- Email: [email protected]
The effect of heparegen on antioxidant enzyme activities in ethanol-induced liver injury in rats
Alcohol, Vol. 9, pp. 403-407, 1992
0741-8329/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
Printed in the U.S.A. All rights reserved.
The Effect of Heparegen on Antioxidant Enzyme Activities in Ethanol-Induced Liver Injury in Rats R. F A R B I S Z E W S K I , * A. RADECKA,I" M. C H W I E C K O * A N D A. H O L O W N I A *
•Department of Inorganic and Analytical Chemistry and tDepartment o f Drug Chemistry and Analysis, Medical Academy, 15-230 8ialystok 8, Poland Received 16 January 1992; Accepted 8 April 1992 FARBISZEWSKI, R., A. RADECKA, M. CHWIECKO AND A. HOLOWNIA. The effect of heparegen on antioxidant enzyme activitiesin ethanol-induced liver injury in rats. ALCOHOL 9(5) 403-407, 1992.-SOD, CAT, GSH-Px, and sulfhydryl compounds MDA contents in liver of rats treated with heparegen for 7, 14, and 21 days after alcoholicliver injury have been investigated. After use of this drug, we found beneficial effects on GSH-Px activity, snifhydrylcompounds (total and nonprotein), and MDA content and a partially beneficial effect on SOD and CAT activities. These enzyme activities after 21 days of drug administration were restored. Furthermore, heparegen shortens the time necessary for the return of AIAT and GGTP to normal value. This enzymaticdata are supported by histological studies in light microscopy. Heparegen
Ethanol
Antioxidantenzymes
SH groups
BOTH in humans and animals, chronic ethanol intake causes many cellular alterations, particularly in the liver. In postalcoholic liver injury, biochemical studies revealed an increase of reactive oxygen intermediates generation (4,7,28) and a significantly higher lipoperoxide content (2,6). The dangerously acting superoxide anion radical is eliminated by superoxide dismutase (SOD) (E.C.l.15.1.1), and is produced in high amounts in metabolically active tissues such as liver. This free radical is generated during the univalent reduction of oxygen. The widespread occurrence in nature of superoxide dismutase enzymes and their importance to the growth and survival of organisms in aerobic environment gives considerable support to the view that O~ is a toxic species
(14). SOD enzyme accelerates this dismutation reaction, forming hydrogen peroxide, which is converted by catalase (CAT) (E.C.l.ll.l.6) to water and oxygen. Glutathione peroxidase (GSH-Px) (E.C.l.ll.l.9) also plays an important role in the removal of hydrogen peroxide and organic hydroperoxides (14,17). The efficient removal of the superoxide anion radical and hydrogen peroxide prevents the formation of the powerful oxidant hydroxyl radical (OH.) in the Fenton and HaberWeiss reaction (7). The defect of endogenous protective antioxidant mechanisms after prolonged ethanol intake promotes lipid peroxidation, structural protein modification, subsequent proteolytic digestion, and finally, serious liver cell injury. These processes lead to the formation of multiple aldehydes, including easily detectable malondialdehyde (MDA). Among radical scavengers there are natural antioxidants
MDA
Rats
like reduced glutathione, c~-tocopherol, and ascorbic acid, eliminating reactive oxygen species (9, l 0,1 I, 17,18,24,27). Heparegen (thiazolidine-4-carboxylic acid) is a new synthetic compound that is intramitochondrially transformed into sulfhydryl containing N-formylcysteine. Heparegen, when given orally, can be accumulated in the liver cells and subsequently enzymatically hydrolyzed to replenish the appropriate thiol level essential in cell biochemistry. It is well known that intoxication with ethanol produces marked decrease in liver thiols leading to several abnormalities (8,9,17). The aim of the present study is the evaluation of the status of hepatic antioxidant defense, after heparegen treatment in ethanol-induced liver injury in rats. METHOD
Male Wistar rats (approximately 200 g b.wt.) fed on a standard LSM diet (containing no antioxidants but 0.55% of cystine and methionine) were divided into five groups, each group containing 15 rats. Control rats (group l) were fed a standard diet and had free access to water. The animals of groups 2, 3, 4, and 5 had free access to drinking of 20e/0 ethanol for 3 months (approximately 12.5 g/kg of b.wt./d). Group 2 animals were sacrificed after this time. Groups 3, 4, and 5 were divided into two subgroups, A and B. Groups 3A, 4A, and 5A were administered heparegen (Syntex, Switzerland) in a daily dose of 2.5 mg/rat per day (by gastric tube in solution of saline on an empty stomach). Group 3A received the drug for 7 days, group 4A for 14 days, and group 5A for 403
404
FARBISZEWSKI ET AL.
21 days. Animals of groups 3B, 4B, and 5B did not receive the drug. Groups 3B, 4B, and 5B were sacrificed 7, 14, and 21 days after ethanol cessation, respectively. The animals of all groups were killed under ether anesthesia. Blood samples were collected by cardiac puncture into a tube containing 0.1 M sodium citrate (9: 1) and immediately centrifuged at 1000g for 10 rain. Plasma was used for estimation of aianine (AIAT) and aspartarte (AspAT) aminotransferase activities, as described by Reitman and Frankel (22), and gamma-glutamyltransferase activity (GGT) was assayed by the method of Szewczuk and Kuropatwa (26). After exsanguination, the liver tissue was quickly removed and 5-ram longitudinal slices (six from each liver) from the left and right lobes were taken and fixed in 10% buffered formalin and in Carnoy's fluid. Seven-micron sections were cut and stained with hematoxylin and eosin. Also, liver slices were stained with oil red O and, after diastase digestion, subjected to Schiff reaction with periodic acid with dimedon blocking acetyl groups. Histological evaluation by light microscopy consisted of the blind and systematic grading of steatosis, inflammation, and necrosis in sections obtained from multiple liver lobes. The remaining liver tissue was placed into iced 0.15 M NaCl, blotted on filter paper and homogenized using a glass homogenizer with Teflon pestle. Sorensen phosphate buffer (0.05 M), pH 7.2, containing 0.25 M sucrose, was used to prepare the liver homogenates for measuring enzymatic activities. Homogenates for GSH-Px and SOD determination were centrifuged at 105.000g for 45 min and, for CAT assay, at 9000g for 10 min to obtain cytosol and postmitochondrial fraction respectively. Liver slices were homogenized in 0.15 M KCI for determination of SH groups and malondialdehyde. Cu,Zn-superoxide dismutase activity (SOD) was measured by the epinephrine assay of Sykes et al. (25). One unit of SOD activity was defined as the amount of the enzyme required for 50070 inhibition of the oxidation of epinephrine to adrenochrome, and was calculated per gram of liver tissue.
