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Home , Disproportionation, Oxidative stress, Radical (chemistry), Reactive oxygen species

, , and Free Damage

Defeng Wu, Ph.D., and Arthur I. Cederbaum, Ph.D.

Reactive species (ROS) are small, highly reactive, oxygen-containing that are naturally generated in small amounts during the body’s metabolic reactions and can react with and damage complex cellular molecules such as fats, , or DNA. Alcohol promotes the generation of ROS and/or interferes with the body’s normal defense mechanisms against these compounds through numerous processes, particularly in the . For example, alcohol breakdown in the liver results in the formation of molecules whose further in the to ROS production. Alcohol also stimulates the activity of called cytochrome P450s, which contribute to ROS production. Further, alcohol can alter the levels of certain metals in the body, thereby facilitating ROS production. Finally, alcohol reduces the levels of agents that can eliminate ROS (i.e., ). The resulting state of the cell, known as oxidative stress, can to cell . ROS production and oxidative stress in liver cells play a central role in the development of alcoholic . KEY WORDS: alcoholic liver disorder; oxidative stress; free radicals; ; chronic AODE (alcohol and other drug effects); NAD; NADH ; ; peroxidation; metals; proteins; DNA; ; peroxidase; biochemical mechanism; survey of research

s described throughout the terms and concepts will be defined and radicals, ROS, and oxidative stress is articles in this issue of Alcohol explained in more detail in the follow- followed by a review of the alcohol-related A Research & Health, alcohol acts ing sections.) Particularly important are cellular systems involved in ROS pro- through numerous pathways to affect the actions of a class of oxygen-containing duction. Next, the article explains why the liver and other organs and to lead free radicals known as reactive oxygen ROS are toxic to cells and what systems to the development of alcoholic liver species (ROS). ROS can damage or have evolved to help cells protect them- disease (ALD) (for summaries of many cause complete degradation (i.e., perox- selves against ROS. Finally, the role of of these pathways, see Cederbaum 2001; idation) of essential complex molecules ROS and oxidative stress in alcohol- Bondy 1992; Nordmann et al. 1992). in the cells, including fat molecules induced cell injury is discussed, with No single process or underlying mecha- (i.e., lipids), proteins, and DNA. Both suggestions about future directions for nism can account for all the effects of acute and chronic alcohol exposure research in this field. Although this dis- alcohol on an organism or even on one can increase production of ROS and cussion focuses on the role of oxidative specific organ; instead, many mechanisms enhance peroxidation of lipids, , stress in , alcohol- act in concert, reflecting the spectrum and DNA, as has been demonstrated in induced oxidative stress also occurs in of the organism’s response to a myriad a variety of systems, cells, and species, and damages other tissues (e.g., muscle, of direct and indirect actions of alcohol. including . pancreas, and nerve cells). One factor that has been suggested as Researchers have learned much about playing a central role in many pathways alcohol metabolism and the various DEFENG WU, PH.D., is a research associate of alcohol-induced damage, and which enzymes and pathways involved, as well professor, and ARTHUR I. CEDERBAUM, has been the focus of much research, is as about the role of peroxidation PH.D., is a professor, both in the Department the excessive generation of molecules and oxidative stress in alcohol toxicity. of Pharmacology and Biological , called free radicals, which can result in This article summarizes some of these Mount Sinai School of Medicine, New a state called oxidative stress. (These findings. A detailed description of free York, New York.

