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Effects of chronic ethanol feeding and thiamin deficiency on antioxidant defenses in kidney and lung of rats
Nutrition Research 22 (2002) 835– 845 www.elsevier.com/locate/nutres
Effects of chronic ethanol feeding and thiamin deficiency on antioxidant defenses in kidney and lung of rats L.H. Chena,*, V. Thielena, R. Cicciab, P.J. Langlaisb a
Graduate Center for Nutritional Sciences, University of Kentucky, Lexington, Kentucky 40506, USA Behavioral Neurobiology Laboratory, San Diego State University, San Diego, California 92120, USA
b
Received 24 June 2001; received in revised form 3 April 2002; accepted 6 April 2002
Abstract The effects of a long-term ethanol consumption and thiamin deficiency on the antioxidant defense systems of the kidney and lung were studied in 32 male Sprague-Dawley rats. They were divided into four groups: Control, Ethanol (EtOH), Thiamin-Deficient (TD), and EtOH/TD. Rats in the EtOH and EtOH/TD groups received 20% ethanol in the drinking water, while rats in the TD and EtOH/TD groups received three bouts of the TD diet in order to mimic human behavior of binge drinking. The rats were sacrificed after 30 weeks, and the kidney and lung were excised for biochemical determinations. The results demonstrated that GSH levels and SOD activity were suppressed in both tissues, and catalase and GPx activities in the kidney was increased by EtOH or EtOH/TD diet when compared to the control. The results suggest that long-term ingestion of EtOH, but not mild thiamin deficiency, moderately increases the susceptibility of the kidney and lung to oxidative stress. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Ethanol; Thiamin Deficiency; Glutathione; Catalase; Superoxide Dismutase
1. Introduction Increasing evidence supports the hypothesis that ethanol-induced tissue damage may be a consequence of oxidative stress and nutritional deficiencies, including thiamin and other vitamins. Alcoholics can develop thiamin deficiency due to thiamin malabsorption [1] and
* Corresponding author. Graduate Center for Nutritional Sciences, 204 Funkhouser Building, University of Kentucky, Lexington, Kentucky, 40506-0054. Tel.: ⫹1-859-257-3288; fax: ⫹1-859-257-3288. E-mail address: [email protected] (L.H. Chen). 0271-5317/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 2 ) 0 0 3 9 8 - 6
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poor diet, and it is generally accepted as the primary etiologic factor responsible for the Wernike-Korsakoff syndrome in these individuals. A decrease in the concentration of the coenzyme of thiamin, thiamin pyrophosphate (TPP), has also been observed in alcoholics [2,3]. The activity of thiamin pyrophosphokinase (the TPP synthesizing enzyme) significantly decreases, while the activities of thiamin pyrophosphatase (the enzyme dephosphorylating TPP) and thiamin monophosphatase (the enzyme dephosphorylating thiamin monophosphate) significantly increase in the liver and kidney of rats with chronic ethanol intake [4]. One of the three major TPP-dependent enzymes is transketolase (TK), a key enzyme of the hexose monophosphate shunt. TK is responsible for the generation of NADPH and maintenance of the levels of reduced glutathione (GSH), since NADPH is the coenzyme for glutathione reductase (GR) catalyzed reaction. Extensive studies have shown that ethanol elicits an enhancement in the production of reactive oxygen species (ROS) in the microsomes [5] and mitochondria [6] that may cause oxidative stress [5,7,8]. Oxidative stress is defined as the inability of the cells to defend against ROS through imbalance of the prooxidants and antioxidant defenses toward prooxidants. The cellular antioxidant defense systems mainly include glutathione (GSH) and antioxidant defense enzymes. GSH, a tripeptide, is considered one of the most important non-enzymatic antioxidants [9] and also serves as the substrate for glutathione peroxidase (GPx). Many proteins require their thiol groups in the reduced form in order to be functional, and GSH protects the thiol groups by removing peroxides that can oxidize sulfhydryl groups. The major enzymatic antioxidant system includes superoxide dismutase (SOD), catalase, GPx and GR. SOD catalyzes the dismutation of superoxide anion (O⫺䡠 2 ) to form hydrogen peroxide (H2O2) [10]. Further reduction of H2O2 can be carried out by catalase-dependent reaction. GPx catalyzes the reduction of H2O2 and peroxides using GSH as the substrate and producing oxidized glutathione (GSSG). In the presence of NADPH, GR catalyzes the regeneration of GSH from GSSG, and is therefore essential to the maintenance of an appropriate intracellular GSH redox status. Chronic ethanol ingestion causes toxic effects to the kidney and lung as well as the liver. Unlike the well-studied hepatotoxic effects of ethanol, the mechanisms of pathogenesis in the kidney and lung are not clearly understood. Ethanol may cause functional abnormalities in the kidney and lung depending on the quantity ingested and the duration of drinking. A possible link between alcoholism and glomerulonephritis [11] has been reported. Alcohol may also cause acute tubular necrosis [12]. In addition, nephrotoxic effects of acetaldehyde should not be ruled out because increased activities of alcohol dehydrogenase and catalase in the kidney [13] have been found in chronic alcoholism. Ethanol-fed rats have larger kidneys with significant increases in protein and lipid contents, and show reduction in creatinine clearance and osmotic clearance [14]. Furthermore, varying degrees of cellular injury are found in renal epithelial cells, particularly in the distal tubules and Henle’s loops [14]. Chronic respiratory disorders, including bronchitis, emphysema, pulmonary fibrosis, bronchietactsis and chronic airway obstruction are more common in alcoholics than in non-alcoholics [15]. The high incidence of chronic lung diseases in alcoholics is usually explained on the basis of poor nutrition, frequent infections in the respiratory tract and excessive smoking. While there is no doubt these factors contribute significantly, an association of pulmonary disease and direct damage of the lung by ethanol has been postulated
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[16] since the alveolar epithelial surface is under constant pressure of high oxygen pressure that makes the lung highly susceptible to ROS generation. Thiamin deficiency has been found to increase the production of ROS in the brain [17]. Therefore, it is important to study the effects of ethanol alone or in combination with TD diet because thiamin deficiency is a consequence of chronic alcoholism. We previously reported that long-term ethanol ingestion suppressed antioxidant defenses in the liver, and thiamin deficiency further suppressed those defenses [18]. The present work was undertaken to investigate whether long-term EtOH administration and thiamin deficiency, alone or combined, would affect the antioxidant defense systems in the kidney and lung of rats.
