THE EFFECT OF MELATONIN ADMINISTRATION ON ETHANOL-INDUCED LIPID PEROXIDATION IN RATS

Pharmacological Research, Vol. 37, No. 1, 1998

THE EFFECT OF MELATONIN ADMINISTRATION ON ETHANOL-INDUCED LIPID PEROXIDATION IN RATS ˙ ¨ ¨ U , YILDIZ ONER-IYIDOGAN ¨ ˙ ˙ ˇ SEMA GENC ¸ , FIGEN GURDOL and ˙ILHAN ONARAN1 Department of Biochemistry, Istanbul Faculty of Medicine, C ¸apa 34390, Istanbul, Turkey and 1 Department of Medical and Biological Sciences, Istanbul Uni®ersity, Cerrahpas¸a, Istanbul, Turkey Accepted 4 No®ember 1997

This study was carried out in order to determine the role of melatonin in preventing lipid peroxidation due to acute ethanol intoxication. Male Wistar Albino rats, 2.5]3 months old, were divided into two groups. Melatonin Žin 1% ethanol, 2 mg kgy1 body weight. was given intraperitoneally Ži.p.. for 21 days to experimental rats whereas controls received 1% ethanol only. On day 21, 6 g kgy1 body weight ethanol was injected to half of the animals in each group and the remainder were kept as corresponding controls. Animals were killed 5 h after ethanol injection. Malondialdehyde ŽMDA., reduced glutathione ŽGSH. and the antioxidant enzyme activities Žsuperoxide dismutase, glutathione peroxidase and catalase . were determined in liver tissue homogenates. MDA levels were increased whereas GSH levels tend to decrease following alcohol injection. Melatonin administration prior to ethanol did not alter MDA and GSH levels of tissue and among antioxidant defence enzymes studied, only CuZn]SOD was found to be increased in animals that received melatonin q ethanol. According to the findings of this study, melatonin did not appear to have any direct effect on alcohol-induced lipid peroxidation. Q 1998 The Italian Pharmacological Society KEY

WORDS:

melatonin, lipid peroxidation, liver, ethanol.

INTRODUCTION The biological effects of free radicals are neutralized in ®i®o by antioxidative defence mechanisms, which include vitamins C and E, carotenoids, glutathione and antioxidant enzymes. Among these enzymes, superoxide dismutase ŽSOD, EC 1.15.1.1. converts a . . into a reactive oxygen intermedifree radical Ž Oy 2 ate ŽH 2 O 2 ., catalase ŽCat, EC 1.11.1.6. detoxifies H 2 O 2 and glutathione peroxidase ŽGPx, EC 1.11.1.9. catalyses the breakdown of H 2 O 2 and lipid hydroperoxides to non-toxic products. Recently, the pineal hormone melatonin was shown to be a very efficient neutralizer of free radicals and its hydroxyl and peroxyl scavenging ability was found to be significantly more effective than both glutathione and vitamin E w1x. It has been shown that melatonin stimulates the antioxidative enzyme GPx in the brain, thus providing indirect protection against free radical attack w2x. In animal experiments, melatonin preU

Corresponding author.

1043]6618r98r010037]04r$25.00r0rfr970263

vented the induction of free radical damage by a variety of conditions including ingestion of toxins, ionizing radiation, ischaemiarreperfusion and excessive exercise w3]7x. Although these reports revealed direct scavenging ability of melatonin, there are studies in which melatonin acted as a weak protector against prooxidants and exhibited very limited direct antioxidant activity w8x. Lipid peroxidation and associated membrane damage is a key feature of alcoholic liver injury w9x. Acute and chronic ethanol, either administered orally or intraperitoneally, lead to a free radical increase and lipid peroxide formation w10x and markedly decrease the levels of reduced glutathione ŽGSH., which is the most abundant non-protein thiol w11x. Since the effect of melatonin on ethanolinduced liver damage has not been studied, this study was undertaken to examine if melatonin is capable of reducing free radical generation in the liver of rats treated with toxic doses of ethanol. Antioxidant enzyme activities, reduced glutathione levels and the concentrations of malondialdehyde were used as indices of oxidative stress. Q1998 The Italian Pharmacological Society