Glutathione peroxidase activity (GSH-Px) was measured spectrophotometrically using a technique based on Paglia and Valentine (20), wherein GSSG formation is assessed by measurement of conversion of N A D P H to NADP. One unit of enzyme activity was defined as the amount of the enzyme catalyzing the conversion of 1 ~tmol of N A D P / m i n per milligram of protein. Catalase activity (CAT) was measured after 30-rain preincubation of the postmitochondrial fraction of liver homogehate with 1°70 Triton X-100 by following the decrease in absorbance of hydrogen peroxide at 240 nm (1). The rates were determined at 25°C using 10 mM hydrogen peroxide and the activity was expressed as micromoles of H202 decomposed/ min per milligram of protein. SH groups were determined according to Ellman (8) using 5,5 '-dithio-bis(-2-nitrobenzoic acid) (DTNB) in previously deproteinized (with perchloric acid) liver homogenates. Hepatic malondialdehyde content was measured by using the technique based on Buege and Aust (3). Protein levels in homogenates were determined by the method of Lowry et al. (16) using bovine serum albumin as a standard. Results were expressed as mean + SD. Statistical analysis was performed by Student's t test for unpaired data, and values o f p < 0.05 were considered statistically significant. RESULTS Table 1 shows the effect of heparegen administration after 7, 14, and 21 days on the liver SOD, CAT, and GSH-Px activities of rats in ethanol-induced liver injury. Following ethanol ingestion (group 2) the activities of the three studied enzymes were significantly decreased. After heparegen administration for 7 days (group 3A) the activities of SOD and CAT were similar to those in group 2, but the activity of GSH-Px returned to normal value. After 7 days of ethanol cessation (group 3B), the liver activities of all enzymes were still significantly lower, in comparison with control group (group 1).
TABLE 1 THE EFFECT OF HEPAREGEN ON THE LIVER SOD, CAT, AND GSH-Px ACTIVITIES IN ETHANOL-INDUCED LIVER INJURY IN RATS
Group
SOD U/g of Tissue
CAT #moi/min per Milligram of Protein
GSH-Px ~nol NADP/min. per Milligramof Protein
Control (1) Ethanol (2)
598.2 ± 53.2 321.2 ± 29.7
225.4 + 28.3 141.2 + 16.7
68.3 + 5.5 59.8 ± 8.8
399.4 ± 35.5 382.1 ± 36.1
169.6 ± 19.5 143.3 ± 11.2
62.6 ± 9.2 59.4 ± 8.1
501.9 ± 29.0 415.3 ± 31.9
187.1 + 14.4 161.3 ± 12.8
68.1 ± 7.6 63.9 ± 7.8
583.2 + 33.3 458.1 ± 28.8
228.2 ± 28.1 203.6 ± 17.2
68.5 ± 6.3 62.7 ± 8.0
7 days after: Heparegen treatment (3A) Ethanol cessation (3B) 14 days after: Heparegen treatment (4A) Ethanol cessation (413) 21 days after: Heparegen treatment (5A) Ethanol cessation (5B)
Chosen statistically significant differences (p < 0.05) between groups: S O D - 1-2, I-3A, I-3B, I4A, I-4B, I-5B, 2-3A, 2-3B, 2-4A, 2-4B, 2-5A, 2-5B; CAT-1-2, I-3A, I-3B, I-4A, I-4B, I-5B, 2-3A, 2--4A, 2-4, 2-5A, 2-5B, 3A-3B, 4A-4B, 5A-5B; GSH-Px-1-2, I-3B, 1-5B, 2-4A, 2-5A, 5A5B.
HEPAREGEN AND ANTIOXIDANT ENZYME ACTIVITY
405
TABLE 2 THE EFFECT OF HEPAREGEN ON LIVER MDA PROTEIN AND NONPROTEIN SULFHYDRYLCOMPOUNDS IN ETHANOL-INDUCEDLIVER INJURY IN RATS SulfhydrylCompounds/~mol/gof Tissue Group Control(l) Ethanol(2) 7 days after: Heparegen treatment (3A) Ethanolcessation (3B) 14 days after: Heparegen treatment (4A) Ethanolcesssation (4B) 21 days after: Heparegentreatment (SA) Ethanol cessation (5B)
MDA nmol/gof Tissue
Total
Nonprotein
112.5 + 17.3 187.3 + 13.2
22.3 + 3.1 15.6 + 2.8
5.4 :t: 0.6 3.1 + 1.0
121.6 + 9.3 138.2 + 11.0
19.5 + 1.9 16.9 + 2.3
4.2 + 0.7 3.6 + 0.8
109.8 + 13.4 111.3 + 10.8
23.1 + 2.8 17.7 + 3.5
5.4 + 0.6 3.9 + 0.8
110.6 + 15.4 114.7 + 16.1
22.9 + 1.9 19.1 + 2.2
5.5 + 0.4 4.4 + 0.4
Chosen statistically significant differences (p < 0.05) between groups: MDA-1-2, 1-3B, 2-3A, 2-3B, 2-4A, 2-4B, 2-5A, 2-5B, 3A-3B; nonprotein S H - 1-2, I-3A, I-3B, I--4A, I-4B, 1-5B, 2-3A, 2-4A, 2-4B, 2-5A, 3A-3B, 4A--4B, 5A-5B; total SH-1-2, I-3A, 1-3B, I-4A, I-4B, I-5B, 2-3A, 24A, 2-4B, 2-5A, 2-5B, 3A-3B, 4A-4B, 5A-5B.
Heparegen used for 14 days (group 4A) slightly increased the liver activities of SOD and CAT. Just after 21 days of heparegen treatment (group 5A), activities of SOD and CAT in the liver were normal and amounted to 583.2 and 228.2, respectively. No normalization of the activities of studied enzymes in ethanol-induced fiver injury was observed in rats without heparegen (groups 4B and 5B). The effect of heparegen administration for 7, 14, and 21 days on the total and nonprotein sulfhydryl compounds and MDA content in fiver is listed in Table 2. Following ethanol ingestion (group 2) total SH groups and nonprotein SH in the fiver were significantly decreased while MDA content was increased in comparison with the control group (group 1).