2. Materials and methods 2.1. Treatment of animals The treatment of rats was the same as that described by Chen et al. [18]. Male SpragueDawley rats, 2 months old, were used in this study. The animals were fed and treated at the Behavioral Neurobiology Laboratory at San Diego State University. All animals were individually housed and maintained on a regular lab chow diet and water ad lib. After a 3-day acclimation period, the rats began the experimental phase of this study. The dietary manipulation phase of the experiment lasted 30 weeks. A total of 32 rats were randomly assigned to one of the following treatment groups: Control, Ethanol (EtOH), Thiamin-Deficient (TD), and Ethanol/Thiamin-Deficient (EtOH/TD) groups. During the 30 weeks of treatment, all animals received regular lab chow, except during the three TD bouts when they received a diet deficient in thiamin in order to mimic a human pattern of alcohol bingeing. The three TD bouts began on weeks 10, 18 and 26 and lasted approximately four weeks. Except during the TD bouts, the rats received 1.0 mg/kg body weight of thiamin intraperitoneally (i.p.) three times a week. The TD diet (Tekled Diets) was a modified AIN-76 diet excluding thiamin, and consisted of casein, 19.1%; sucrose, 51.8%; corn starch, 15%; corn oil, 5%; mineral mix, 3.5% and vitamin mix except thiamin, 2.19 g/kg diet. Control and TD rats had tap water as the drinking fluid, whereas EtOH and EtOH/TD rats received an aqueous ethanol solution as the sole drinking fluid. The ethanol dose was gradually increased by 2% (v/v) increments every other day until a 20% (v/v) ethanol concentration was reached (approximately three weeks). During each TD bout, all animals received TD diet and continued to drink the same fluid they had been consuming. Also during the TD bouts, animals from the control and the EtOH groups were injected i.p. with thiamin (1 mg/kg body weight), while animals in the TD and the EtOH/TD groups received i.p injections of 0.9% normal saline instead of thiamin three times a week. Rats were examined and weighed weekly. Physical and behavioral changes such as slowed activity, wide stance, ataxic gait, opisthotonus, circling, retropulsion, hypersensitivity, impaired righting reflex and decreased grooming were monitored as indicators of thiamin deficiency. Animals that displayed more than one symptom of thiamin deficiency were monitored every day until reversed. If an animal showed a severely impaired righting reflex, the TD bout was reversed with a one time injection of thiamin (100 mg/kg body wt) and by changing the TD diet to regular lab chow. After Bout 1 and Bout 2, all
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remaining animals were reversed no later than four weeks using the same procedure as previously described. The animals were not reversed after Bout 3. At the end of the experimental period while the rats were still on TD diet, rats were anesthetized by CO2 inhalation (15 to 20 seconds), and killed by decapitation. The kidney and lung, were excised rapidly and frozen in dry ice. Specimens were frozen at -80°C, delivered in dry ice by air to the University of Kentucky, and kept frozen at -80°C until tissue preparation was performed. 2.2. Biochemical assays A 10% (w/v) tissue homogenate was prepared in 0.1 M phosphate buffer, pH 7.4, and immediately used for the determinations of GSH and protein concentrations. The remaining tissue homogenates were centrifuged at 3000 x g for 15 min at 4°C, to yield the supernatant which was used for the determinations of the activities of SOD, catalase, GPx, GR and GST as well as protein concentrations. GSH was determined by the method of Owen and Belcher [19] and SOD activity was determined by the method of McCord and Fridovich [20]. Catalase activity was determined by the method of Aebi [21], while GPx activity was determined by the method of Paglia and Valentine [22] using cumene hydroperoxide as the substrate. GR activity was determined by the method of Racker [23]. Protein concentrations in the tissue homogenate and supernatant were determined by the method of Lowry et al. [24].
Fig. 1. Effect of chronic consumption of ethanol (EtOH) and thiamin deficiency (TD) on reduced glutathione (GSH) levels. Means with different letters are significantly different at p ⬍ 0.05.
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Fig. 2. Effect of chronic consumption of ethanol (EtOH) and thiamin deficiency (TD) on superoxide dismutase (SOD) activity. Means with different letters are significantly different at p ⬍ 0.05.
2.3. Statistical analysis Data were analyzed by two way analysis of variance. When analysis of variance indicated significant differences, Fisher’s Least Significance Difference Test [25] was used to compare the treatment means. A probability of p ⱕ 0.05 was considered significant.