Pharmacological Research, Vol. 37, No. 1, 1998

38

MATERIALS AND METHODS

Thirty-six male Wistar Albino rats, 2.5]3 months old, were divided into two groups. Melatonin ŽSigma. was dissolved in absolute ethanol Žthe ethanol concentration in the final solution was 1%. and given intraperitoneally Ž2 mg kgy1 body weight. for 21 days to experimental rats whereas controls received 1% ethanol only. The animals were maintained under a 12-h alternating lightrdark cycle and were fed standard rat chow and tap water ad libitum. Melatonin was administered at the same time each day. On day 21, 30% ethanol Ž6 g kgy1 body weight. was injected to one-half of the animals in each group Ž n s 9. in two doses and the remainder was kept as control which received two doses of saline through i.p. injections 18 h apart. Ethanol injections were also made with an 18 h-interval and animals were killed under light ether anesthaesia 5 h after the second ethanol injection w12x. The livers were quickly removed, washed three times with ice-cold 0.9% NaCl, dried on a filter paper and were kept at y808C until studied. A piece of tissue was minced and homogenized in 2 ml of 0.05 M potassium phosphate buffer ŽpH 7.0. over ice with a 1:9 wrv ratio. The homogenates were centrifuged at 480 g for 5 min with a refrigerated centrifuge at 48C. The supernatant was removed and the pellet was resuspended and centrifuged again at 480 g; then the supernatants were combined and centrifuged at 6230 g for 30 min w13x. The final supernatant was referred to as the cytosolic fraction and used for the mea-

surement of enzyme activities and for the fluorometric determination of MDA levels w14x. The CuZn]SOD, Cat and GPx activities were measured kinetically w15]17x. Cumen hydroperoxide was used as a substrate for the determination of GPx. The protein content of the cytosolic fraction was measured by the method of Lowry et al. w18x and enzyme activities were expressed as U gy1 protein. For the measurement of GSH levels, liver tissues were homogenized in 0.15 M KCl Žwrv. and the method for the determination of total-SH groups was assayed in deproteinized homogenates w19x. The Student’s t-test with Bonferroni correction was used for the statistical evaluation of the data.

RESULTS

The data obtained from two ethanol-injected groups with or without melatonin and their corresponding controls at the end of the experimental period are shown in Table I. Ethanol injection caused significant increases in MDA levels in both groups, regardless of pre-treatment with melatonin Ž P- 0.001.. The decrement in liver GSH levels was significant in ethanol-treated rats only, when compared to the controls Ž P- 0.05.. There was a slight but non-significant decrease in GSH levels of animals that received melatonin prior to ethanol injections ŽTable I.. In control animals, Cat and GPx activities remained unchanged following ethanol injection.

Table I Antioxidant enzyme activities, MDA and GSH levels in liver tissues of control and experimental rats ŽI.

ŽII.

Normal diet 21 days (a)

Normal diet 21 days q6 g kg y 1 ethanol (b)

Melatonin injection i.p. (a)

MDA m mol mg y1 cytosolic protein

65.8" 14.5

172 " 65UU

57.5" 9.4

135 " 47‡

GSH m mol g y1 protein

7.17" 1.3

4.62" 2.1U

6.31" 1.52

5.03" 1.99

GPx U g y1 cytosolic protein

1579 " 594

1350 " 519

1702 " 566

1290 " 659

Cat U gy1 cytosolic protein

1176 " 322

965 " 397

1509 " 389

1092 " 295

SOD U g y1 cytosolic protein

5122 " 834

6134 " 1813

5481 " 1708

8662 " 1935†§

Each value represents the mean Ž"SD. from nine rats. UU In comparison to Ia: P- 0.05 P- 0.001. †In comparison to IIa: P- 0.01 ‡ P - 0.001. §In comparison to Ib: P- 0.01.