This low level of SH groups and MDA content was maintained for at least 7 days after cessation of ethanol treatment (group 3B). After heparegen administration for 7 days (group 3A), the values of SH groups were slightly higher, but the differences were significantlydecreased (p < 0.05), when compared to liver SH groups in control animals. The rats which received heparegen for 14 days after cessation of ethanol ingestion (group 4A) possessed normal liver values of total and nonprotein SH groups and MDA concentration. Results of plasma enzyme tests suggest damage of parenchymai fiver cells in rats receiving ethanol (Table 3). The activity of all three enzymes examined were significantly increased after 3 months of ethanol ingestion in comparison with the
TABLE 3 THE EFFECT OF HEPAREGEN ON THE ACTIVITIESOF AIAT, #.spAT, AND GGTP IN PLASMA OF RATS IN ETHANOL-INDUCEDLIVER INJURY Group Control(l) Ethanol(2) 7 days after Heparegen treatment (3A) Ethanol cessation (3B) 14 days after Heparegentreatment (4A) Ethanol cessation (4B) 21 days after Heparegen treatment (5A) Ethanol cessation (513)
AIAt(UL)
AspAT(UL)
GGTP(UL)
87.3 + 16.2
140.6 + 22.1
9.2 + 1.6
126.7 + 26.7
207.9 + 37.8
23.4 + 4.3
92.5 + 14.8 90.8 + 20.2
168.2 + 28.8 173.5 + 23.0
10.3 + 1.5 12.7 + 2.0
93.2 + 20.6 86.1 + 17.8
147.8 + 21.1 156.5 + 20.7
8.3 :t: 2.7 8.6 + 1.9
83.4 + 15.5 90.8 + 20.0
131.5 + 18.8 145.3 + 23.2
8.0 + 1.9 9.3 + 1.6
Chosen statistically significant differences (/7 < 0.05) between groups: AIAT--1-2, 2-3A, 2-3B, 2-4A, 2-4B, 2-5A, 2-5B; AspAT-1-2, I-3A, I-3B, 2-3A, 2-3B, 2--4A, 2-4B, 2-5A, 2-5B, 3A-4A, 3A-5A, 3B-4A, 3B-4B, 3B--5A, 3B--5B, 4A-5A, 4B-SA; GGTP-1-2, 1-3B, 2-3A, 2-3B, 2--4A, 24B, 2-5A, 2-5B, 3A-3B, 3B-4B.
406
FARBISZEWSKI ET AL.
control group. However, in rats treated with heparegen for just 7 days (group 3A), an increase in AIAT, AspAT, and GGTP was observed, when compared with group 2. Histopathological examination (Table 4) of the liver sections obtained from rats intoxicated with ethanol revealed focal steatosis which was predominantly located in central perivenous part of hepatic lobule. Large droplet steatosis was prominent resulting in enlarged hepatocytes. It was seen focal hepatocyte necrosis with infiltration o f neutrophiles and mononuclear phagocytes. The presence of Mallory bodies was only found rarely. Pathological changes in the liver of rats treated with heparegen for 7 days (group 3A) were much less expressive than in group 3B rats, and particularly after 14 days of heparegen treatment (group 4A). Neither cell necrosis nor polymorphonuclear leukocytes were found. In the livers of group 5A, fatty degeneration of hepatocytes, cell necrosis, or Mallory bodies were not observed.
portance of the NADPH-GSH-Px system in scavenging toxic species of oxygen. The superoxide anion radical is the primary free radical species involved in various tissue injuries. It undergoes spontaneous dismutation to produce hydrogen peroxide and, in the presence of iron ions, hydroxyl radicals (23). These radicals are known to be the most reactive species of oxygen radicals because, once generated, they will react with any molecules in their immediate surroundings. It is also the most widely proposed initiator of lipid peroxidation, though other radicals like the ferryl or perferryl iron are also involved in those processes that are widely evaluated by measurement of the level of thiobarbituric acid-reactive substances. The result of the present study shows that ethanol treatment elicits an increase in hepatic malondialdehyde content (measured as TBA reactive substances). On day 7 after cessation of ethanol, the hepatic malondialdehyde level decreases; however, this normalization is significantly more evidenced in rats receiving heparegen. Malondialdehyde is metabolized in living organisms in many nonenzymatic and enzymatic pathways. Among enzymes participating in transformation of malondialdehyde and malondialdehyde-like substances, the most important role of aldehyde dehydrogenase and xanthine oxidase should be stressed. Free SH groups released from N-formyl cysteine may influence xanthine oxidase activity converting this enzyme to xanthine dehydrogenase that is inactive against aldehydic products of lipid peroxidation. Therefore, an increase in the level of thiobarbituric-reactive substances may also result from inhibition of degradation processes. Superoxide anion radicals are less reactive, however, they can diffuse away from the sites of formation, leading to hydroxyl radical generation. Therefore, hydroxyl radicals can he damaging because of their high reactivity, whereas superoxide anion radicals can also be damaging because of their diffusing properties (13). We have observed that the diminution of SOD activity in liver injury is coupled with the decrease of CAT activity. This is consistent with other studies of authors showing that the protective action of SOD is dependent on the presence of CAT (15,21). The increase of SOD activity observed in our studies after beparegen treatment is accompanied by higher CAT activity. It is well known that reactive oxygen radicals can dam-
DISCUSSION The effect of heparegen, thiazolidine-4-carboxylic acid, is well documented in the pharmacotherapy of human and animal liver damage, irrespective of the origin. The drug is converted inside mitochondria into a compound containing free SH groups with a beneficial action in severe toxic liver injury, including ethanol-induced liver damage. Those effects are confirmed by basic biochemical research, morphological studies, and in most clinical examinations, but the influence of this drug on SOD, CAT, and GSH-Px activities and lipid peroxidation in damaged liver has not been evaluated. A direct connection between heparegen and the formation of superoxide anion radicals in ethanol-induced liver injury has not been established either. It has been demonstrated in recent studies that the use of heparegen in rats intoxicated simultaneously with ethanol leads to the diminution of blood ammonia concentration and decreases glutamate dehydrogenase activity (10). Our studies reveal that heparegen administration in the rats for 7, 14, and 21 days gradually restores the decreased activities of liver antioxidant enzymes to normal values. SOD selectively scavenges superoxide anion radicals, and the resulting hydrogen peroxide is removed from the cell mainly by the GSH-Px system (12), and this suggests the im-
TABLE 4 THE EFFECT OF HEPAREGEN ON THE LIVER HISTOLOGY OF RATS IN ETHANOL-INDUCED LIVER INJURY Group Control (1) Ethanol (2) 7 days after Heparegen treatment (3A) Ethanol cessation (3B) 14 days after Heparegen treatment (4A) Ethanol cessation (4B) 21 days after Heparegen treatment ( 5 A ) Ethanol
cessation
(5B)
Steatosis*
Inflammations
Necrosist
+++
+++
++
++ +++
+ ++
+ ++
+ ++
+ ++
+
-
-
-
+
+
-
*+, 0-25% of hepatocytes with fat deposits; + +, 25-50%; + + + , >50%. 1 +, < 1 focus per low power view; + +, > 1 focus; + + +, >2 focus.