3. Results The body weights of the rats were similar in all four groups [18]. All data in the present study are expressed as mean ⫾ SE. As shown in Fig. 1, chronic consumption of ethanol significantly decreased GSH levels in the kidney (23%) and lung (32%) when compared to the respective control group. The combination of both ethanol consumption and TD diet also significantly suppressed GSH levels in the kidney (29%) and lung (35%) when compared to the control group, respectively. There was no significant difference between the GSH levels of the EtOH group and those of the EtOH/TD group in either tissue. Feeding a TD diet did not affect GSH levels in the two tissues. Chronic ethanol administration alone elicited a significant suppression of SOD activity (16%) when compared to the respective control group in both the kidney and lung (Fig. 2). The combination of ethanol and the TD diet also significantly suppressed SOD activity in the kidney (19%) and lung (17%) when compared to the respective control group. However, there was no significant difference between the SOD activity of the EtOH group and that of the EtOH/TD group in either tissue. Feeding TD diet alone did not produce changes on SOD activity in the tissues studied. As depicted in Fig. 3, catalase activity was substantially
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Fig. 3. Effect of chronic consumption of ethanol (EtOH) and thiamin deficiency (TD) on catalase activity. Means with different letters are significantly different at p ⬍ 0.05.
different between the kidney and lung, not only in the control group but also in response to the treatments. Catalase activity in the kidney was approximately 10 fold greater than that in the lung. In the kidney, catalase activity was significantly increased not only by ethanol (15%) but also by the combination of ethanol and TD diet (19%) when compared to the control group. However, there was no significant difference between the catalase activity of the EtOH group and that of the EtOH/TD group. Whereas EtOH consumption alone, TD diet alone or the combination of the two treatments did not affect catalase activity in the lung. Fig. 4 shows that GPx activity was significantly increased by ethanol consumption (17%) or ethanol consumption in combination with TD diet (21%) in the kidney. However, in the lung neither ethanol nor EtOH/TD diet affected GPx activity. In Fig. 5, it can be seen that no alteration on GR activity was observed in both tissues of rats fed ethanol alone, TD diet alone or combination of the two treatments.
4. Discussion This study examined a much longer period (30 weeks) of alcohol ingestion on tissue antioxidant defense systems of the kidney and lung than most reported studies. To our knowledge this is the first paper that examined the combined effects of ethanol consumption and mild thiamin deficiency on GSH and the antioxidant defense enzymes of these tissues. GSH plays an important role in maintaining the integrity of mitochondria, cell membranes and cell systems. Therefore, a decrease in GSH levels can decrease the effectiveness of the antioxidant defense systems and lead to oxidative injury. The results from the present study showed that GSH levels were significantly suppressed after chronic ethanol consumption in
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Fig. 4. Effect of chronic consumption of ethanol (EtOH) and thiamin deficiency (TD) on glutathione peroxidase (GPx) activity.
both tissues, and they were suppressed to about the same levels with combined treatment of ethanol and TD diet. Thus, the suppressive effects appear to be mainly attributed to ethanol consumption, but not to TD diet. Our results are consistent with those of Fernandez and Videla that chronic ethanol feeding decreased GSH levels in the kidney [26]. However, Rikans and Gonzalez reported that GSH levels in the lung were not affected by chronic
Fig. 5. Effect of chronic consumption of ethanol (EtOH) and thiamin deficiency (TD) on glutathione reductase (GR) activity.
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ethanol inhalation [27]. The different results in the lung can be explained by the different mode of ethanol administration (drinking versus inhalation) and the length of experimental period (30 weeks versus 35 days). The suppression of GSH appears to be a consequence of oxidative stress in rats fed ethanol when ROS are generated in large amount [5,28]. Generation of ROS during ethanol oxidation enhances the demand in the utilization of the antioxidant. Increased oxidation of GSH [29,30] and binding of GSH or its metabolite cysteinyl-glycine with acetaldehyde (the major metabolite of ethanol oxidation) through its cysteine residue [26,31] might also contribute to the suppression of GSH levels. Enhanced alcohol dehydrogenase activity and consequent increase in acetaldehyde production in the kidney were observed by Orellana et al. after rats were fed 20% ethanol solution for 10 weeks [13]. To our knowledge, data on ethanol-induced alterations on SOD activity in the kidney and lung after long-term consumption of ethanol have not been reported. However, Rikans and Gonzalez have reported a significant increase in SOD activity in the lung of rats exposed to ethanol vapors for 35 days [27]. The increase in SOD activity is explained as an adaptive response to mild oxidative stress [27]. It is well established that oxidant gases, such as ozone, nitrogen dioxide and 100% oxygen, cause an induction in lung antioxidant defense enzymes [32]. The discrepancies in the results of our study and those of Rikans and Gonzalez can again be explained by different modes of ethanol administration and treatment periods. Chronic ethanol consumption significantly decreased SOD activity in other tissues, including the liver [18,33] and the brain [34]. Taking into account that SOD is a sulfhydryl enzyme, suppression of its activity could be a consequence of oxidative damage [35] since excessive levels of H2O2 produced after ethanol oxidation could oxidize the sulfhydryl enzyme [36]. One important consequence of the decreased activity of SOD might be the increased 䡠 production of O⫺䡠 2 and further formation of OH (hydroxyl radical) through an iron-catalyzed Haber-Weiss reaction. The hydroxyl radical produced may lead to structural and functional disturbance of mitochondria [28] and increased susceptibility to oxidative injury. Therefore, with a decrease in GSH levels and SOD activity, the kidney and lung might become more vulnerable to oxidative stress. Contrasting with the observed decrease in GSH levels and SOD activity, the activity of catalase in the kidney was significantly increased after rats were fed either ethanol or the combination of ethanol and TD diet. This result is consistent with the increase of catalase in the kidney shown by Orellana et al. [13]. Chen et al. also reported that catalase activity was significantly increased in the liver of rats fed ethanol [18,33]. Catalase not only removes H2O2 produced by SOD-catalyzed reaction, but it is also a pathway for ethanol oxidation and a component of the peroxisomal fatty acid oxidation system. Therefore, increased catalase activity might be caused by increased substrates: H2O2 and ethanol. In fact, ethanol consumption enhances extra-mitochondrial oxidation of fatty acids in the kidney and consequently it may also increase the production of H2O2 [13]. Our study demonstrated that ethanol consumption significantly increased GPx activity in the kidney, but not in the lung. Since catalase activity was significantly increased in the kidney, the production of H2O2 could also be increased, and thus might induce GPx due to the increase in the substrate level. Rikans and Gonzalez have reported that there is no change in GPx activity in the lung of rats exposed to ethanol vapors for 35 days [27]. The