U

Melatonin injection i.p. q6 g kg y 1 ethanol (b)

Pharmacological Research, Vol. 37, No. 1, 1998

39

Among animals which received either melatonin or melatonin q alcohol, relatively lower levels of enzymatic activity for Cat and GPx were seen in the latter. In this group, CuZn]SOD activity showed a 58% increase following ethanol injections Ž P- 0.01., whereas only a 19% increment was found in animals which were treated solely with alcohol. As a result, there was a significant difference between the two ethanol-treated groups, with regard to SOD activity Ž P- 0.01..

the tissue examined and the dose applied. Furthermore, the route of application of this compound may be of importance in order to evaluate its antioxidative properties, since little is known about the absorption and kinetics of this hormone. In view of the preliminary experiments, whether melatonin should be used either at a single high dose, or for a long period at low doses for its scavenging ability against prooxidants needs to be evaluated by in ®i®o experiments.

DISCUSSION

REFERENCES

Ethanol is capable of generating oxygen radicals, inhibiting GSH synthesis, producing GSH loss from the tissue, increasing MDA levels and impairing antioxidative defence systems in humans and experimental animals. It has been shown that marked decreases in the glutathione pool occur in many tissues after acute and chronic ethanol intoxications w12x, which is partly due to the binding of acetaldehyde with cysteine andror glutathione w10x. Severe GSH depletion is known to associate with lipid peroxidation w20x. Our results are consistent with the studies in which GSH content decreased by acute ethanol treatment w21, 22x. Since the role of free radicals in the toxicity of alcohol is widely supported, many researchers have tried to use the antioxidant defence enzymes as biological markers of alcoholism. No variation in the activities of erythrocyte GPx and Cat was observed in alcoholics w23x and only plasma GPx was increased significanty due to alcohol consumption w24x. On the other hand, several groups reported an increase in SOD activity both in plasma and erythrocytes w25]27x. In another study, an approximately 40% increase of SOD activity following one large dose Ž5 g kgy1 body weight. ethanol was observed w28x. In our study, ethanol injection increased liver CuZn]SOD activity by approximately 20%, whereas the other antioxidant enzymes were found to be affected in the opposite way. It is interesting to note that ethanol administration significantly altered CuZn]SOD activity in animals pre-treated with melatonin, revealing a 58% increase when compared to that in melatonin-receiving controls. Although preliminary evidence suggests that melatonin may protect against lipid peroxidation caused by several exogenous agents and conditions w29x, there is no data to our knowledge to assign its protective role in acute ethanol intoxication. According to our findings, melatonin seems to increase the susceptibility of antioxidant defence enzymes, namely SOD, to ethanol when administered in ®i®o. However, no effect on the levels of MDA and GSH was observed. Therefore, melatonin’s effect as a free radical scavenger which was reported previously seems to depend on both

1. Pieri C, Marra MF, Moroni F, Recchioni R, Marcheselli F. Melatonin: a peroxyl radical scavenger more effective than vitamin E. Life Sci 1994; 55: 272]6. 2. Barlow-Walden LR, Reiter RJ, Abe M, Pablos A, Menendez-Pelaez LD, Chen B, Poeggeler B. Melatonin stimulates brain glutathione peroxidase activity. Neurochem Int 1995; 26: 497]502. 3. Sewerynek E, Melchiorri D, Reiter RJ, Ortiz GG, Lewinsky A. Lipopolysaccharide-induced hepatotoxicity is inhibited by the antioxidant melatonin. Eur J Pharmacol 1995; 293: 327]34. 4. Melchiorri D, Reiter RJ, Attia AM, Hara M, Burgos A, Nistico G. Potent protective effect of melatonin on in vivo paraquat-induced oxidative damage in rats. Life Sci 1994; 56: 83]9. 5. Blinkenstaff RT, Brandstadter SM, Reddy S, Witt R. Potential radioprotective agents. 1. Homologs of melatonin. J Pharm Sci 1994; 83: 216]8. 6. Sewerynek E, Reiter RJ, Melchiorri D, Ortiz GG, Lewinsky A. Oxidative damage in the liver induced by ischemia-reperfusion: protection by melatonin. Hepatogastroenterology 1996; 43: 898]905. 7. Reiter RJ. Functional aspects of the pineal hormone melatonin in combating cell and tissue damage induced by free radicals. Eur J Endocrinol 1996; 134: 412]20. 8. Marshall KA, Reiter RJ, Poeggeler B, Aruoma OI, Halliwell B. Evaluation of the antioxidant activity of melatonin in vitro. Free Radical Biol Med 1996; 21: 307]15. 9. Diluzio NR, Stege TE. The role of ethanol metabolites in hepatic lipid peroxidation. In: MM Fisher, JG Rankin. eds. Alcohol and the li®er. New York, London: Plenum Press, 1976: pp. 45]59. 10. Lieber CS. Mechanisms of ethanol-drug-nutrition interactions. Clin Toxicol 1994; 32: 631]81. 11. Hirano T, Kaplowitz N, Tsukamoto H, Kanimura S, Fernandez-Checa JC. Hepatic mitochondrial glutathione depletion and progression of experimental alcoholic liver disease in rats. Hepatology 1992; 6: 1423]7. 12. Guerri C, Grisolia S. Changes in glutathione in acute and chronic alcohol intoxication. Pharmacol Biochem Beha® 1980; 13: 53]61. 13. Ji LL, Dillon D, Wu E. Myocardial aging: antioxidant enzyme systems and related biochemical properties. Am J Physiol 1991; 261: R386]92. 14. Conti M, Morand PC, Levillain P, Lemonnier A. Improved fluorometric determination of malonaldehyde. Clin Chem 1991; 37: 1273]5. 15. Suttle NF, Mcmurray CH. Use of erytrocyte copper: zinc superoxide dismutase activity and hair or fleece copper concentrations in the diagnosis of hypocuprosis in ruminants. Res Vet Sci 1983; 35: 47]52.