Vol. 27, No. 4, 2003 277 What Are Free Radicals several molecules of ATP for each such as acute or chronic alcohol expo­ and ROS? that passes through the respiratory chain. sure, ROS production is enhanced Molecular oxygen can accept a total and/or the level or activity of antioxi­ A free radical is an , , or of four , one at a time, and the dants is reduced. The resulting state— compound that is highly unstable because corresponding number of to which is characterized by a disturbance of its atomic or molecular structure (i.e., generate two molecules of . During in the balance between ROS produc­ the distribution of electrons within the this process, different oxygen radicals tion on one hand and ROS removal molecule). As a result, free radicals are are successively formed as intermediate •– and repair of damaged complex very reactive as they attempt to pair up products, including (O2 ); = molecules (such as proteins or DNA) with other molecules, , or even peroxide (O2 ), which normally exists on the other—is called oxidative stress individual electrons to create a stable in cells as (H2O2); • (Halliwell 1999). Oxidative stress is compound. To achieve a more stable and the ( OH). Super­ associated with numerous deleterious oxide, peroxide, and the hydroxyl radi­ state, free radicals can “steal” a hydro­ consequences for the cell (e.g., lipid gen atom from another molecule, bind cal are considered the primary ROS to another molecule, or interact in vari­ and have sparked major research on ous ways with other free radicals (see the role of free radicals in biology and 3 Reactions Involving the textbox). medicine. However, because they are One frequently unstable and rapidly react with addi­ Free Radicals involved in free radical formation is oxy­ tional electrons and protons, most of these ROS are converted to water Free radicals are highly unstable gen. Molecular oxygen (O2) is essential molecules that attempt to for cell function because it plays a piv­ before they can damage cells. It has been estimated that only about 2 to 3 achieve a more stable state by otal role in a series of biochemical reac­ reacting with other atoms or tions occurring in the respiratory chain, percent of the O2 consumed by the res­ piratory chain is converted to ROS molecules in the cell. The which is responsible for most of the four primary types of chemical production of (Chance et al. 1979). Nevertheless, the toxic effects of oxygen in biological reactions that free radicals (ATP), which provides the energy required undergo are: for a multitude of cellular reactions and systems—such as the breakdown (i.e., oxidation) of lipids, inactivation of functions. (For more information on enzymes, introduction of changes (i.e., the respiratory chain and ATP produc­ •Hydrogen abstraction, in ) in the DNA, and destruc­ tion, see the article by Cunningham which a radical interacts with tion of cell membranes and, ultimately, another molecule that has a and Van Horn in this issue.) cells—are attributable to the reduction In the respiratory chain, which takes free hydrogen atom (i.e., a of O2 to ROS (Toykuni 1999; de hydrogen donor). As a result, place in membrane-enclosed cell struc­ Groot 1994; Nakazawa et al. 1996). tures called mitochondria, an electron the radical binds to the hydro­ and a (H+) are removed from a gen atom and becomes stable, whereas the hydrogen donor helper molecule (i.e., cofactor) called What Is Oxidative Stress? reduced nicotinamide adenine dinu­ is converted to a free radical. 1 cleotide (NADH). The electron is Because ROS form naturally during transferred to the first component of • Addition, in which the radical many metabolic processes, cells have binds to another, originally the respiratory chain, and the proton is developed several protective mecha­ released into the surrounding fluid. stable molecule, converting nisms to prevent ROS formation or to the combined molecule into Chemically speaking, NADH is oxidized detoxify the ROS. These mechanisms + a radical. to NAD in this reaction, whereas the employ molecules called antioxidants, respiratory chain, component that accepts which will be discussed in more detail the electron is reduced.2 The NAD+ • Termination, in which two in the section “Protection Against ROS radicals react with each other subsequently can be used again to accept Toxicity.” Under certain conditions, new hydrogen atoms that are generated to form a stable compound. during the metabolism of sugars (e.g., 1NADH is generated in the fluid filling the cell (i.e., the • , in which glucose) and other nutrients. The reduced cytosol) and then moves to the mitochondria. two identical radicals react respiratory chain component, in turn, 2 Oxidation reactions are those that add oxygen to a with each other, with one of passes the electron on to other molecules molecule or remove hydrogen or an electron from a in the respiratory chain until it is finally molecule. The reverse reactions (i.e., removal of oxygen the radicals donating an elec­ or addition of hydrogen or electrons) are called reductions. tron to the other so that two transferred to O2, which then interacts 3 different molecules are formed, with protons in cells to generate water. Superoxide can react with itself to produce H2O2. Thus, systems producing superoxide also will result in formation each of which is stable. This series of electron transfer reactions of H2O2. Technically, H2O2 is not a free radical, but it is generates sufficient energy to produce commonly included among the ROS.

