Blackwell Publishing AsiaMelbourne, AustraliaJGHJournal of Gastroenterology and Hepatology0815 9319 2006 The Authors; Journal compilation 2006 Journal

l of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd? 200621S3S26S29Original Article Alcohol and oxidative stressD Wu
et al.

doi:10.1111/j.1440-1746.2006.04589.x

O X I D A N T S T R E S S , I N F L A M M AT I O N A N D G E N E T I C S

Alcohol-induced oxidative stress and cell responses

Dongmei Wu, Qiwei Zhai and Xianglin Shi
Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

Abstract
Epidemiological and animal studies have demonstrated that alcohol abuse is directly associated with the increase of multiple organ diseases, such as liver injury, cardiovascular diseases, and neurological disorders. While the mechanisms of alcohol-induced cell injury and disease remain to be investigated, recent studies indicate that reactive oxygen species (ROS) may play an important role. Reactive oxygen species are able to cause various cellular injuries, such as DNA damage, lipid peroxidation and protein modication. Cellular systems are protected from ROS-induced cell injuries by an array of defenses composed of various anti-oxidants with different functions. When the ROS present in the cellular system overpower the defense systems, they will cause oxidative stress or cell injury, leading to the development of diseases. This article reviews recent literature on alcoholinduced ROS production, oxidative stress, signal transduction, and cellular responses. The implication of these processes in alcohol-related diseases is also discussed.

Shi X Key words cell injury, free radicals, oxidative stress, signal transduction. Correspondence Xianglin Shi, Institute for Nutritional Sciences, Chinese Academy of Sciences, 294 Taiyuan Road, Shanghai 200031, China. Email: xlshi@sibs.ac.cn

system. This radical can be converted to H2O2 through dismutation and to OH in the presence of certain transition metal ions by HaberWeiss reaction and Fenton-like reactions:15,16 M(n+1)+ + O2 Mn+ + O2 M + H2O2 M
n+ (n+1)+

(1)

+ OH + OH (Fenton-like reaction)

(2)

Reactive oxygen species and oxidative stress

While the mechanisms of alcohol-related cellular damage remain to be investigated, excessive generation of molecules or species called free radicals is believed to play a central role in many pathways of alcohol-induced damage.113 Free radicals can result in a state called oxidative stress, which is characterized by a disturbance in the balance between free radical generation and free radical scavenging, including repair of damaged molecules. A free radical is a cluster of atoms containing at least one unpaired electron. Most of the free radical species are unstable. In this conguration, the radicals tend to react with surrounding molecules or radicals to achieve a stable conguration.14 Any free radical having at least one oxygen in its structure can be dened as reactive oxygen species (ROS), representing a class of molecules mainly including superoxide anion radical (O2), hydrogen peroxide (H2O2) and highly reactive hydroxyl radical (OH). Hydrogen peroxide is not a free radical and it does not have free electron in its structure. However, this molecule can be easily converted to OH in the presence of a transition metal and is considered to be the precursor of OH. It is an important member of the ROS family. The O2 plays a central role in the generation of other ROS, such as H2O2 and OH and is continuously produced in the cellular
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By adding equations (1) and (2), one obtains O 2 + H 2 O 2 M /M O 2 + OH + OH (Haber Weiss-like reaction)
n+ (n+1)+

(3)

where M(n+1)+ represents an oxidized metal and Mn+ represents the reduced one. Among the ROS family, OH radicals are the most short-lived and the most reactive. Cellular systems develop enzymatic and non-enzymatic mechanisms called anti-oxidant systems, for the removal of ROS. Among the cellular anti-oxidants, superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase are important. The function of SOD is to convert O2 to H2O2 and molecular oxygen, and that of GPx and catalase, to convert H2O2 to water.17,18 Normally, ROS are produced from endogenous sources as byproducts of normal and essential metabolic reactions, such as mitochondria energy generation or the detoxication reactions involving the cytochrome P-450 enzyme system.19 Exogenous sources include exposure to cigarette smoke,20 consumption of alcohol in excess13 and exposure to ionizing radiation.21 Reactive oxygen species are able to (i) cause permanent structural changes in DNA as base-pair mutations, deletions, insertions, rearrangements, and sequence amplication; (ii) initiate lipid peroxidation; (iii) activate cytoplasmic and nuclear signal transduction