Pharmacological Research, Vol. 37, No. 1, 1998

40

16. Beers RF, Faser IW. A spectrophotometric method for measuring the breakdown of H 2 O 2 by catalase. J Biol Chem 1952; 195: 133]40. 17. Paglia OE, Valentine WN. Studies on the quantitative characterization of erythrocyte glutathione peroxidase. J Lab Clin 1967; 70: 158]69. 18. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265]75. 19. Beutler E. Reduced Glutathione (GSH) in Red Cell Metabolism: A Manual of Biochemical Methods. New York: Grune and Stratton, 1971: pp. 103]5. 20. Videla LA, Fernandez V, Ugarte G, Valenzuela A. Effect of acute ethanol intoxication on the content of reduced glutathione in the liver in relation to its lipoperoxidative capacity in the rat. FEBS Lett 1980; 11: 6]10. 21. Speisky H, Mac Donald A, Giles G, Orrego H, Israel Y. Increased loss and decreased synthesis of hepatic glutathione after acute ethanol administration. Biochem J 1985; 225: 565]72. 22. Chen LH, Hu N, Huang TL. Effects of acute alcohol intoxication on liver antioxidant defense systems in rats. Biochem Arch 1992; 8: 95]100. 23. Tanner AR, Bantock I, Hinks L, Lloyd B, Turner NR, Wright R. Depressed selenium and vitamin E

24.

25. 26.

27.

28.

29.

levels in alcoholic population. Possible relationship to hepatic injury through increased lipid peroxidation. Dig Dis Sci 1986; 31: 1307]12. Guemouri L, Artur Y, Herberth B, Jeandel C, Cuny G, Siest G. Biological variability of superoxide dismutase, glutathione peroxidase and catalase in blood. Clin Chem 1991; 37: 1932]7. Del Vilano BC, Miller SI, Schacter LP, Tischfield JA. Elevated superoxide dismutase in black alcoholics. Science 1980; 207: 991]3. Kubota S, Sato N, Matsumura T, Kamada T. Chemiluminescence and superoxide dismutase in the plasma in patients with alcoholic and non-alcoholic liver injuries. Alcohol 1985; 2: 469]72. Ledig M, Doffoel M, Doffoel S, Kopp P, Bockel R, Mandel P. Blood cell superoxide dismutase and enolase activities as markers of alcoholic and nonalcoholic liver diseases. Alcohol 1988; 5: 387]91. Valenzuela A, Fernandez N, Fernandez V, Ugarte G, Videla LA, Guerra R, Villanueva A. Effect of acute ethanol ingestion on lipoperoxidation and on the activity of the enzymes related to peroxide metabolism in rat liver. FEBS Lett 1980; 111: 11]13. Tan DX, Chen LD, Poeggeler B, Manchester LC, Reiter RJ. Melatonin: a potent endogenous hydroxyl radical scavenger. Endocr J 1993; 1: 57]60