaging it, possibly inducing a mutation. The second is the interaction of high-energy radiation with intracellular water. Such an interaction results in the generation of hydroxyl radicals (from the splitting of water). Of all the free radicals, I might argue the hydroxyl radicals are about, if not the, most damgerous. Antioxidants A variety of naturally occurring endogenous and exogenous substances have substantial antioxidant (anti free radical) properties. These can include vitamins A (beta carotene), C, and E, the enzymes superoxide dismutase and glutathione peroxidase, coenzyme Q, Se (contained in garlic), and a variety of other substances. Their mode of action is really quite simple. They collide with the free radicals, a hydrogen is extracted from the antioxidant, leaving the antioxidant a free radical, which can then combine and “neutralize” the other missing electron of opposite spin of the original free radical. (That is, as long as the antioxidant itself isn’t transformed into a free radical! Oh yes, that can happen. See the next paragraph.) As for the exogenous antioxidants, the difficult thing is to know where they will actually go at the cell level, once in the body, for how long, and in what quantity to be effective as antioxidants. Also, another yet-to-be-totally resolved question - what about interactions between various antioxidants? This later question is quite interesting. I would like to explain with a specific example. We all understand that when LDL is oxidized in the blood, it can be taken up by arterial smooth muscle cells and macrophages via the scavenger receptor system, which takes up the lipid without

any feedback inhibition. It is believed that free radicals contribute to the production of the oxidized form of LDL. Therefore, experiments were conducted to enrich LDL particles (in the test tube) with alpha-tocopherol (vitamin E - actually vitamin E is a mixture of tocopherols) and to determine whether or not such enrichment would diminish experimentally-induced oxidation of the LDL. It did not. In fact, it made the oxidation more likely! Why? This occurred because the vitamin E itself became a free radical. Thus, the experiments were repeated with both vitamin E and CoQ (acting as a second agent to be reduced), and the oxidation of the LDL was greatly diminished. How one would extrapolate these data to the human condition is difficult to say. However, it sounds like maybe (only conjecture here) antioxidants should be taken more than one at a time. Antioxidant structures One of most important naturally occurring antioxidants that can be synthesized by the body and can also be obtain from the diet is glutathione. This is a simple tripeptide of glycine, cysteine, and glutamic acid. The cysteine is in the middle (look at the structure in Devlin). The antioxidant properties of this critically important tripeptide reside in the sulfhydryl group of the cysteine where the H becomes extracted from the SH group to combine with and neutralize the free radical. This leaves the sulfur atom as an S. , which will immediately combine with the electron of another sulfur atom from another glutathione molecule that had also undergone interaction with a free radical. This leaves the two sulfur atoms combined to form a disulfide linkage. The glutathione has now gone from its original reduced state (SH) to an oxidized form linking two glutathione via a disulfide bond. See the figure below to observe the reduced and oxidized states of glutathione. The oxidized form of glutathione (now no longer an antioxidant) must be changed back to the reduced form in order to regenerate the original antioxidant form where the disulfide bond is now split. This process requires the enzyme glutathione reductase, and the coenzyme is the allimportant NADPH. Do you remember where NADPH comes from? Sure you do – the oxidative part of the pentose pathway. This is one of the top few reasons why we need the oxidative part of the pentose pathway, so that our antioxidant form of glutathione can be regenerated. If you really think back, you might remember that the first enzyme utilized in the oxidative pentose pathway is glucose-6-P dehydrognase. In patients who are deficient with this enzyme, NADPH production can be quite insufficient, and so will the ability of such individuals to regenerate the reduced form of glutathione from the oxidized cross-linked state. This compromises the red cell antioxidant system. Therefore, the hemoglobin of patients afflicted with this inborn error of metabolism will be subject to oxy radical-induced cross linkage at the sulfhydryl groups, producing Heinz bodies within the red cells. Like sickle cell anemia, the cell membranes will collapse, producing a hemolytic anemia. That such cross linkage of hemoglobins at the cysteine sulfhydryl group would occur makes sense, because the free radicals produced in the oxygen-rich environment of the red cell not be quenched by the glutathione system and would thus easily target those sulfhydryl groups. One interesting point about glutathione from a nutritional point is that this peptide has its glutamic acid “peptide” bonded to the cysteine, not by the usual alpha carboxyl group, but rather by the carboxyl at the end of the R group of the glutamic acid. What this does is protect the glutathione

(found in muscle meat, for example) from digestion after it enters the G.I. tract. There are so many naturally occurring antioxidants. Generally, the actions are very similar, in that they donate a hydrogen to neutralize the free radical. We see this in the figure below for ascorbate (vitamin C). Please become familiar with that figure, which includes the ascorbate interaction, indirectly, with glutathione, and which includes a diagram of the mechanism by which that vitamin C molecule quenches free radicals. Final Note: In discussing vitamins, one enters a huge topic, which I have not tried to explain here. What I have tried to achieve is to talk briefly about one critical role of some of the vitamins - their antioxidant nature. This antioxidant role for these particular vitamins, some water soluble and some fat soluble, is critical to the survival of the cell, the tissue, the organ, and thus ultimately the organism. If you doubt this, just look at the reticulocyte, one cell stage in the differentiation and maturation of the red blood cell line of development. At the reticulocyte stage, the cell has a nucleus and numerous mitochondria. Through a series of reactions catalyzed by oxygenase enzymes, a large quantity of free radicals are generated. However, this is a controlled set of reactions, wherein the mitochondria and the nucleus are destroyed. Can you imagine what would happen to the cell if these reactions were uncontrolled? The answer would most likely be cell death. Instead, this free radical damage becomes important to the maturation of the reticulocyte differentiating into the erythrocyte - the final end-product of the erythroid line. Another example of the potential destruction free radicals can deliver is seen in macrophages, as part of the immune response. The oxygen bursts from high-level metabolism generate a huge free radical output. These radicals participate in the destruction of the cells that are targeted by the macrophage. Thus, free radicals are sometimes part of a natural, biochemical-physio- logicallynormal process. In other instances, they are stochastic events, that is, random reactions that do indiscriminate damage to various cell compartments. Finally, we remember that LDL oxidation can occur, often mediated by free radicals. This oxidation renders these lipoproteins sufficiently changed, so as to not be recognized by the standard feedback inhibition receptor system. Instead, they easily gain entrance to the arterial wall, because they are now recognized by a “scavenger receptor” system that is not subject to feedback inhibition, but rather just keeps taking up more and more of the oxidized LDL’s into the arterial smooth muscle cells and macrophages. This, of course, is an important aspect of atherogenesis.

α-tocopherol (vitamin E)

Mechanism of ascorbate antioxidant action from Rx above. R. = free radical Below is a figure showing the structure of the antioxidant, vitamin E (α-tocopherol). Hydrogen extraction from (most likely) the methyl group is the probable source of its antioxidant action. and that of lycopene (found in tomatos) and β-carotene. Also shown are their food


To close, I need to mention that not all free radical reactions are harmful. Many metabolic sequences depend on free radicals being stable long enough or to produce the proper product for normal metabolism. As one of many examples, cobalamin (vitamin B12) is a coenzyme that mediates methyl group transfer; and when proprionyl CoA is produced from βoxidation of odd chain fatty acids, it is converted to succinyl CoA (which enters the Kreb’s cycle). The steps involved in this conversion are free radicalmediated of reactions. Also, I remind you of free radical action in red blood cell

maturation from the reticulocyte.

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