278 Alcohol Research & Health Alcohol, Oxidative Stress, and Free Radical Damage

peroxidation or even cell ), and tion of certain signaling molecules Systems Producing ROS alcohol-induced oxidative stress may called , which in turn lead play a significant role in the develop­ to the activation of an array of bio­ As implied in the previous section, ment of ALD. chemical processes. (For more infor­ numerous cellular systems can produce Many processes and factors are mation on alcohol’s effect on ROS. The major source of ROS pro­ involved in causing alcohol-induced duction in the cell is the mitochondrial oxidative stress, including: production and its consequences, respiratory chain, which, as described see the article in this issue by Neuman.) earlier, utilizes approximately 80 to 90 + • Changes in the NAD /NADH ratio percent of the O2 a person consumes. in the cell as a result of alcohol •Alcohol-induced increase in the Thus, even though only a small per­ metabolism. Alcohol is metabolized ability of the bacterial molecule centage of that oxygen is converted to in two steps. First, the alco­ endotoxin to enter the bloodstream ROS, the mitochondrial respiratory hol dehydrogenase converts alcohol and liver, where it can activate cer­ chain in all cells generates most of the to , a toxic and reactive tain immune cells. (For more infor­ ROS produced in the body. molecule. Next, the enzyme mation on the role of endotoxin in Another major source of ROS, espe­ cially in the liver, is a group of enzymes dehydrogenase converts the acetalde­ liver damage, see the article by hyde to . Each of these reac­ called the cytochrome P450 mixed- tions leads to formation of one Wheeler in this issue.) function oxidases. Many different variants molecule of NADH, thereby pro­ of these -containing enzymes exist, viding more starting material and •Alcohol-induced increases in the some of which are responsible for thus enhanced activity of the respi­ activity of the enzyme cytochrome removing or detoxifying a variety of ratory chain, including heightened P450 2E1 (CYP2E1), which (as compounds present in our environment O2 use and ROS formation. described in the section “Systems and ingested (e.g., foods or drugs), Producing ROS”) metabolizes alco­ including alcohol. Some cytochrome •Production of acetaldehyde during hol and other molecules and gener­ P450 enzymes also are important for alcohol metabolism, which through ates ROS in the process. metabolizing substances that naturally its interactions with proteins and occur in the body, such as fatty acids, lipids also can lead to radical forma­ cholesterol, , or bile acids. The •Alcohol-induced increases in the tion and cell damage. (For informa­ biochemical reactions spurred (i.e., tion on acetaldehyde and its detri­ levels of free iron in the cell (i.e., catalyzed) by the cytochrome P450 mental effects, see the article in this iron that is not bound to various molecules use molecular oxygen, and issue by Tuma and Casey.) proteins), which can promote ROS during these reactions small amounts generation, as described in the sec­ of ROS are generated. The extent of •Damage to the mitochondria result­ tion “Role of Metals.” ROS generation may vary considerably ing in decreased ATP production. depending on the compound to be •Effects on enzymes and degraded and on the cytochrome •Effects on cell structure (e.g., the chemicals, particularly a molecule P450 molecule involved. One type of membranes) and function caused by called glutathione (GSH), as described cytochrome molecule that is especially alcohol’s interactions with either mem­ active in producing ROS is known as brane components (i.e., phosphate- in the section “Protection Against CYP2E1. This enzyme is of particular containing lipids [phospholipids]) ROS Toxicity.” interest when investigating alcohol- or enzymes and other protein com­ induced oxidative stress because its ponents of the cells. •Biochemical reactions generating activity increases after heavy alcohol an alcohol-derived radical (i.e., the exposure and because CYP2E1 itself •Alcohol-induced oxygen deficiency 1-hydroxyethyl radical). also metabolizes alcohol (Lieber 1997). (i.e., ) in tissues, especially ROS also are produced by a variety in certain areas of the liver lobules • Conversion of the enzyme xanthine of oxidative enzymes present in cells, (i.e., the pericentral region), where dehydrogenase into a form called such as the previously mentioned xan­ extra oxygen is required to metabolize , which can gener­ thine oxidase. Under normal physiolog­ the alcohol. (For more information ate ROS. ical conditions, xanthine oxidase acts on alcohol-induced hypoxia in the as a dehydrogenase—that is, it removes liver and its consequences, see the hydrogen from xanthine or hypoxanthine article by Cunningham and Van Many of these processes operate and attaches it to NAD, thereby gener­ Horn in this issue.) concurrently, and it is likely that sev­ ating NADH. However, under certain eral, indeed many, systems contribute conditions, such as the disruption of • Alcohol’s effects on the immune sys­ to the ability of alcohol to induce a flow to a , xanthine dehydroge­ tem, which lead to altered produc- state of oxidative stress. nase is converted to a ROS-producing