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Alcohol and oxidative stress

pathways; and (iv) modulate the activity of stress proteins and stress genes that regulate effector genes related to growth, differentiation, and cell death. Cellular systems are protected from ROS-induced cell injuries by various anti-oxidants with different functions. They suppress the generation of ROS (rst-line defense), scavenge and remove ROS (second-line defense), and detoxify, repair and reconstitute the damage (third-line defense). When the ROS present in the cellular system overpower the defense systems, they will cause cell injury, or oxidative stress, leading to the development of various diseases.

Alcohol induces oxidative stress

Alcohol is metabolized in two steps. First, alcohol dehydrogenase converts alcohol to acetaldehyde, a toxic and reactive molecule. Next, aldehyde dehydrogenase converts acetaldehyde to acetate. Each of these reactions leads to the formation of one molecule of nicotinamide adenine dinucleotide, reduced form (NADH), enhancing the activity of the respiratory chain, including increased O2 consumption and ROS formation.22 In the setting of alcoholism, other sources of ROS could be NADH-dependent cytochrome C reductase, aldehyde oxidase, xanthine oxidase, and neutrophil nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase. Alcohol consumption increases iron absorption from the gut with its consequent accumulation in the liver. Most of the cellular sources of ROS are associated with the generation of O2 and H2O2, which are precursors of the OH radical. The direct reaction between O2 and H2O2 is slow. Transition metals such as iron, chromium, and copper will catalyze this reaction. Due to the extremely reactive nature of this radical, it reacts with essentially any chemical species and will not travel a long distance from the site of its generation. If the metal ion is at or near the target, the OH radical will generate at or near the target and causes damage through a so-called site-specic reaction. It has been reported that iron is able to enhance ROS production in the liver.23 These reactive oxygen species, particularly the OH radical, could cause oxidative stress and play an important role in alcohol-induced liver damage. Anti-oxidant intervention could be an approach for protection of alcohol-induced liver injury through inhibition of oxidative stress. Previous studies have demonstrated that alcohol increases the activity of cytochrome P450 2E1 (CYP2E1), which can metabolize alcohol and generate ROS. While detailed studies remain to be done to understand the role of CYP2E1, it is believed that ethanol upregulates the CYP2E1 protein level and its activity by stabilizing the enzyme against proteasome-mediated degradation. The CYP2E1 constitutes the microsomal ethanol oxidizing system, which is inducible by higher amounts of ethanol and other xenobiotics.24 The degree of CYP2E1 induction can be correlated with generation of ROS, in particular hydroxyethyl radicals and lipid peroxides.25 In the presence of oxygen, O2 and H2O2 are produced during the CYP2E1 catalytic cycle.26 In the presence of a transition metal, these reactive oxygen species will be converted to OH radicals, leading to protein oxidation and lipid peroxidation. During these processes, lipid- and lipid peroxide-derived free radicals will be produced. It is likely that all of these reactive species contribute to the CYP2E1 toxicity and mitochondrial injury.6 These reactive species may interact with stellate cells to initiate a brogenic response.6