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discrepancies in the results of our study and those of Rikans and Gonzalez can again be explained by different modes of ethanol administration and treatment periods. When compared with reports in different tissues, GPx activity in the liver was decreased [18], but that in the myocardium was not altered [37] by ethanol administration. The present study showed no change on GR activity in the kidney or the lung, although chronic consumption of ethanol has been found to increase the activity of this enzyme in the liver [18] that is the major organ to synthesize GSH. GR is an inducible enzyme that responds to suppressed GSH levels. Even though GSH levels were significantly suppressed in both tissues in this study, the extent of suppression was moderate (23–32%), and it appears that the GSH levels was not low enough to induce GR. Although simultaneous administration of ethanol and the TD diet caused a significant decrease of GSH levels or SOD activity in the kidney and lung, the effects are mainly due to ethanol, because thiamin deficiency in combination with ethanol did not further suppress GSH levels or SOD activity. Since thiamin deficiency was found to be synergistic to ethanol in suppressing GSH levels and SOD activity in the liver in our previous report [18], thiamin deficiency appears to only affect the antioxidant defenses of the liver, but not those of the kidney and lung. Zimatkin and Zimatkina [38] reported significant decreases on TK activity in the liver and brain of rats fed 15% alcohol, while the effect of chronic ingestion of ethanol on TK activity in the kidney and lung has not been reported. Given the fact that thiamin, as coenzyme of TK, is required for the regeneration of GSH from GSSG by providing NADPH, we can assume that thiamin deficiency could increase alcohol toxicity by suppressing GSH levels in the liver and brain, but not in the kidney and lung. Our earlier study on rats with the same treatments showed that the antioxidant defenses of the liver was significantly weakened [18]. When compared to the liver, there were lesser alterations of antioxidant defenses caused by ethanol administration in the kidney and lung. The differences in these tissues can be explained by the observation that liver microsomes can oxidize ethanol at rates several fold greater than the kidney and lung microsomes [39]. In summary, data in the present investigation revealed that long-term consumption of ethanol, but not thiamin deficiency, suppressed GSH levels and SOD activity in the kidney and lung. In addition, chronic consumption of ethanol increased catalase and GPx activities of the kidney. The results suggest that chronic consumption of ethanol, but not mild thiamin deficiency, moderately decreases the antioxidant defense capability of the kidney and lung; and the effects are greater in the kidney than the lung. Acknowledgments This work was supported by NIAAA Grant No. 10473 and KYAES S-521, Paper No. 01–10-55. References [1] Thomson AD, Baker H, Leevy CM. Patterns of 35S-thiamin hydrochloride absorption in the malnourished alcoholic patient. J Lab Clin Med 1970;76:34 – 45.
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[2] Herve C, Beyne P, Letteron, Delacoux E. Comparison of erythrocyte transketolase activity with thiamin and thiamin phosphate ester levels in chronic alcoholic patients. Clin Chim Acta 1995;234:91–100. [3] Poupon RE, Gervaise G, Riant P, Houin G, Tillement JP. Blood thiamin and thiamin phosphate concentrations in excessive drinkers with or without peripheral neuropathy. Alcohol Alcohol 1990;25:605–11. [4] Laforenza U, Patrini C, Gastaldi G, Rindi G. Effects of acute and chronic ethanol administration on thiamin metabolizing enzymes in some brain areas and in other organs of the rat. Alcohol Alcohol 1990;25:591– 603. [5] Cederbaun AI. Oxygen radical generation by microsomes: Role of iron and implications for alcohol metabolism and toxicity. Free Rad Biol Med 1989;7:537–9. [6] Freidovich I. Oxygen radicals from acetaldehyde. Free Rad Biol Med 1989;7:557– 8. [7] Shaw S, Jayatilleke E, Ross WA, Gorden ER, Lieber CS. Ethanol-induced lipid peroxidation: Potentiation by long-term alcohol feeding and attenuation by methionine. J Lab Clin Med 1981;98:417–24. [8] Diluzio DR, Hartman AD. Role of lipid peroxidation in the pathogenesis of alcohol-induced fatty liver. Fed Proc 1967;26:1436 – 42. [9] Mannervick B. The enzymes of glutathione metabolism: an overview. Biochem Soc Trans 1987;15:717–9. [10] Slot JW, Genze HI, Freeman BA, Crapo JD. Intracellular localization of copper-zinc and manganese superoxide dismutase in rat parenchymal cells. Lab Invest 1986;545:363–71. [11] Keller CK, Andrassy K, Waldherr R, Ritz E. Post infectious glomerulonephritis: is there a link to alcoholism? Quart J Med 1994:87–97. [12] Hirsch DJ, Jindal KK, Trillo A, Cohen AD. Acute renal failure after binge drinking. Nephr Dial Transplant 1994;9:330 –1. [13] Orellana M, Valdes E, Fernandez J, Rodrigo R. Effects of chronic ethanol consumption on extra-mitochondria fatty acid oxidation and ethanol metabolism by rat kidney. Gen Pharmacol 1998;30:719 –23. [14] Van Thiel DH, Williams Jr, WD, Gavaler JS, Little JM, Estes LW, Rabin BS. Ethanol-its nephrotoxic effect in the rat. Am J Pathol 1977;89:67– 84. [15] Krumpe PE, Commiskey JM, Lillington GA. Alcohol and the respiratory tract. Med Clin North Am 1984;68:201–19. [16] Burch GE, De Pasquale NP. Alcoholic lung diseases. An hypothesis. Am Heart J 1967;73:147– 8. [17] Langlais PJ, Anderson G, Guo SX, Bondy SC. Increased celebral free radical production during thiamin deficiency. Metab Brain Dis 1997;12:137– 43. [18] Chen LH, Zhao Y, Ciccia R, Langlais PJ. Effects of alcohol feeding and thiamin deficiency on antioxidant defenses in the liver and spleen of rats. Biochem Arch 1999;15:103– 6. [19] Owens CWI, Belcher RV. A colorimetric micro-method for the determination of glutathione. Biochem J 1965;94:705–11. [20] McCord JM, Fridovich F. Superoxide dismutase. An enzymatic function for erythrocuprein (hemocuprien). J Biochem 1969;244:6049 –55. [21] Aebi H. Catalase in vitro. Meth Enzymol 1984;105:121– 6. [22] Paglia DE, Valentine WN. Studies on the qnantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70:158 – 69. [23] Racker E. Glutathione reductase. Meth Enzymol 1955;2:722–5. [24] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with Folin phenol reagent. J Biol Chem 1951;193:265–75. [25] Senedecor AA. Statistical Methods. 7th ed. Ames, Iowa: Iowa State Press, 1974. [26] Fernandez V, Videla LA. Effects of acute and chronic ethanol ingestion on the content of reduced glutathione of various tissues of the rat. Experientia 1981;37:392– 4. [27] Rikans LE, Gonzalez LP. Antioxidant protection systems of rat lung after chronic ethanol inhalation. Alcohol Clin Exp Res 1990;14:872– 6. [28] Nordmann R, Ribiere C, Rouach H. Implication of free radical mechanisms in ethanol-induced cellular injury. Free Rad Biol Med 1992;12:219 – 40. [29] Morton S, Mitchell MC. Effects of chronic ethanol feeding on glutathione turnover in the rat. Biochem Pharmacol 1985;34:1559 – 63.