Vol. 27, No. 4, 2003 279 oxidase form. Alcohol consumption also Role of Metals Why Are ROS Toxic? may promote the conversion of xanthine Most of the systems for the production dehydrogenase to xanthine oxidase ROS are toxic to cells because they can of ROS described above produce super­ react with most cellular macro­ (Sultatos 1988), which can generate oxide radicals or hydrogen peroxide. ROS, thereby enhancing oxidative stress. molecules, including proteins, lipids, Earlier studies suggested the possibility and DNA. Other sources of ROS in the body that these two radicals could interact are two types of immune cells called Proteins perform numerous crucial with each other to produce the most functions in the cell, primarily in the and , which • reactive ROS, the hydroxyl radical ( OH). form of enzymes that mediate most help defend the body against invading Under normal physiological condi­ biochemical reactions required for cel­ microorganisms. In this case, however, tions, direct interaction between these lular functions. Proteins are made up of ROS production is beneficial and even two radicals is not likely to play a sig­ approximately 20 different building essential to the organism because it nificant role in generating hydroxyl blocks called amino acids, which differ plays a central role in destroying for­ radicals. However, in the presence of in their sensitivity to interactions with eign (Rosen et al. 1995). certain metals, particularly free iron or ROS. For example, the amino acids Macrophages and neutrophils contain , a sequence of two reaction cysteine, , and are a group of enzymes called the NADPH steps can occur that results in hydroxyl especially sensitive to attack and oxidation oxidase complex, which, when activated, radical generation. In the first step, by the hydroxyl radical. Accordingly, generates superoxide radicals and hydro­ hydrogen peroxide can produce the enzymes in which these amino acids gen peroxide. Hydrogen peroxide then hydroxyl radical by removing an elec­ are located at positions that are critical interacts with chloride ions present in tron from the participating metal .4 to the enzyme’s activity will become inactivated by the interaction with ROS. the cells to produce hypochlorite (the In the second step, involving the super­ •– Alternatively, the ROS-induced oxida­ active ingredient in bleach), which in oxide radical (O2 ), the original metal turn destroys the . The NADPH tion of proteins can lead to changes in ions are regenerated so that they are again the proteins’ three-dimensional structure oxidase complex and the resulting ROS available for reaction with the hydrogen as well as to fragmentation, aggregation, production are critical to the body’s peroxide. This combination of two or cross-linking of the proteins. Finally, defense against all kinds of diseases, as chemical reactions appears to account protein oxidation often will make the is evident in patients with a condition for most of the hydroxyl radical pro­ marked protein more susceptible to called chronic granulomatous disease, duction in biological systems and explains, degradation by cellular systems respon­ in which ROS production by the at least in part, why metals such as iron sible for eliminating damaged proteins NADPH oxidase complex is drastically and copper produce oxidative stress from the cell. reduced. Patients with this condition and ROS-induced injury in cells. Lipids that contain phosphate groups are highly sensitive to and Because of iron’s critical contribution (i.e., phospholipids) are essential com­ usually die at an early age. to hydroxyl radical formation, anything ponents of the membranes that sur­ Besides the ROS generation that that increases the levels of free iron in round the cells as well as other cellular occurs naturally in the body, humans the cells promotes ROS generation and structures, such as the nucleus and are constantly exposed to environmen­ oxidative stress. Chronic alcohol con­ mitochondria. Consequently, damage tal free radicals, including ROS, in the sumption has been shown to increase to the phospholipids will compromise form of , UV light, , iron levels in the body not only when the viability of the cells. The complete degradation (i.e., peroxidation) of lipids , and certain compounds iron-rich alcoholic beverages, such as red wine, are consumed, but also because is a hallmark of oxidative damage. The referred to as cycling agents, polyunsaturated fatty acids5 present in chronic alcohol consumption enhances which include some pesticides, but also the membranes’ phospholipids are par­ certain medications used for iron absorption from food (see Nanji ticularly sensitive to attack by hydroxyl treatment. The toxicity of these medi­ and Hiller-Sturmhöfel 1997). Similarly, radicals and other oxidants. A single cations against tumor cells (as well as adding iron to alcohol-containing diets hydroxyl radical can result in the perox­ normal body cells) results from the fact has been shown to exacerbate liver idation of many polyunsaturated fatty that the compounds are modified by injury in animal studies (Tsukamoto et acid molecules because the reactions cellular enzymes to an unstable inter­ al. 1995), whereas administration of involved in this process are part of a mediate, which then reacts with molec­ agents that capture free iron can pre­ ular oxygen to produce the original vent or ameliorate alcohol’s toxic effects 5Unsaturated fatty acids are those that contain a double on the liver (Sadrzadeh et al. 1994). bond between two of the carbon atoms making up the product plus a superoxide radical. Thus, backbone of the fatty acid molecule. These double bonds a vicious cycle of chemical reactions can easily be opened in chemical reactions and interact involving these compounds continually 4This reaction can generate other products as well, but with other substances. Fatty acids containing only one the hydroxyl radical appears to be the primary oxidant such double bond are called monounsaturated; fatty acids produces ROS. generated (McCord 1998). with two or more double bonds are called polyunsaturated.