In neutrophils and some other cell types, the small GTPase Rac has been found to be associated with production of ROS. A number of studies using different cell types have identied small GTP-binding proteins as targets of alcohol toxicity. For example, ethanol exposure increases the GTP-bound forms of several GTPases including ras in mouse liver, RhoA in fetal rat astrocytes, and cdc42 in SVEC410 cells.26 Some other factors may also have an effect on oxidative stress induced by alcohol in the mammalian cell. For example, it has been reported that ethanol is able to generate ROS in the cells overexpressing ErbB2. Using the spin trapping method with 5,5dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap, a recent study has shown that the cells overexpressing ErbB2 generated OH radicals at a yield much higher than that using wild-type cells. ErbB2 belongs to the ErbB family of receptor kinases, which includes three other closely related members: epidermal growth factor receptor (EGFR or ErbB1), ErbB3, and ErbB4. The aforementioned studies indicate that the receptor kinases may play a role in alcohol-induced ROS generation. Although various studies have indicated that alcohol exposure can lead to the generation of ROS in vitro, the in vivo generation has not been demonstrated. The detection of free radical generation in living animals is important to demonstrate the role of ROS in the mechanism of alcohol-induced cell injury. The difculty of in vivo radical detection may be due to their very short lifetime and very low steady-state concentration, as well as lack of appropriate electron spin resonance (ESR) instrumentation and techniques to detect these transient species. The ESR method is the preferred choice for the detection of free radicals, in general, due to its specicity for free radicals. In the past several years, new spin trapping agents have been developed and the sensitivity of ESR instrumentation has been very much improved. Now, the previously undetectable and unidentiable free radicals generated in cellular and in vivo systems may become detectable and identiable. This in vivo ESR technique could be used to examine free radical generation in living animals as a whole or in specic organs of animals exposed to ethanol.

Cell signaling in alcohol-induced oxidative stress

Oxidative stress may lead to the stimulation of inammatory processes involving secretion of chemotactic factors, growth factors, proteolytic enzymes, lipoxygenases, cycloxygenase, inactivation of antiproteolytic enzymes, and the release of signaling proteins.27 The ROS may act as second messengers and activate intracellular signal cascade. Ethanol is a tumor promoter and may enhance the invasion and metastasis of breast cancer, colon cancer, and liver cancer. Multiple mechanisms are involved in alcohol-associated cancer development, including the effect of acetaldehyde (AA), which is the rst metabolite of ethanol oxidation and the induction of CYP2E1, which generates ROS and enhances procarcinogen activation.28 Matrix metalloproteinase-2 (MMP-2) is also a target of ethanol and its expression is associated with the status of ErbB2. Ethanol activated MMP-2 in human mammary epithelial cells (HB2) overexpressing ErbB2, but not in wild-type HB2 cells. It enhances the cleavage of the inactive form of MMP-2 (72 kDa) to an active form (62 kDa). The activation was dependent on c-jun N-terminal
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Alcohol and oxidative stress