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[30] Altomare E, Vendemiale G, Grattagliano I, Albano, OJ. Increased plasma levels of glutathione and malonaldehyde after acute ethanol ingestion in humans. Hepatol 1989;9:359 – 65. [31] Lieber CS, Biochemical, and molecular basis of alcohol-induced injury to liver, and other tissues. N Engl J Med 1988;319:1639 –50. [32] Plaa GL, Witsch H. Chemicals, drugs, and lipid peroxidation. Ann Rev Pharmacol Toxicol 1976;16:125– 41. [33] Chen LH, Xi S, Cohen DA. Liver antioxidant defenses in mice fed ethanol and AIN-76A diet. Alcohol 1995;5:453–7. [34] Ledig M, M’pavia JR, Mandel P. Superoxide dismutase activity in rat brain during acute and chronic alcohol intoxication. Neurochem Res 1981;6:385–91. [35] Bondy SC, Lee DK. Oxidative stress induced by glutamate receptor agonist. Brain Res 1993;610:229 –33. [36] Hodgson EK, Fridovich I. The interaction of bovine erythrocyte superoxide dismutase with water: inactivation on the enzyme. Biochemistry 1975;14:5294 –9. [37] Ribiere C, Hininger I, Rouach H, Nordmann R. Effects of chronic ethanol administration on free radical defense in rat myocardium. Biochem Pharmacol 1992;44:1495–500. [38] Zimatkin SM, Zimatkina TI. Thiamin deficiency as predisposition to, and consequence of, increased alcohol consumption. Alcohol Alcohol 1996;31:421–7. [39] Winston GW, Cederbaum AI. A correlation between hydroxy radical generation, and ethanol oxidation by liver, lung, and kidney microsomes. Biochem Pharmacol 1982;31:2031–7.
Effects of chronic ethanol feeding and thiamin deficiency on antioxidant defenses in kidney and lung of rats L.H. Chena,*, V. Thielena, R. Cicciab, P.J. Langlaisb a
Graduate Center for Nutritional Sciences, University of Kentucky, Lexington, Kentucky 40506, USA Behavioral Neurobiology Laboratory, San Diego State University, San Diego, California 92120, USA
b
Received 24 June 2001; received in revised form 3 April 2002; accepted 6 April 2002
Abstract The effects of a long-term ethanol consumption and thiamin deficiency on the antioxidant defense systems of the kidney and lung were studied in 32 male Sprague-Dawley rats. They were divided into four groups: Control, Ethanol (EtOH), Thiamin-Deficient (TD), and EtOH/TD. Rats in the EtOH and EtOH/TD groups received 20% ethanol in the drinking water, while rats in the TD and EtOH/TD groups received three bouts of the TD diet in order to mimic human behavior of binge drinking. The rats were sacrificed after 30 weeks, and the kidney and lung were excised for biochemical determinations. The results demonstrated that GSH levels and SOD activity were suppressed in both tissues, and catalase and GPx activities in the kidney was increased by EtOH or EtOH/TD diet when compared to the control. The results suggest that long-term ingestion of EtOH, but not mild thiamin deficiency, moderately increases the susceptibility of the kidney and lung to oxidative stress. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Ethanol; Thiamin Deficiency; Glutathione; Catalase; Superoxide Dismutase
1. Introduction Increasing evidence supports the hypothesis that ethanol-induced tissue damage may be a consequence of oxidative stress and nutritional deficiencies, including thiamin and other vitamins. Alcoholics can develop thiamin deficiency due to thiamin malabsorption [1] and
* Corresponding author. Graduate Center for Nutritional Sciences, 204 Funkhouser Building, University of Kentucky, Lexington, Kentucky, 40506-0054. Tel.: ⫹1-859-257-3288; fax: ⫹1-859-257-3288. E-mail address: [email protected] (L.H. Chen). 0271-5317/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 2 ) 0 0 3 9 8 - 6
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poor diet, and it is generally accepted as the primary etiologic factor responsible for the Wernike-Korsakoff syndrome in these individuals. A decrease in the concentration of the coenzyme of thiamin, thiamin pyrophosphate (TPP), has also been observed in alcoholics [2,3]. The activity of thiamin pyrophosphokinase (the TPP synthesizing enzyme) significantly decreases, while the activities of thiamin pyrophosphatase (the enzyme dephosphorylating TPP) and thiamin monophosphatase (the enzyme dephosphorylating thiamin monophosphate) significantly increase in the liver and kidney of rats with chronic ethanol intake [4]. One of the three major TPP-dependent enzymes is transketolase (TK), a key enzyme of the hexose monophosphate shunt. TK is responsible for the generation of NADPH and maintenance of the levels of reduced glutathione (GSH), since NADPH is the coenzyme for glutathione reductase (GR) catalyzed reaction. Extensive studies have shown that ethanol elicits an enhancement in the production of reactive oxygen species (ROS) in the microsomes [5] and mitochondria [6] that may cause oxidative stress [5,7,8]. Oxidative stress is defined as the inability of the cells to defend against ROS through imbalance of the prooxidants and antioxidant defenses toward prooxidants. The cellular antioxidant defense systems mainly include glutathione (GSH) and antioxidant defense enzymes. GSH, a tripeptide, is considered one of the most important non-enzymatic antioxidants [9] and also serves as the substrate for glutathione peroxidase (GPx). Many proteins require their thiol groups in the reduced form in order to be functional, and GSH protects the thiol groups by removing peroxides that can oxidize sulfhydryl groups. The major enzymatic antioxidant system includes superoxide dismutase (SOD), catalase, GPx and GR. SOD catalyzes the dismutation of superoxide anion (O⫺䡠 2 ) to form hydrogen peroxide (H2O2) [10]. Further reduction of H2O2 can be carried out by catalase-dependent reaction. GPx catalyzes the reduction of H2O2 and peroxides using GSH as the substrate and producing oxidized glutathione (GSSG). In the presence of NADPH, GR catalyzes the regeneration of GSH from GSSG, and is therefore essential to the maintenance of an appropriate intracellular GSH redox status. Chronic ethanol ingestion causes toxic effects to the kidney and lung as well as the liver. Unlike the well-studied hepatotoxic effects of ethanol, the mechanisms of pathogenesis in the kidney and lung are not clearly understood. Ethanol may