280 Alcohol Research & Health Alcohol, Oxidative Stress, and Free Radical Damage

cyclic chain reaction. In addition to (SODs), , and glutathione per­ tors glutathione (GSH) and reduced damaging cells by destroying mem­ oxidase. SODs catalyze the rapid removal nicotinamide adenosine dinucleotide branes, can result in of superoxide radicals. In mammals phosphate (NADPH).7 Together, these the formation of reactive products that there are several types of SODs, which molecules effectively remove hydrogen themselves can react with and damage differ with respect to their location in peroxide. GSH, which consists of three proteins and DNA. (For more infor­ the cells and the metal ions they require amino acids, is an essential component mation regarding the actions of such for their function. For example, a copper– of this system and serves as a cofactor reactive products, see the article by Tuma SOD is present in the fluid filling for an enzyme called glutathione trans­ and Casey in this issue.) the cell (i.e., the cytosol) and in the ferase, which helps remove certain drugs DNA is the cell’s genetic material, and space between the two membranes and chemicals as well as other reactive any permanent damage to the DNA surrounding the mitochondria. Further­ molecules from the cells. Moreover, can result in changes (i.e., mutations) more, a -containing SOD GSH can interact directly with certain in the proteins encoded in the DNA, is present in the mitochondrial interior ROS (e.g., the hydroxyl radical) to which may lead to malfunctions or (i.e., matrix). Both of these enzymes are detoxify them, as well as performing complete inactivation of the affected critical for prevention of ROS-induced other critical activities in the cell. proteins. Thus it is essential for the toxicity (Fridovich 1997).6 The effects viability of individual cells or even the of chronic alcohol exposure on the cellu­ Nonenzymatic Mechanisms entire organism that the DNA remain lar content or activity of SODs are con­ intact. The building blocks of DNA troversial, with reports of increases, no Because of all its functions, GSH is molecules are called nucleotides; they changes, or decreases, depending on probably the most important antioxi­ consist of a sugar component and an the model, diet, amount, and time of dant present in cells. Therefore, enzymes organic base. Each DNA molecule con­ alcohol feeding. Studies employing a that help generate GSH are critical to sists of two strands of nucleotides held commonly used model in which alcohol the body’s ability to protect itself against together by weak chemical bonds. Changes is administered directly into the stom­ oxidative stress. Alcohol has been shown in the nucleotides in one strand can ach of laboratory animals (i.e., the to deplete GSH levels, particularly in result in mismatches with the nucleotides intragastric infusion model, used most the mitochondria, which normally are in the other strand, yielding subsequent commonly with and mice) found characterized by high levels of GSH mutations. ROS are a major source of decreases in SOD activity in the liver needed to eliminate the ROS generated DNA damage, causing strand breaks, (Polavarapu et al. 1998) (see the article during activity of the respiratory chain. removal of nucleotides, and a variety of by Nanji and French in this issue). Mitochondria cannot synthesize modifications of the organic bases of the Catalase and the glutathione peroxi­ GSH but import it from the cytosol nucleotides. Although cells have devel­ dase system both help to remove hydrogen using a carrier protein embedded in the oped repair mechanisms to correct nat­ peroxide. Catalase is an iron-containing membrane surrounding the mitochon­ urally occurring changes in the DNA, enzyme found primarily in the small dria. Alcohol appears to interfere with additional or excessive changes caused membrane-enclosed cell components the function of this carrier protein, by ROS or other agents can lead to called ; it serves to detoxify thereby leading to the depletion of permanent changes or damage to the hydrogen peroxide and various other mitochondrial GSH (Fernandez-Checa DNA, with potentially detrimental effects molecules. One way that catalase elimi­ et al. 1997). for the cell. nates hydrogen peroxide is by catalyz­ NADPH is involved in a much ing a reaction between two hydrogen more diverse range of reactions in the peroxide molecules, resulting in the cell than GSH. Nevertheless, because Protection Against ROS formation of water and O2. In addition, of its role in the Toxicity catalase can promote the interaction of system, NADPH or the enzymes that hydrogen peroxide with compounds generate this compound are sometimes Because ROS production is a naturally that can serve as hydrogen donors so considered antioxidants. occurring process, a variety of enzymatic that the hydrogen peroxide can be con­ In addition to GSH and NADPH, and nonenzymatic mechanisms have verted to one molecule of water, and numerous other nonenzymatic anti­ evolved to protect cells against ROS (Yu the reduced donor becomes oxidized oxidants are present in the cells, most 1994). At least some of these mechanisms (a process sometimes called the peroxi­ prominently (α-) are impaired after long-term alcohol datic activity of catalase). Compounds and (ascorbate). Vitamin E consumption and may therefore contribute that can provide these hydrogen atoms is a major antioxidant found in the lipid to damage to the liver and other organs. include beverage alcohol (i.e., )