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kinases (JNK) and ROS. In contrast, ethanol affects neither the expression nor the activation of MMP-9.29 Ethanol activated conventional protein kinase C (PKC) and JNK in broblasts. Inhibitors of PKC (Go6850 and Go6976) and JNK (SP600125) signicantly decreased ethanol-mediated MMP-2 activation as well as cell invasion, indicating that PKC and JNK play a role in ethanolinduced MMP-2 activation and cell invasion in vitro. Ethanolpromoted breast cancer cell invasion may be mediated by the modulation of broblastic MMP-2, which is dependent on JNK, ROS, and PKC. Meanwhile, ethanol-stimulated invasion of cells overexpressing ErbB2 was mediated, at least partially, by MMP-2 activation.30 Ethanol can stimulate cell proliferation independent of ErbB2 levels. In the cells overexpressing ErbB2, ethanol was more effective in the activation of JNK and p38 mitogen-activated protein kinase (p38) than the cells with normal ErbB2 expression.12 Ethanol promotes phosphorylation of conventional PKC in broblasts, demonstrating that the PKCMMP-2 pathway plays an important role in ethanol-stimulated cell invasion. A recent study has shown that ethanol stimulation altered the integrity of actin laments and increased the formation of lamellipodia and lopodia in a mouse lymph node endothelial cell line (SVEC410) cells.13 Further experiments demonstrated that ethanol stimulation increased cell migration and invasion and induced in vitro angiogenesis in SVEC4-10 cells. Mechanistically, ethanol stimulation activated Cdc42 and produced H2O2 a reactive oxygen species intermediate in SVEC4-10 cells. Measurement of the time course of Cdc42 activation and H2O2 production upon ethanol stimulation showed that the Cdc42 activation and the increase of H2O2 lasted more than 3 h, which indicates the long duration effects of ethanol on the cells. Furthermore, either overexpression of a constitutive dominant negative Cdc42 or inhibition of H2O2 production abrogated the effects of ethanol on SVEC4-10 cells, indicating that both the activation of Cdc42 and the production of H2O2 are essential for the actions of ethanol. The overexpression of a constitutive dominant positive Cdc42 itself was sufcient to produce H2O2 and to induce in vitro angiogenesis. Taken together, these results suggest that ethanol stimulation can induce H2O2 production through the activation of Cdc42, which results in reorganizing actin laments and increasing cell motility and in vitro angiogenesis. While Cdc42 is important in ethanol-induced ROS generation, it appears that NADPH oxidase is not involved in this process because Cdc42 cannot activate NADPH oxidase.31 Further studies remain to be done to elucidate the Cdc42-regulated ROS generation after alcohol exposure. Growth factor receptors, particularly receptor tyrosine kinases, play an important role in modulating many biological effects of ethanol. The effect of ethanol on the activity of transcription factor activator protein-1 (AP-1) is dependent on the expression of epidermal growth factor receptor (EGFR). Transcription factor activator protein-1 is an oxidant-responsive protein and is a transcription factor that controls the transcription of many genes responsible for the regulation of cell proliferation, differentiation, transformation, and survival. It consists of a family of Jun/Fos dimers that include different Jun proteins (c-Jun, JunB and JunD) and Fos proteins (c-Fos, FosB, Fra-1, Fra-2 and FosB2). Ethanol inhibited AP-1 activity in broblast cells that expressed either a wild-type EGFR or a kinase-inactive receptor in a concentrationdependent manner, but had little effect on AP-1 activity in the broblast cells devoid of EGFR. Ethanol inhibited EGF-induced
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EGFR autophosphorylation, phosphorylation of extracellular signal-regulated kinase (ERK) as well as AKT/PKB and its substrate glycogen synthase kinase 3 beta (GSK-3), and subsequently blocked EGF-stimulated AP-1 activation. In contrast, ethanol had little effect on EGF-stimulated JNK activation. Ethanol also inhibited 12-O-teradecanoyl-phorbol-13acetate (TPA)-induced AP-1 activation in an EGFR-dependent manner, but to a much lesser extent. Ethanol selectively inhibited TPA-induced phosphorylation of ERK and PKC, and modestly suppressed TPA-stimulated AP-1 activation. Thus, EGFR plays a critical role in the interaction between ethanol and AP-1. It has also been demonstrated that EGFR is a critical signaling component, which determines cellular susceptibility to ethanol exposure.32 Transcription factor activator protein-1 is a known target of ethanol exposure during neuronal development. It has been shown that ethanol can also block insulin-stimulated phosphoinositide-3-kinase (PI3K)/Akt activation in cerebellar neurons.33 Ethanol selectively downregulates the expression of certain cyclins, such as cyclin A and D, in the postnatal cerebellum and cultured cerebellar granule progenitors.34,35 Protein kinase C plays an important regulatory role in astrocyte function. Chronic exposure to ethanol for 4 days resulted in an increase in Ca2+dependent PKC activity in the supernatant fraction of astrocyte homogenates.36 It should be noted that most of the signal proteins mentioned here, such as JNK, PKC, p38, Cdc42, AP-1, EGFR, PI3K, Akt, and cyclin A and D, are oxidative responsive proteins. Their activities could be regulated by alcohol-induced oxidative stress. Investigation of alcohol-induced signal transduction through oxidative stress could be very important in understanding the mechanism of alcohol-induced diseases.