cause functional abnormalities in the kidney and lung depending on the quantity ingested and the duration of drinking. A possible link between alcoholism and glomerulonephritis [11] has been reported. Alcohol may also cause acute tubular necrosis [12]. In addition, nephrotoxic effects of acetaldehyde should not be ruled out because increased activities of alcohol dehydrogenase and catalase in the kidney [13] have been found in chronic alcoholism. Ethanol-fed rats have larger kidneys with significant increases in protein and lipid contents, and show reduction in creatinine clearance and osmotic clearance [14]. Furthermore, varying degrees of cellular injury are found in renal epithelial cells, particularly in the distal tubules and Henle’s loops [14]. Chronic respiratory disorders, including bronchitis, emphysema, pulmonary fibrosis, bronchietactsis and chronic airway obstruction are more common in alcoholics than in non-alcoholics [15]. The high incidence of chronic lung diseases in alcoholics is usually explained on the basis of poor nutrition, frequent infections in the respiratory tract and excessive smoking. While there is no doubt these factors contribute significantly, an association of pulmonary disease and direct damage of the lung by ethanol has been postulated
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[16] since the alveolar epithelial surface is under constant pressure of high oxygen pressure that makes the lung highly susceptible to ROS generation. Thiamin deficiency has been found to increase the production of ROS in the brain [17]. Therefore, it is important to study the effects of ethanol alone or in combination with TD diet because thiamin deficiency is a consequence of chronic alcoholism. We previously reported that long-term ethanol ingestion suppressed antioxidant defenses in the liver, and thiamin deficiency further suppressed those defenses [18]. The present work was undertaken to investigate whether long-term EtOH administration and thiamin deficiency, alone or combined, would affect the antioxidant defense systems in the kidney and lung of rats.
2. Materials and methods 2.1. Treatment of animals The treatment of rats was the same as that described by Chen et al. [18]. Male SpragueDawley rats, 2 months old, were used in this study. The animals were fed and treated at the Behavioral Neurobiology Laboratory at San Diego State University. All animals were individually housed and maintained on a regular lab chow diet and water ad lib. After a 3-day acclimation period, the rats began the experimental phase of this study. The dietary manipulation phase of the experiment lasted 30 weeks. A total of 32 rats were randomly assigned to one of the following treatment groups: Control, Ethanol (EtOH), Thiamin-Deficient (TD), and Ethanol/Thiamin-Deficient (EtOH/TD) groups. During the 30 weeks of treatment, all animals received regular lab chow, except during the three TD bouts when they received a diet deficient in thiamin in order to mimic a human pattern of alcohol bingeing. The three TD bouts began on weeks 10, 18 and 26 and lasted approximately four weeks. Except during the TD bouts, the rats received 1.0 mg/kg body weight of thiamin intraperitoneally (i.p.) three times a week. The TD diet (Tekled Diets) was a modified AIN-76 diet excluding thiamin, and consisted of casein, 19.1%; sucrose, 51.8%; corn starch, 15%; corn oil, 5%; mineral mix, 3.5% and vitamin mix except thiamin, 2.19 g/kg diet. Control and TD rats had tap water as the drinking fluid, whereas EtOH and EtOH/TD rats received an aqueous ethanol solution as the sole drinking fluid. The ethanol dose was gradually increased by 2% (v/v) increments every other day until a 20% (v/v) ethanol concentration was reached (approximately three weeks). During each TD bout, all animals received TD diet and continued to drink the same fluid they had been consuming. Also during the TD bouts, animals from the control and the EtOH groups were injected i.p. with thiamin (1 mg/kg body weight), while animals in the TD and the EtOH/TD groups received i.p injections of 0.9% normal saline instead of thiamin three times a week. Rats were examined and weighed weekly. Physical and behavioral changes such as slowed activity, wide stance, ataxic gait, opisthotonus, circling, retropulsion, hypersensitivity, impaired righting reflex and decreased grooming were monitored as indicators of thiamin deficiency. Animals that displayed more than one symptom of thiamin deficiency were monitored every day until reversed. If an animal showed a severely impaired righting reflex, the TD bout was reversed with a one time injection of thiamin (100 mg/kg body wt) and by changing the TD diet to regular lab chow. After Bout 1 and Bout 2, all
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remaining animals were reversed no later than four weeks using the same procedure as previously described. The animals were not reversed after Bout 3. At the end of the experimental period while the rats were still on TD diet, rats were anesthetized by CO2 inhalation (15 to 20 seconds), and killed by decapitation. The kidney and lung, were excised rapidly and frozen in dry ice. Specimens were frozen at -80°C, delivered in dry ice by air to the University of Kentucky, and kept frozen at -80°C until tissue preparation was performed. 2.2. Biochemical assays A 10% (w/v) tissue homogenate was prepared in 0.1 M phosphate buffer, pH 7.4, and immediately used for the determinations of GSH and protein concentrations. The remaining tissue homogenates were centrifuged at 3000 x g for 15 min at 4°C, to yield the supernatant which was used for the determinations of the activities of SOD, catalase, GPx, GR and GST as well as protein concentrations. GSH was determined by the method of Owen and Belcher [19] and SOD activity was determined by the method of McCord and Fridovich [20]. Catalase activity was determined by the method of Aebi [21], while GPx activity was determined by the method of Paglia and Valentine [22] using cumene hydroperoxide as the substrate. GR activity was determined by the method of Racker [23]. Protein concentrations in the tissue homogenate and supernatant were determined by the method of Lowry et al. [24].