and . 6Another type of SOD (ECÐSOD) is found outside the cells. The glutathione peroxidase system Protective Enzymes 7 consists of several components, includ­ Glutathione peroxidase contains an amino acid that is modified by addition of a molecule of the metal selenium; Enzymes involved in the elimination ing the enzymes glutathione peroxidase therefore, low amounts of selenium are critical for the of ROS include superoxide dismutases and and the cofac­ body’s antioxidant defense.

Vol. 27, No. 4, 2003 281 phase of membranes and, like other with ALD commonly exhibit reduced a variety of diseases (see the chemically related molecules, acts as a vitamin E levels (see Nanji and Hiller- sidebar). What is the evidence that powerful terminator of lipid peroxidation. Sturmhöfel 1997). alcohol-induced oxidative stress plays a During the reaction between vitamin E role in cell injury, particularly damage and a lipid radical, the vitamin E radical to the liver cells? Many studies have is formed, from which vitamin E can Alcohol, Oxidative Stress, demonstrated that alcohol increases be regenerated in a reaction involving and Cell Injury lipid peroxidation as well as the modifi­ GSH and ascorbate. Alcohol also appears cation of proteins; however, it is not to interfere with the body’s normal Excess levels of ROS and the resulting always clear if these changes are the vitamin E content because patients oxidative stress have been implicated in causes rather than consequences of