References
1 Purohit V, Khalsa J, Serrano J. Mechanism of alcohol-associated cancers: introduction and summary of the symposium. Alcohol 2005; 35: 15560. 2 Petersen DR. Alcohol, iron-associated oxidative stress, and cancer. Alcohol 2005; 35: 2439. 3 Navasumrit P, Ward TH, Dodd NJF, OConnor J. Ethanol-induced free radicals and hepatic DNA strand breaks are prevented in vivo by antioxidants: effects of acute and chronic ethanol exposure. Carcinogenesis 2000; 21: 939. 4 Luczaj W, Waszkiewicz E, Skrzydlewska E, Roszkowska-Jakimiec R. Green tea protection against age-dependent ethanol-induced oxidative stress. J. Toxicol. Environ. Health 2004; 67: 595606. 5 Cohen-Kerem R, Koren G. Antioxidants and fetal protection against ethanol tetragenicity, I: review of the experimental data and implications to humans. Neurotoxicol. Teratol. 2003; 25: 19. 6 Wu D, Cederbaum AI. Oxidative stress mediated toxicity exerted by ethanol-inducible CYP2E1. Toxicol. Appl. Pharmacol. 2005; 207: S570S76. 7 Wu D, Cederbaum AI. Alcohol, oxidative stress, and free radical damage. Alcohol Res. Health 2003; 27: 27784. 8 Badger TM, Ronis MJ, Seitz HK, Albano E, Ingelman-Sundberg M, Lieber CS. Alcohol metabolism: role in toxicity and carcinogenesis. Alcohol. Clin. Exp. Res. 2003; 27: 33647. 9 Deugnier Y. Iron and liver cancer. Alcohol 2003; 30: 14550. 10 Andican G, Gelisgen R, Unal E et al. Oxidative stress and nitric oxide in rats with alcohol-induced acute pancreatitis. World J. Gastroenterol. 2005; 11: 23405.

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D Wu et al.

Alcohol and oxidative stress

11 Campos SC, Moreira DA, Nunes TD, Colepicolo P, Brigagao MR. Oxidative stress in alcohol-induced rat parotid sialadenosis. Arch. Oral Biol. 2005; 50: 6618. 12 Ma C, Lin H, Leonard SS, Shi X, Ye J, Luo J. Overexpression of ErbB2 enhances ethanol-stimulated intracellular signaling and invasion of human mammary epithelial and breast cancer cells in vitro. Oncogene 2003; 22: 528190. 13 Qian Y, Luo J, Leonard Y et al. Hydrogen peroxide formation and actin lament reorganization by Cdc42 are essential for ethanolinduced in vitro angiogenesis. J. Biol. Chem. 2003; 278: 16 18997. 14 Karlsson J. Introduction to nutraology and radical formation. In: Antioxidants and Exercise. Illinois: Human Kinetics Press, 1997; 1143. 15 Halliwell B, Gutteridge JMC. Oxygen toxicity, oxygen radicals, transition metals, and disease. J. Biochem. 1984; 219: 114. 16 Chen F, Shi X. Intracellular signal transduction of cells in response to carcinogenic metals. Crit. Rev. Oncol. Hematol. 2002; 42: 10521. 17 Li S, Yan T, Yang JQ, Oberley TD, Oberley LW. The role of cellular glutathione peroxidase redox regulation in the suppression of tumor cell growth by manganese superoxide dismutase. Cancer Res. 2000; 60: 392739. 18 Amstad P, Peskin A, Shah G et al. The balance between Cu,Znsuperoxide dismutase and catalase affects the sensitivity of mouse epidermal cells to oxidative stress. Biochemistry 1991; 30: 930513. 19 Paolini M, Perocco P, Canistro D et al. Induction of cytochrome P450, generation of oxidative stress and in vitro cell-transforming and DNAdamaging activities by glucoraphanin, the bioprecursor of the chemopreventive agent sulforaphane found in broccoli. Carcinogenesis 2004; 25: 617. 20 Serikov VB, Leutenegger C, Krutilina R et al. Cigarette smoke extract inhibits expression of peroxiredoxin v and increases airway epithelial permeability. Inhal. Toxicol. 2006; 18: 7992. 21 Martinez-Solano JR, Sanchez-Bel P, Egea I, Olmos E, Hellin E, Romojaro F. Electron beam ionization induced oxidative enzymatic activities in pepper (Capsicum annuum L.), associated with ultrastructure cellular damages. J. Agric. Food Chem. 2005; 53: 85935. 22 Halliwell B. Antioxidant defense mechanisms: from the beginning to the end. Free Radic. Res. 1999; 31: 26172. 23 Tsukamoto H, Horne W, Kamimura S et al. Experimental liver cirrhosis induced by alcohol and iron. J. Clin. Invest. 1995; 96: 62030.