HEPAREGEN AND ANTIOXIDANT ENZYME ACTIVITY age enzymatic proteins and thereby decrease SOD, CAT, and GSH-Px activities. The improvement of liver function with heparegen follows an increase of hepatic antioxidane enzymes. Results of enzymatic indicators in plasma (Table 3) and histological studies (Table 4) gave additional evidence supporting this statement. There is increasing evidence that reactive oxygen species, especially hydroxyethyl radical, mediate oxidation of sulfhydryl compounds in rats with ethanol-induced liver
407 damage (5,9,19). The antioxidant activity of GSH-Px is coupled with the oxidation of GSH to oxidized GSSG, which can subsequently be reduced by GSSG reductase using NADPH as the reducing agent (14,17). In conclusion, the ethanol-induced changes reported here are partially normalized by oral heparegen treatment. It is also evident that this drug possesses antioxidant and, consequently, hepatoprotective properties.
REFERENCES 1. Aebi, H. Catalase in vitro. Meth. Enzymol. 105:121-126; 1984. 2. Antonenkov, V. D.; Pirozhkov, S. V.; Popova, S. V.; Panchenko, L. F. Effect of chronic ethanol treatment on lipid peroxidation in rat liver homogenate and subcellular fractions. Int. J. Biochem. 21:1191-1195; 1989. 3. Buege, J. A.; Aust, S. D. Microsomal lipid peroxidation. Meth. Enzymol. 51:302-310; 1978. 4. Cederbanm, A. I. Oxygen radical generation by microsomes: Role of iron and implications for alcohol and toxicity. Free Rad. Biol. Med. 7:559-567; 1989. 5. Chwiecko, M.; Holownia, A.; Pawlowska, D.; Farbiszewski, R. The effect of immunostimulatory drugs on sulfhydryl compounds in plasma, liver and brain after ethanol-induced liver injury in rats. Alcohol 8:179-181; 1991. 6. Dianzani, M. U. Lipid peroxidation in ethanol poisoning: A critical reconsideration. Alcohol Alcohol. 20:161-173; 1985. 7. Dicker, E.; Cederbaum, A. I. Hydroxyl radical generation by microsomes after chronic ethanol consumption. Alcohol. Clin. Exp. Res. 11:309-314; 1987. 8. Ellman, G. L. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82:70-77; 1959. 9. Farbiszewski, R.; Holownia, A.; Chwiecko, M. Changes in sulfhydryl compounds in plasma, fiver and brain after acute and chronic ethanol administration in rats. Drug Alcohol Depend. 20: 129-133; 1987. 10. Farbiszewski, R.; Holownia, A.; Chwiecko, M.; Pawlowska, D. The effects of heparegen and D-penicillamine on the activity of some ammonia metabolizing enzymes in liver and brain of rats intoxicated with ethanol. Drug Alcohol Depend. 27:69-72; 1991. 11. Fernandez-Checa, J.; Ookhtens, M.; Kaplowitz, N. Effect of chronic ethanol feeding on rat hepatocytic glutathione. J. Clin. Invest. 83:1247-1252; 1989. 12. Fridovich, I.; Freeman, B. Antioxidant defenses in the lung. Ann. Rev. Physiol. 48:693-702; 1986. 13. Halliwel, B.; Gutteride, J. M. C. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J. 219:1-14; 1984. 14. Hinder, R. A.; Stein, H. J. Oxygen-derived free radicals. Arch. Surg. 126:104-105; 1991. 15. Koerner, J. E.; Anderson, B. A.; Dage, R. C. Protection against postischemic myocardial dysfunction in anesthetized rabbits with scavengers of oxygen-derived free radicals: superoxide dismutase
plus catalase, N-2-mercapto-propionyl glycine and captopril. J. Cardiovasc. Pharmacol. 17:185-191; 1991. 16. Lowry, O. H.; Rosenbrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with Folin phenol reagent. J. Biol. Chem. 153:265-275; 1951. 17. Munoz, M. E.; Martin, M. I.; Fermoso, J.; Gonzalez, J.; Esteller, A. Effect of chronic ethanol feeding on glutathione and glutathione related enzyme activities in rat liver. Drug Alcohol Depend. 20:221-226; 1987. 18. Njus, D.; Kelley, P. M. Vitamins C and E donate single hydrogen atoms in vivo. FEBS Lett. 284:147-151; 1991. 19. Nordmann, R.; Ribiere, C.; Rouach, H. Alcool et radicaux libres: donnees actuelles. Medecine Sci. 6:336--345; 1988. 20. Paglia, E. D.; Valentine, W. N. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70:158-169; 1967. 21. Przyklenk, K.; Kloner, R. A. Superoxide dismutase plus catalase improve contractile function in the canine model of the "stunned myocardium." Circ. Res. 58:148-156; 1986. 22. Reitman, S.; Frankel, S. A colorimetric method for determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol. 28:56-62; 1957. 23. Southon, P. A.; Powis, G. Free radicals in medicine. Mayo Clin. Proc. 63:381--408;1988. 24. Speisky, H.; Kera, Y.; Pentilla, K. E.; Israel, Y.; Lindros, K. O. Depletion of hepatic glutathione by ethanol occurs independently Of ethanol metabolism. Alcohol. Clin. Exp. Res. 12:224-228; 1988. 25. Sykes, J. A.; McCormack, F. X.; O'Brien, T. J. A preliminary study of the superoxide dismutase content of some human tumors. Cancer Res. 38:2759-2762; 1978. 26. Szewczuk,A.; Kuropatwa, M. A. A new diagnostic 7-GT-test for estimation of V-glutamyltransferaseactivity in serum. Diag. Lab. (in Polish) 21:289-295; 1985. 27. Takenaka, Y.; Miki, M.; Yasuda, H.; Mino, M. The effect of c~-tocopherol as antioxidant on the oxidation of membrane protein thiols induced by free radicals generated in different sites. Arch. Biochem. Biophys. 285:344-350; 1991. 28. Younes, M.; Strubelt, O. Alcohol-induced hepatotoxicity: A role for oxygen free radicals. Free Rad. Res. Commun. 3:19-26; 1987.