Fig. 1. Effect of chronic consumption of ethanol (EtOH) and thiamin deficiency (TD) on reduced glutathione (GSH) levels. Means with different letters are significantly different at p ⬍ 0.05.
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Fig. 2. Effect of chronic consumption of ethanol (EtOH) and thiamin deficiency (TD) on superoxide dismutase (SOD) activity. Means with different letters are significantly different at p ⬍ 0.05.
2.3. Statistical analysis Data were analyzed by two way analysis of variance. When analysis of variance indicated significant differences, Fisher’s Least Significance Difference Test [25] was used to compare the treatment means. A probability of p ⱕ 0.05 was considered significant.
3. Results The body weights of the rats were similar in all four groups [18]. All data in the present study are expressed as mean ⫾ SE. As shown in Fig. 1, chronic consumption of ethanol significantly decreased GSH levels in the kidney (23%) and lung (32%) when compared to the respective control group. The combination of both ethanol consumption and TD diet also significantly suppressed GSH levels in the kidney (29%) and lung (35%) when compared to the control group, respectively. There was no significant difference between the GSH levels of the EtOH group and those of the EtOH/TD group in either tissue. Feeding a TD diet did not affect GSH levels in the two tissues. Chronic ethanol administration alone elicited a significant suppression of SOD activity (16%) when compared to the respective control group in both the kidney and lung (Fig. 2). The combination of ethanol and the TD diet also significantly suppressed SOD activity in the kidney (19%) and lung (17%) when compared to the respective control group. However, there was no significant difference between the SOD activity of the EtOH group and that of the EtOH/TD group in either tissue. Feeding TD diet alone did not produce changes on SOD activity in the tissues studied. As depicted in Fig. 3, catalase activity was substantially
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Fig. 3. Effect of chronic consumption of ethanol (EtOH) and thiamin deficiency (TD) on catalase activity. Means with different letters are significantly different at p ⬍ 0.05.
different between the kidney and lung, not only in the control group but also in response to the treatments. Catalase activity in the kidney was approximately 10 fold greater than that in the lung. In the kidney, catalase activity was significantly increased not only by ethanol (15%) but also by the combination of ethanol and TD diet (19%) when compared to the control group. However, there was no significant difference between the catalase activity of the EtOH group and that of the EtOH/TD group. Whereas EtOH consumption alone, TD diet alone or the combination of the two treatments did not affect catalase activity in the lung. Fig. 4 shows that GPx activity was significantly increased by ethanol consumption (17%) or ethanol consumption in combination with TD diet (21%) in the kidney. However, in the lung neither ethanol nor EtOH/TD diet affected GPx activity. In Fig. 5, it can be seen that no alteration on GR activity was observed in both tissues of rats fed ethanol alone, TD diet alone or combination of the two treatments.
4. Discussion This study examined a much longer period (30 weeks) of alcohol ingestion on tissue antioxidant defense systems of the kidney and lung than most reported studies. To our knowledge this is the first paper that examined the combined effects of ethanol consumption and mild thiamin deficiency on GSH and the antioxidant defense enzymes of these tissues. GSH plays an important role in maintaining the integrity of mitochondria, cell membranes and cell systems. Therefore, a decrease in GSH levels can decrease the effectiveness of the antioxidant defense systems and lead to oxidative injury. The results from the present study showed that GSH levels were significantly suppressed after chronic ethanol consumption in
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Fig. 4. Effect of chronic consumption of ethanol (EtOH) and thiamin deficiency (TD) on glutathione peroxidase (GPx) activity.
both tissues, and they were suppressed to about the same levels with combined treatment of ethanol and TD diet. Thus, the suppressive effects appear to be mainly attributed to ethanol consumption, but not to TD diet. Our results are consistent with those of Fernandez and Videla that chronic ethanol feeding decreased GSH levels in the kidney [26]. However, Rikans and Gonzalez reported that GSH levels in the lung were not affected by chronic
Fig. 5. Effect of chronic consumption of ethanol (EtOH) and thiamin deficiency (TD) on glutathione reductase (GR) activity.