Diseases Involving Excessive ROS Levels

In addition to contributing to the development of Finally, increasing evidence suggests that aging may ALD, ROS have been implicated in many other major be a consequence of the normal, long-term exposure diseases that plague humans. A partial listing of these to ROS and the accumulation of oxidized, damaged conditions (Knight 1998; Kehrer 1993) includes: molecules within the cell—a process that could be likened to a lifetime of “rusting away.” • The toxic effects of O2 itself, such as the oxidation of Accordingly, the health benefits of administering lipids and proteins, generation of mutations in the antioxidants such as vitamins E and C or other com­ DNA, and destruction of cell membranes. pounds are the subject of much current research, and • Cardiovascular diseases. clinical trials employing antioxidants in the treatment of various conditions are under way. For example, some •. therapeutic interventions with antioxidants have shown success or promise in the treatment of Parkinson’s dis­ •Various types of cancer. ease and in reducing the toxicity of the cancer medi­ cation adriamycin. •. Not all instances of ROS production are detrimen­ tal to the organism, however. One beneficial effect, as •Neurodegenerative diseases, including Parkinson’s the main article describes, is the production of ROS by disease and Alzheimer’s disease. certain immune cells in order to destroy invading foreign •Toxicity of (e.g., iron). organisms (Rosen et al. 1995). Furthermore, recent evidence suggests that ROS, especially hydrogen perox­ •Radiation injury. ide, may be important in mecha­ nisms in cells and thus may be an integral component •Vitamin deficiency. of cellular and metabolism (Lander 1997).

•Toxicity of certain medications. —Defeng Wu and Arthur I. Cederbaum

•, such as the destruction of joints, the References synovial fluid that lubricates joints and one of its

components (i.e., hyaluronic acid), as well as activa­ KEHRER, J.P. Free radicals as mediators of tissue injury and disease. tion of inflammation-promoting signaling molecules Critical Reviews in Toxicology 23:21–48, 1993. called cytokines. KNIGHT, J.A. Free radicals: Their history and current status in aging •Toxic effects of tobacco smoke. and disease. Annals of Clinical and Laboratory Science 28:331–346, 1998. LANDER, H.M. An essential role for free radicals and derived species in •Emphysema. signal transduction. FASEB Journal 11:118–124, 1997.

•Cataracts. ROSEN, G.M.; POU, S.; RAMOS, C.L.; ET AL. Free radicals and phago­ cytic cells. FASEB Journal 9:200–209, 1995.