0741-8329/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
Printed in the U.S.A. All rights reserved.
The Effect of Heparegen on Antioxidant Enzyme Activities in Ethanol-Induced Liver Injury in Rats R. F A R B I S Z E W S K I , * A. RADECKA,I" M. C H W I E C K O * A N D A. H O L O W N I A *
•Department of Inorganic and Analytical Chemistry and tDepartment o f Drug Chemistry and Analysis, Medical Academy, 15-230 8ialystok 8, Poland Received 16 January 1992; Accepted 8 April 1992 FARBISZEWSKI, R., A. RADECKA, M. CHWIECKO AND A. HOLOWNIA. The effect of heparegen on antioxidant enzyme activitiesin ethanol-induced liver injury in rats. ALCOHOL 9(5) 403-407, 1992.-SOD, CAT, GSH-Px, and sulfhydryl compounds MDA contents in liver of rats treated with heparegen for 7, 14, and 21 days after alcoholicliver injury have been investigated. After use of this drug, we found beneficial effects on GSH-Px activity, snifhydrylcompounds (total and nonprotein), and MDA content and a partially beneficial effect on SOD and CAT activities. These enzyme activities after 21 days of drug administration were restored. Furthermore, heparegen shortens the time necessary for the return of AIAT and GGTP to normal value. This enzymaticdata are supported by histological studies in light microscopy. Heparegen
Ethanol
Antioxidantenzymes
SH groups
BOTH in humans and animals, chronic ethanol intake causes many cellular alterations, particularly in the liver. In postalcoholic liver injury, biochemical studies revealed an increase of reactive oxygen intermediates generation (4,7,28) and a significantly higher lipoperoxide content (2,6). The dangerously acting superoxide anion radical is eliminated by superoxide dismutase (SOD) (E.C.l.15.1.1), and is produced in high amounts in metabolically active tissues such as liver. This free radical is generated during the univalent reduction of oxygen. The widespread occurrence in nature of superoxide dismutase enzymes and their importance to the growth and survival of organisms in aerobic environment gives considerable support to the view that O~ is a toxic species
(14). SOD enzyme accelerates this dismutation reaction, forming hydrogen peroxide, which is converted by catalase (CAT) (E.C.l.ll.l.6) to water and oxygen. Glutathione peroxidase (GSH-Px) (E.C.l.ll.l.9) also plays an important role in the removal of hydrogen peroxide and organic hydroperoxides (14,17). The efficient removal of the superoxide anion radical and hydrogen peroxide prevents the formation of the powerful oxidant hydroxyl radical (OH.) in the Fenton and HaberWeiss reaction (7). The defect of endogenous protective antioxidant mechanisms after prolonged ethanol intake promotes lipid peroxidation, structural protein modification, subsequent proteolytic digestion, and finally, serious liver cell injury. These processes lead to the formation of multiple aldehydes, including easily detectable malondialdehyde (MDA). Among radical scavengers there are natural antioxidants
MDA
Rats
like reduced glutathione, c~-tocopherol, and ascorbic acid, eliminating reactive oxygen species (9, l 0,1 I, 17,18,24,27). Heparegen (thiazolidine-4-carboxylic acid) is a new synthetic compound that is intramitochondrially transformed into sulfhydryl containing N-formylcysteine. Heparegen, when given orally, can be accumulated in the liver cells and subsequently enzymatically hydrolyzed to replenish the appropriate thiol level essential in cell biochemistry. It is well known that intoxication with ethanol produces marked decrease in liver thiols leading to several abnormalities (8,9,17). The aim of the present study is the evaluation of the status of hepatic antioxidant defense, after heparegen treatment in ethanol-induced liver injury in rats. METHOD
Male Wistar rats (approximately 200 g b.wt.) fed on a standard LSM diet (containing no antioxidants but 0.55% of cystine and methionine) were divided into five groups, each group containing 15 rats. Control rats (group l) were fed a standard diet and had free access to water. The animals of groups 2, 3, 4, and 5 had free access to drinking of 20e/0 ethanol for 3 months (approximately 12.5 g/kg of b.wt./d). Group 2 animals were sacrificed after this time. Groups 3, 4, and 5 were divided into two subgroups, A and B. Groups 3A, 4A, and 5A were administered heparegen (Syntex, Switzerland) in a daily dose of 2.5 mg/rat per day (by gastric tube in solution of saline on an empty stomach). Group 3A received the drug for 7 days, group 4A for 14 days, and group 5A for 403
404
FARBISZEWSKI ET AL.
21 days. Animals of groups 3B, 4B, and 5B did not receive the drug. Groups 3B, 4B, and 5B were sacrificed 7, 14, and 21 days after ethanol cessation, respectively. The animals of all groups were killed under ether anesthesia. Blood samples were collected by cardiac puncture into a tube containing 0.1 M sodium citrate (9: 1) and immediately centrifuged at 1000g for 10 rain. Plasma was used for estimation of aianine (AIAT) and aspartarte (AspAT) aminotransferase activities, as described by Reitman and Frankel (22), and gamma-glutamyltransferase activity (GGT) was assayed by the method of Szewczuk and Kuropatwa (26). After exsanguination, the liver tissue was quickly removed and 5-ram longitudinal slices (six from each liver) from the left and right lobes were taken and fixed in 10% buffered formalin and in Carnoy's fluid. Seven-micron sections were cut and stained with hematoxylin and eosin. Also, liver slices were stained with oil red O and, after diastase digestion, subjected to Schiff reaction with periodic acid with dimedon blocking acetyl groups. Histological evaluation by light microscopy consisted of the blind and systematic grading of steatosis, inflammation, and necrosis in sections obtained from multiple liver lobes. The remaining liver tissue was placed into iced 0.15 M NaCl, blotted on filter paper and homogenized using a glass homogenizer with Teflon pestle. Sorensen phosphate buffer (0.05 M), pH 7.2, containing 0.25 M sucrose, was used to prepare the liver homogenates for measuring enzymatic activities. Homogenates for GSH-Px and SOD determination were centrifuged at 105.000g for 45 min and, for CAT assay, at 9000g for 10 min to obtain cytosol and postmitochondrial fraction respectively. Liver slices were homogenized in 0.15 M KCI for determination of SH groups and malondialdehyde. Cu,Zn-superoxide dismutase activity (SOD) was measured by the epinephrine assay of Sykes et al. (25). One unit of SOD activity was defined as the amount of the enzyme required for 50070 inhibition of the oxidation of epinephrine to adrenochrome, and was calculated per gram of liver tissue.