24 Koop DR, Crump BL, Nordblom GD, Coon MJ. Immunochemical evidence for induction of the alcohol-oxidizing cytochrome P-450 of rabbit liver microsomes by diverse agents: ethanol, imidazole, trichloroethylene, acetone, pyrazole, and isoniazid. Proc. Natl Acad. Sci. USA 1985; 82: 40659. 25 Albano E, Clot P, Morimoto M, Tomasi A, Ingelman-Sundberg M, French SW. Role of cytochrome P4502E1-dependent formation of hydroxyethyl free radical in the development of liver damage in rats intragastrically fed with ethanol. Hepatology 1996; 23: 15563. 26 Sebastian BM, Nagy LE. Decreased insulin-dependent glucose transport by chronic ethanol feeding is associated with dysregulation of the Cbl/TC10 pathway in rat adipocytes. Am. J. Physiol. Endocrinol. Metab. 2005; 289: 107784. 27 Leonard SS, Harris GK, Shi X. Metal-induced oxidative stress and signal transduction. Free Radic. Biol. Med. 2004; 37: 192142. 28 Poschl G, Seitz HK. Alcohol and cancer. Alcohol 2004; 39: 15565. 29 Ke Z, Lin H, Fan Z et al. MMP-2 mediates ethanol-induced invasion of mammary epithelial cells over-expressing ErbB2. Int. J. Cancer 2006; 119: 816. 30 Aye MM, Ma C, Lin H, Bower KA, Wiggins RC, Luo J. Ethanolinduced in vitro invasion of breast cancer cells: the contribution of MMP-2 by broblasts. Int. J. Cancer 2004; 112: 73846. 31 Werner E. GTPases and reactive oxygen species: switches for killing and signaling. J. Cell Sci. 2004; 117: 14353. 32 Ma C, Bower KA, Lin H et al. The role of epidermal growth factor receptor in ethanol-mediated inhibition of activator protein-1 transactivation. Biochem. Pharmacol. 2005; 69: 178594. 33 de la Monte S, Wands JR. Chronic gestational exposure to ethanol impairs insulin-stimulated survival and mitochondrial function in cerebellar neurons. Cell. Mol. Life Sci. 2002; 59: 88293. 34 Li Z, Lin H, Zhu Y, Wang M, Luo J. Disruption of cell cycle kinetics and cyclin-dependent kinase system by ethanol in cultured cerebellar granule progenitors. Brain Res. Dev. Brain Res. 2001; 132: 4758. 35 Li Z, Miller MW, Luo J. Effects of prenatal exposure to ethanol on the cyclin-dependent kinase system in the developing rat cerebellum. Brain Res. Dev. Brain Res. 2002; 139: 23745. 36 Smith TL, Bitrick MS. Ethanol enhances the in situ phosphorylation of MARCKS and protein kinase C activity in primary cultures of astrocytes. Life Sci. 1996; 58: 85560.

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