282 Alcohol Research & Health Alcohol, Oxidative Stress, and Free Radical Damage

alcohol-induced tissue injury. Nevertheless, continuously had been fed alcohol humans will be a difficult task because numerous investigations have found indicated that alcohol metabolism via ROS production and antioxidant status that administering antioxidants, agents the enzyme alcohol dehydrogenase in humans are affected by numerous that reduce the levels of free iron, or results in increased ROS production, nutritional, environmental, and drug agents that replenish GSH levels can hepatocyte injury, and a type of cell influences that are difficult to repro­ prevent or ameliorate the toxic actions death known as . Moreover, duce in animals. To date, scattered data of alcohol. For example, in the intra- all of these reactions could be blocked suggest that the blood of human alco­ gastric infusion model, the antioxidant by the administration of antioxidants holics can contain lipids modified by vitamin E; the chemical ebselen, which (Adachi and Ishii 2002; Bailey and radicals and other reactive molecules as mimics the actions of glutathione per­ Cunningham 2002). Finally, studies well as immune molecules targeted at oxidase; the copper–zinc or manganese using an established hepatocyte cell line such modified lipids and proteins. SODs; or a GSH precursor—all pre­ that contains the alcohol-metabolizing These data indicate that ROS and vented ALD (Iimuro et al. 2000; Nanji and ROS-producing enzyme CYP2E1 other reactive molecules are indeed et al. 1996; Kono et al. 2001; Wheeler demonstrated that adding alcohol, formed in human alcoholics. (For more et al. 2001a,b). polyunsaturated fatty acids, or iron, as information on the presence of such The most convincing data indicat­ well as reducing GSH, resulted in cell compounds in humans, see the article ing that oxidative stress contributes to toxicity, increased oxidative stress, and by Tuma and Casey in this issue.) ALD come from studies using the intra- mitochondrial damage (Wu and Other questions that should be gastric infusion model. In these studies, Cederbaum 1999). Furthermore, all of addressed in future research include ALD was associated with enhanced these reactions could be prevented by the following: lipid peroxidation, protein modification, administering antioxidants. Taken formation of the 1-hydroxyethyl radical together, these findings indicate that •Do reactive species (e.g., and lipid radicals, and decreases in the alcohol-induced oxidative stress is a ) play a role in alcohol- hepatic antioxidant defense, particularly pivotal factor in the development of ALD. induced oxidative stress in addition GSH levels (Knecht et al. 1995; Tsuka­ to ROS? moto and Lu 2001; Iimuro et al. 2000; Nanji et al. 1994; Morimoto et al. 1994). Future Directions for • What is the impact of possible interac­ Moreover, changes in the animals’ diets Research tions between alcohol and environ­ that helped promote or reduce oxidative mental influences such as smoking, stress led to corresponding changes in Although researchers already have gained use of other drugs or medications, the extent of liver injury. For example, substantial insight into the mechanisms and viral infections (e.g., hepatitis C) when polyunsaturated fats (which are and consequences of alcohol-induced on ROS production, oxidative stress, required for lipid peroxidation to occur) oxidative stress, additional studies are and tissue injury? These interactions were replaced with saturated fats or required to further clarify how alcohol must be better defined because most other types of fats (i.e., medium-chain produces oxidative stress in various tis­ alcoholics are exposed to one or triglycerides), lipid peroxidation as sues. For example, more detailed infor­ more of these influences in addition well as ALD were reduced or prevented mation is needed on the mechanisms to alcohol. completely, indicating that both alcohol involved in some of the major proposed and polyunsaturated fats must be pre­ pathways (e.g., how alcohol-derived •How is oxidative stress affected by sent for ALD to occur. The extent of NADH leads to ROS production either interactions between alcohol and the ALD was further exacerbated when directly or during the passage of NADH- nutritional factors, such as the levels iron—which, as mentioned earlier, is derived electrons through the mito­ and specific types of fats ingested? required for the generation of the chondrial respiratory chain). Other And how much iron is “safe” in a hydroxyl radical and therefore pro­ mechanisms remain highly controver­ heavy drinker? motes oxidative stress—was added to sial, such as the role of CYP2E1 or of these diets (Tsukamoto et al. 1995). various cytokines in alcohol-induced •What are the effects of antioxidants Conversely, the addition of antioxidants oxidative stress. Additional analyses (e.g., vitamin E, vitamin C, or such as vitamin E, SOD, or GSH pre­ need to determine the role of alcohol ) in heavy drinkers? This cursors prevented the development of metabolism and its byproducts (e.g., question is important because some ALD, as mentioned above. acetaldehyde) in the production of antioxidants can be toxic under cer­ In addition to these studies conducted ROS. Finally, it still is unclear how tain conditions. with intact animals (i.e., in vivo), studies alcohol-induced oxidative stress is pro­ with liver cells (i.e., hepatocytes) grown duced in tissues where only limited The ability of alcohol to promote in culture also showed that alcohol can alcohol metabolism occurs. oxidative stress and the role of free radi­ produce oxidative stress and hepatocyte Many of these issues can be studied cals in alcohol-induced tissue injury toxicity. Studies with hepatocytes iso­ using animal models; however, extrapo­ clearly are important areas of research lated from control rats or from rats that lation of findings from animals to in the alcohol field, particularly because

Vol. 27, No. 4, 2003 283 such information may be of major HALLIWELL, B. Antioxidant defense mechanisms: POLAVARAPU, R.; SPITZ, D.R.; SIM, J.E.; ET AL. From the beginning to the end. Free Radical Research Increased lipid peroxidation and impaired antioxi­ therapeutic significance in attempts to 31:261–272, 1999. dant enzyme function is associated with pathologi­ prevent or ameliorate alcohol’s toxic cal liver injury in experimental alcoholic liver dis­ effects. As basic information continues IIMURO, Y.; BRADFORD, B.U.; YAMASHINA, S.; ET ease in rats fed diets high in corn oil and fish oil. AL. The glutathione precursor L-2-oxothiazolidine- Hepatology 27:1317–1323, 1998. to emerge regarding the role of oxidative 4- protects against liver injury due to stress in disease development and the chronic enteral ethanol exposure in the . Hepatology ROSEN, G.M.; POU, S.; RAMOS, C.L.; ET AL. Free mechanisms underlying ROS-related 31:391–398, 2000. radicals and phagocytic cells. 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