Glutathione peroxidase activity (GSH-Px) was measured spectrophotometrically using a technique based on Paglia and Valentine (20), wherein GSSG formation is assessed by measurement of conversion of N A D P H to NADP. One unit of enzyme activity was defined as the amount of the enzyme catalyzing the conversion of 1 ~tmol of N A D P / m i n per milligram of protein. Catalase activity (CAT) was measured after 30-rain preincubation of the postmitochondrial fraction of liver homogehate with 1°70 Triton X-100 by following the decrease in absorbance of hydrogen peroxide at 240 nm (1). The rates were determined at 25°C using 10 mM hydrogen peroxide and the activity was expressed as micromoles of H202 decomposed/ min per milligram of protein. SH groups were determined according to Ellman (8) using 5,5 '-dithio-bis(-2-nitrobenzoic acid) (DTNB) in previously deproteinized (with perchloric acid) liver homogenates. Hepatic malondialdehyde content was measured by using the technique based on Buege and Aust (3). Protein levels in homogenates were determined by the method of Lowry et al. (16) using bovine serum albumin as a standard. Results were expressed as mean + SD. Statistical analysis was performed by Student's t test for unpaired data, and values o f p < 0.05 were considered statistically significant. RESULTS Table 1 shows the effect of heparegen administration after 7, 14, and 21 days on the liver SOD, CAT, and GSH-Px activities of rats in ethanol-induced liver injury. Following ethanol ingestion (group 2) the activities of the three studied enzymes were significantly decreased. After heparegen administration for 7 days (group 3A) the activities of SOD and CAT were similar to those in group 2, but the activity of GSH-Px returned to normal value. After 7 days of ethanol cessation (group 3B), the liver activities of all enzymes were still significantly lower, in comparison with control group (group 1).
TABLE 1 THE EFFECT OF HEPAREGEN ON THE LIVER SOD, CAT, AND GSH-Px ACTIVITIES IN ETHANOL-INDUCED LIVER INJURY IN RATS
Group
SOD U/g of Tissue
CAT #moi/min per Milligram of Protein
GSH-Px ~nol NADP/min. per Milligramof Protein
Control (1) Ethanol (2)
598.2 ± 53.2 321.2 ± 29.7
225.4 + 28.3 141.2 + 16.7
68.3 + 5.5 59.8 ± 8.8
399.4 ± 35.5 382.1 ± 36.1
169.6 ± 19.5 143.3 ± 11.2
62.6 ± 9.2 59.4 ± 8.1
501.9 ± 29.0 415.3 ± 31.9
187.1 + 14.4 161.3 ± 12.8
68.1 ± 7.6 63.9 ± 7.8
583.2 + 33.3 458.1 ± 28.8
228.2 ± 28.1 203.6 ± 17.2
68.5 ± 6.3 62.7 ± 8.0
7 days after: Heparegen treatment (3A) Ethanol cessation (3B) 14 days after: Heparegen treatment (4A) Ethanol cessation (413) 21 days after: Heparegen treatment (5A) Ethanol cessation (5B)
Chosen statistically significant differences (p < 0.05) between groups: S O D - 1-2, I-3A, I-3B, I4A, I-4B, I-5B, 2-3A, 2-3B, 2-4A, 2-4B, 2-5A, 2-5B; CAT-1-2, I-3A, I-3B, I-4A, I-4B, I-5B, 2-3A, 2--4A, 2-4, 2-5A, 2-5B, 3A-3B, 4A-4B, 5A-5B; GSH-Px-1-2, I-3B, 1-5B, 2-4A, 2-5A, 5A5B.
HEPAREGEN AND ANTIOXIDANT ENZYME ACTIVITY
405
TABLE 2 THE EFFECT OF HEPAREGEN ON LIVER MDA PROTEIN AND NONPROTEIN SULFHYDRYLCOMPOUNDS IN ETHANOL-INDUCEDLIVER INJURY IN RATS SulfhydrylCompounds/~mol/gof Tissue Group Control(l) Ethanol(2) 7 days after: Heparegen treatment (3A) Ethanolcessation (3B) 14 days after: Heparegen treatment (4A) Ethanolcesssation (4B) 21 days after: Heparegentreatment (SA) Ethanol cessation (5B)
MDA nmol/gof Tissue
Total
Nonprotein
112.5 + 17.3 187.3 + 13.2
22.3 + 3.1 15.6 + 2.8
5.4 :t: 0.6 3.1 + 1.0
121.6 + 9.3 138.2 + 11.0
19.5 + 1.9 16.9 + 2.3
4.2 + 0.7 3.6 + 0.8
109.8 + 13.4 111.3 + 10.8
23.1 + 2.8 17.7 + 3.5
5.4 + 0.6 3.9 + 0.8
110.6 + 15.4 114.7 + 16.1
22.9 + 1.9 19.1 + 2.2
5.5 + 0.4 4.4 + 0.4
Chosen statistically significant differences (p < 0.05) between groups: MDA-1-2, 1-3B, 2-3A, 2-3B, 2-4A, 2-4B, 2-5A, 2-5B, 3A-3B; nonprotein S H - 1-2, I-3A, I-3B, I--4A, I-4B, 1-5B, 2-3A, 2-4A, 2-4B, 2-5A, 3A-3B, 4A--4B, 5A-5B; total SH-1-2, I-3A, 1-3B, I-4A, I-4B, I-5B, 2-3A, 24A, 2-4B, 2-5A, 2-5B, 3A-3B, 4A-4B, 5A-5B.
Heparegen used for 14 days (group 4A) slightly increased the liver activities of SOD and CAT. Just after 21 days of heparegen treatment (group 5A), activities of SOD and CAT in the liver were normal and amounted to 583.2 and 228.2, respectively. No normalization of the activities of studied enzymes in ethanol-induced fiver injury was observed in rats without heparegen (groups 4B and 5B). The effect of heparegen administration for 7, 14, and 21 days on the total and nonprotein sulfhydryl compounds and MDA content in fiver is listed in Table 2. Following ethanol ingestion (group 2) total SH groups and nonprotein SH in the fiver were significantly decreased while MDA content was increased in comparison with the control group (group 1).