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ethanol inhalation [27]. The different results in the lung can be explained by the different mode of ethanol administration (drinking versus inhalation) and the length of experimental period (30 weeks versus 35 days). The suppression of GSH appears to be a consequence of oxidative stress in rats fed ethanol when ROS are generated in large amount [5,28]. Generation of ROS during ethanol oxidation enhances the demand in the utilization of the antioxidant. Increased oxidation of GSH [29,30] and binding of GSH or its metabolite cysteinyl-glycine with acetaldehyde (the major metabolite of ethanol oxidation) through its cysteine residue [26,31] might also contribute to the suppression of GSH levels. Enhanced alcohol dehydrogenase activity and consequent increase in acetaldehyde production in the kidney were observed by Orellana et al. after rats were fed 20% ethanol solution for 10 weeks [13]. To our knowledge, data on ethanol-induced alterations on SOD activity in the kidney and lung after long-term consumption of ethanol have not been reported. However, Rikans and Gonzalez have reported a significant increase in SOD activity in the lung of rats exposed to ethanol vapors for 35 days [27]. The increase in SOD activity is explained as an adaptive response to mild oxidative stress [27]. It is well established that oxidant gases, such as ozone, nitrogen dioxide and 100% oxygen, cause an induction in lung antioxidant defense enzymes [32]. The discrepancies in the results of our study and those of Rikans and Gonzalez can again be explained by different modes of ethanol administration and treatment periods. Chronic ethanol consumption significantly decreased SOD activity in other tissues, including the liver [18,33] and the brain [34]. Taking into account that SOD is a sulfhydryl enzyme, suppression of its activity could be a consequence of oxidative damage [35] since excessive levels of H2O2 produced after ethanol oxidation could oxidize the sulfhydryl enzyme [36]. One important consequence of the decreased activity of SOD might be the increased 䡠 production of O⫺䡠 2 and further formation of OH (hydroxyl radical) through an iron-catalyzed Haber-Weiss reaction. The hydroxyl radical produced may lead to structural and functional disturbance of mitochondria [28] and increased susceptibility to oxidative injury. Therefore, with a decrease in GSH levels and SOD activity, the kidney and lung might become more vulnerable to oxidative stress. Contrasting with the observed decrease in GSH levels and SOD activity, the activity of catalase in the kidney was significantly increased after rats were fed either ethanol or the combination of ethanol and TD diet. This result is consistent with the increase of catalase in the kidney shown by Orellana et al. [13]. Chen et al. also reported that catalase activity was significantly increased in the liver of rats fed ethanol [18,33]. Catalase not only removes H2O2 produced by SOD-catalyzed reaction, but it is also a pathway for ethanol oxidation and a component of the peroxisomal fatty acid oxidation system. Therefore, increased catalase activity might be caused by increased substrates: H2O2 and ethanol. In fact, ethanol consumption enhances extra-mitochondrial oxidation of fatty acids in the kidney and consequently it may also increase the production of H2O2 [13]. Our study demonstrated that ethanol consumption significantly increased GPx activity in the kidney, but not in the lung. Since catalase activity was significantly increased in the kidney, the production of H2O2 could also be increased, and thus might induce GPx due to the increase in the substrate level. Rikans and Gonzalez have reported that there is no change in GPx activity in the lung of rats exposed to ethanol vapors for 35 days [27]. The
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discrepancies in the results of our study and those of Rikans and Gonzalez can again be explained by different modes of ethanol administration and treatment periods. When compared with reports in different tissues, GPx activity in the liver was decreased [18], but that in the myocardium was not altered [37] by ethanol administration. The present study showed no change on GR activity in the kidney or the lung, although chronic consumption of ethanol has been found to increase the activity of this enzyme in the liver [18] that is the major organ to synthesize GSH. GR is an inducible enzyme that responds to suppressed GSH levels. Even though GSH levels were significantly suppressed in both tissues in this study, the extent of suppression was moderate (23–32%), and it appears that the GSH levels was not low enough to induce GR. Although simultaneous administration of ethanol and the TD diet caused a significant decrease of GSH levels or SOD activity in the kidney and lung, the effects are mainly due to ethanol, because thiamin deficiency in combination with ethanol did not further suppress GSH levels or SOD activity. Since thiamin deficiency was found to be synergistic to ethanol in suppressing GSH levels and SOD activity in the liver in our previous report [18], thiamin deficiency appears to only affect the antioxidant defenses of the liver, but not those of the kidney and lung. Zimatkin and Zimatkina [38] reported significant decreases on TK activity in the liver and brain of rats fed 15% alcohol, while the effect of chronic ingestion of ethanol on TK activity in the kidney and lung has not been reported. Given the fact that thiamin, as coenzyme of TK, is required for the regeneration of GSH from GSSG by providing NADPH, we can assume that thiamin deficiency could increase alcohol toxicity by suppressing GSH levels in the liver and brain, but not in the kidney and lung. Our earlier study on rats with the same treatments showed that the antioxidant defenses of the liver was significantly weakened [18]. When compared to the liver, there were lesser alterations of antioxidant defenses caused by ethanol administration in the kidney and lung. The differences in these tissues can be explained by the observation that liver microsomes can oxidize ethanol at rates several fold greater than the kidney and lung microsomes [39]. In summary, data in the present investigation revealed that long-term consumption of ethanol, but not thiamin deficiency, suppressed GSH levels and SOD activity in the kidney and lung. In addition, chronic consumption of ethanol increased catalase and GPx activities of the kidney. The results suggest that chronic consumption of ethanol, but not mild thiamin deficiency, moderately decreases the antioxidant defense capability of the kidney and lung; and the effects are greater in the kidney than the lung. Acknowledgments This work was supported by NIAAA Grant No. 10473 and KYAES S-521, Paper No. 01–10-55. References [1] Thomson AD, Baker H, Leevy CM. Patterns of 35S-thiamin hydrochloride absorption in the malnourished alcoholic patient. J Lab Clin Med 1970;76:34 – 45.
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