This low level of SH groups and MDA content was maintained for at least 7 days after cessation of ethanol treatment (group 3B). After heparegen administration for 7 days (group 3A), the values of SH groups were slightly higher, but the differences were significantlydecreased (p < 0.05), when compared to liver SH groups in control animals. The rats which received heparegen for 14 days after cessation of ethanol ingestion (group 4A) possessed normal liver values of total and nonprotein SH groups and MDA concentration. Results of plasma enzyme tests suggest damage of parenchymai fiver cells in rats receiving ethanol (Table 3). The activity of all three enzymes examined were significantly increased after 3 months of ethanol ingestion in comparison with the
TABLE 3 THE EFFECT OF HEPAREGEN ON THE ACTIVITIESOF AIAT, #.spAT, AND GGTP IN PLASMA OF RATS IN ETHANOL-INDUCEDLIVER INJURY Group Control(l) Ethanol(2) 7 days after Heparegen treatment (3A) Ethanol cessation (3B) 14 days after Heparegentreatment (4A) Ethanol cessation (4B) 21 days after Heparegen treatment (5A) Ethanol cessation (513)
AIAt(UL)
AspAT(UL)
GGTP(UL)
87.3 + 16.2
140.6 + 22.1
9.2 + 1.6
126.7 + 26.7
207.9 + 37.8
23.4 + 4.3
92.5 + 14.8 90.8 + 20.2
168.2 + 28.8 173.5 + 23.0
10.3 + 1.5 12.7 + 2.0
93.2 + 20.6 86.1 + 17.8
147.8 + 21.1 156.5 + 20.7
8.3 :t: 2.7 8.6 + 1.9
83.4 + 15.5 90.8 + 20.0
131.5 + 18.8 145.3 + 23.2
8.0 + 1.9 9.3 + 1.6
Chosen statistically significant differences (/7 < 0.05) between groups: AIAT--1-2, 2-3A, 2-3B, 2-4A, 2-4B, 2-5A, 2-5B; AspAT-1-2, I-3A, I-3B, 2-3A, 2-3B, 2--4A, 2-4B, 2-5A, 2-5B, 3A-4A, 3A-5A, 3B-4A, 3B-4B, 3B--5A, 3B--5B, 4A-5A, 4B-SA; GGTP-1-2, 1-3B, 2-3A, 2-3B, 2--4A, 24B, 2-5A, 2-5B, 3A-3B, 3B-4B.
406
FARBISZEWSKI ET AL.
control group. However, in rats treated with heparegen for just 7 days (group 3A), an increase in AIAT, AspAT, and GGTP was observed, when compared with group 2. Histopathological examination (Table 4) of the liver sections obtained from rats intoxicated with ethanol revealed focal steatosis which was predominantly located in central perivenous part of hepatic lobule. Large droplet steatosis was prominent resulting in enlarged hepatocytes. It was seen focal hepatocyte necrosis with infiltration o f neutrophiles and mononuclear phagocytes. The presence of Mallory bodies was only found rarely. Pathological changes in the liver of rats treated with heparegen for 7 days (group 3A) were much less expressive than in group 3B rats, and particularly after 14 days of heparegen treatment (group 4A). Neither cell necrosis nor polymorphonuclear leukocytes were found. In the livers of group 5A, fatty degeneration of hepatocytes, cell necrosis, or Mallory bodies were not observed.
portance of the NADPH-GSH-Px system in scavenging toxic species of oxygen. The superoxide anion radical is the primary free radical species involved in various tissue injuries. It undergoes spontaneous dismutation to produce hydrogen peroxide and, in the presence of iron ions, hydroxyl radicals (23). These radicals are known to be the most reactive species of oxygen radicals because, once generated, they will react with any molecules in their immediate surroundings. It is also the most widely proposed initiator of lipid peroxidation, though other radicals like the ferryl or perferryl iron are also involved in those processes that are widely evaluated by measurement of the level of thiobarbituric acid-reactive substances. The result of the present study shows that ethanol treatment elicits an increase in hepatic malondialdehyde content (measured as TBA reactive substances). On day 7 after cessation of ethanol, the hepatic malondialdehyde level decreases; however, this normalization is significantly more evidenced in rats receiving heparegen. Malondialdehyde is metabolized in living organisms in many nonenzymatic and enzymatic pathways. Among enzymes participating in transformation of malondialdehyde and malondialdehyde-like substances, the most important role of aldehyde dehydrogenase and xanthine oxidase should be stressed. Free SH groups released from N-formyl cysteine may influence xanthine oxidase activity converting this enzyme to xanthine dehydrogenase that is inactive against aldehydic products of lipid peroxidation. Therefore, an increase in the level of thiobarbituric-reactive substances may also result from inhibition of degradation processes. Superoxide anion radicals are less reactive, however, they can diffuse away from the sites of formation, leading to hydroxyl radical generation. Therefore, hydroxyl radicals can he damaging because of their high reactivity, whereas superoxide anion radicals can also be damaging because of their diffusing properties (13). We have observed that the diminution of SOD activity in liver injury is coupled with the decrease of CAT activity. This is consistent with other studies of authors showing that the protective action of SOD is dependent on the presence of CAT (15,21). The increase of SOD activity observed in our studies after beparegen treatment is accompanied by higher CAT activity. It is well known that reactive oxygen radicals can dam-
DISCUSSION The effect of heparegen, thiazolidine-4-carboxylic acid, is well documented in the pharmacotherapy of human and animal liver damage, irrespective of the origin. The drug is converted inside mitochondria into a compound containing free SH groups with a beneficial action in severe toxic liver injury, including ethanol-induced liver damage. Those effects are confirmed by basic biochemical research, morphological studies, and in most clinical examinations, but the influence of this drug on SOD, CAT, and GSH-Px activities and lipid peroxidation in damaged liver has not been evaluated. A direct connection between heparegen and the formation of superoxide anion radicals in ethanol-induced liver injury has not been established either. It has been demonstrated in recent studies that the use of heparegen in rats intoxicated simultaneously with ethanol leads to the diminution of blood ammonia concentration and decreases glutamate dehydrogenase activity (10). Our studies reveal that heparegen administration in the rats for 7, 14, and 21 days gradually restores the decreased activities of liver antioxidant enzymes to normal values. SOD selectively scavenges superoxide anion radicals, and the resulting hydrogen peroxide is removed from the cell mainly by the GSH-Px system (12), and this suggests the im-
TABLE 4 THE EFFECT OF HEPAREGEN ON THE LIVER HISTOLOGY OF RATS IN ETHANOL-INDUCED LIVER INJURY Group Control (1) Ethanol (2) 7 days after Heparegen treatment (3A) Ethanol cessation (3B) 14 days after Heparegen treatment (4A) Ethanol cessation (4B) 21 days after Heparegen treatment ( 5 A ) Ethanol
cessation
(5B)
Steatosis*
Inflammations
Necrosist
+++
+++
++
++ +++
+ ++
+ ++
+ ++
+ ++
+
-
-
-
+
+
-
*+, 0-25% of hepatocytes with fat deposits; + +, 25-50%; + + + , >50%. 1 +, < 1 focus per low power view; + +, > 1 focus; + + +, >2 focus.
HEPAREGEN AND ANTIOXIDANT ENZYME ACTIVITY age enzymatic proteins and thereby decrease SOD, CAT, and GSH-Px activities. The improvement of liver function with heparegen follows an increase of hepatic antioxidane enzymes. Results of enzymatic indicators in plasma (Table 3) and histological studies (Table 4) gave additional evidence supporting this statement. There is increasing evidence that reactive oxygen species, especially hydroxyethyl radical, mediate oxidation of sulfhydryl compounds in rats with ethanol-induced liver
407 damage (5,9,19). The antioxidant activity of GSH-Px is coupled with the oxidation of GSH to oxidized GSSG, which can subsequently be reduced by GSSG reductase using NADPH as the reducing agent (14,17). In conclusion, the ethanol-induced changes reported here are partially normalized by oral heparegen treatment. It is also evident that this drug possesses antioxidant and, consequently, hepatoprotective properties.
REFERENCES 1. Aebi, H. Catalase in vitro. Meth. Enzymol. 105:121-126; 1984. 2. Antonenkov, V. D.; Pirozhkov, S. V.; Popova, S. V.; Panchenko, L. F. Effect of chronic ethanol treatment on lipid peroxidation in rat liver homogenate and subcellular fractions. Int. J. Biochem. 21:1191-1195; 1989. 3. Buege, J. A.; Aust, S. D. Microsomal lipid peroxidation. Meth. Enzymol. 51:302-310; 1978. 4. Cederbanm, A. I. Oxygen radical generation by microsomes: Role of iron and implications for alcohol and toxicity. Free Rad. Biol. Med. 7:559-567; 1989. 5. Chwiecko, M.; Holownia, A.; Pawlowska, D.; Farbiszewski, R. The effect of immunostimulatory drugs on sulfhydryl compounds in plasma, liver and brain after ethanol-induced liver injury in rats. Alcohol 8:179-181; 1991. 6. Dianzani, M. U. Lipid peroxidation in ethanol poisoning: A critical reconsideration. Alcohol Alcohol. 20:161-173; 1985. 7. Dicker, E.; Cederbaum, A. I. Hydroxyl radical generation by microsomes after chronic ethanol consumption. Alcohol. Clin. Exp. Res. 11:309-314; 1987. 8. Ellman, G. L. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82:70-77; 1959. 9. Farbiszewski, R.; Holownia, A.; Chwiecko, M. Changes in sulfhydryl compounds in plasma, fiver and brain after acute and chronic ethanol administration in rats. Drug Alcohol Depend. 20: 129-133; 1987. 10. Farbiszewski, R.; Holownia, A.; Chwiecko, M.; Pawlowska, D. The effects of heparegen and D-penicillamine on the activity of some ammonia metabolizing enzymes in liver and brain of rats intoxicated with ethanol. Drug Alcohol Depend. 27:69-72; 1991. 11. Fernandez-Checa, J.; Ookhtens, M.; Kaplowitz, N. Effect of chronic ethanol feeding on rat hepatocytic glutathione. J. Clin. Invest. 83:1247-1252; 1989. 12. Fridovich, I.; Freeman, B. Antioxidant defenses in the lung. Ann. Rev. Physiol. 48:693-702; 1986. 13. Halliwel, B.; Gutteride, J. M. C. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J. 219:1-14; 1984. 14. Hinder, R. A.; Stein, H. J. Oxygen-derived free radicals. Arch. Surg. 126:104-105; 1991. 15. Koerner, J. E.; Anderson, B. A.; Dage, R. C. Protection against postischemic myocardial dysfunction in anesthetized rabbits with scavengers of oxygen-derived free radicals: superoxide dismutase
plus catalase, N-2-mercapto-propionyl glycine and captopril. J. Cardiovasc. Pharmacol. 17:185-191; 1991. 16. Lowry, O. H.; Rosenbrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with Folin phenol reagent. J. Biol. Chem. 153:265-275; 1951. 17. Munoz, M. E.; Martin, M. I.; Fermoso, J.; Gonzalez, J.; Esteller, A. Effect of chronic ethanol feeding on glutathione and glutathione related enzyme activities in rat liver. Drug Alcohol Depend. 20:221-226; 1987. 18. Njus, D.; Kelley, P. M. Vitamins C and E donate single hydrogen atoms in vivo. FEBS Lett. 284:147-151; 1991. 19. Nordmann, R.; Ribiere, C.; Rouach, H. Alcool et radicaux libres: donnees actuelles. Medecine Sci. 6:336--345; 1988. 20. Paglia, E. D.; Valentine, W. N. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70:158-169; 1967. 21. Przyklenk, K.; Kloner, R. A. Superoxide dismutase plus catalase improve contractile function in the canine model of the "stunned myocardium." Circ. Res. 58:148-156; 1986. 22. Reitman, S.; Frankel, S. A colorimetric method for determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol. 28:56-62; 1957. 23. Southon, P. A.; Powis, G. Free radicals in medicine. Mayo Clin. Proc. 63:381--408;1988. 24. Speisky, H.; Kera, Y.; Pentilla, K. E.; Israel, Y.; Lindros, K. O. Depletion of hepatic glutathione by ethanol occurs independently Of ethanol metabolism. Alcohol. Clin. Exp. Res. 12:224-228; 1988. 25. Sykes, J. A.; McCormack, F. X.; O'Brien, T. J. A preliminary study of the superoxide dismutase content of some human tumors. Cancer Res. 38:2759-2762; 1978. 26. Szewczuk,A.; Kuropatwa, M. A. A new diagnostic 7-GT-test for estimation of V-glutamyltransferaseactivity in serum. Diag. Lab. (in Polish) 21:289-295; 1985. 27. Takenaka, Y.; Miki, M.; Yasuda, H.; Mino, M. The effect of c~-tocopherol as antioxidant on the oxidation of membrane protein thiols induced by free radicals generated in different sites. Arch. Biochem. Biophys. 285:344-350; 1991. 28. Younes, M.; Strubelt, O. Alcohol-induced hepatotoxicity: A role for oxygen free radicals. Free Rad. Res. Commun. 3:19-26; 1987.