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Ethanol-induced changes in lipid peroxidation and glutathione content in rat brain
Drug and Alcohol Dependence,
Elsevier Scientific Publishers
23 (19891227 - 230 Ireland Ltd.
227
Ethanol-induced changes in lipid peroxidation and glutathione content in rat brain
Miijdat Uysal, Giildal Kutalp, Gi.il ozdemirler Department
of Biochemistry,
Istanbul Faculty of Medicine,
University
and Giilqin Aykaq of Istanbul, Cap% Istanbul (Turkey)
(Received November 24th. 19881
The effect of acute and chronic ethanol administration on brain lipid peroxide and glutathione levels was investigated in rats. Acute ethanol administration (5 g/kg, i.p.1 led to an increase in lipid peroxide levels and a decrease in glutathione levels in whole brain homogenates without cerebellum. However, there was no change in brain lipid peroxide and glutathione levels of rats chronically treated with ethanol. Key words: ethanol; lipid peroxidation;
glutathione:
brain; liver; rat
Introduction It is now generally accepted that lipid peroxides play an important role in the pathogenesis of ethanol-induced hepatotoxicity [1,2]. Most reports indicate that lipid peroxidation is stimulated in ethanol-treated animals [3- 51 and human chronic alcoholics [6- 81. It has been suggested that the changes in lipid peroxidation are not restricted to the liver, but occur also in extrahepatic tissues [4,5,9]. Acute and chronic ethanol treatment in rats have been reported to decrease the brain superoxide dismutase activity [lo]. Therefore, it has been proposed that this may account for an accumulation of cytotoxic superoxide radicals, which are known to initiate lipid peroxidation. However, in our previous study [ll], chronic ethanol treatment was shown to be inefficient in inducing changes in lipid peroxide levels in rat whole brain homogenates. It has also been reported that acute ethanol administration had no effect on brain lipid peroxide levels [12,13]. Furthermore, it has been demonstrated [14,15] that after an acute ethanol load, lipid peroxide 0 1989 Elsevier Scientific Publishers 0376-8716/89/$03.50 Printed and Published in Ireland
levels increased and vitamin E and vitamin C levels decreased in rat cerebellum. It was also proposed that lipid peroxidation may be one of the mechanisms responsible for some of the toxic effects of ethanol on the central nervous system [2,15]. Because of these contradictory results, the present study was designed to investigate the effect of acute ethanol administration on brain lipid peroxidation as well as glutathione levels in rats. In addition, we studied rat brain lipid peroxidation in a chronic model different from that used in our previous study. Materials
and methods
Male Wistar rats (NO- 200 gl were given a single dose of ethanol (5 g/kg, i.p., as a 50% solution in saline) and the control group saline only. The animals were killed 1,2,4,6,9, 12 and 24 h after treatment. Both the saline- and ethanoltreated animals had a fasting period 14 - 16 h prior to decapitation. For chronic treatment, ethanol (5 g/kg per day as 50% solution in saline) was given to WisIreland Ltd.
228
tar rats orally for 6 weeks. The control group received the same volume of saline solution. Both groups were fed on a standard diet ad libiturn. Chronically ethanol-treated rats and controls were decapitated following an overnight fasting. Livers and whole brains, except the cerebellum, were rapidly dissected, washed and homogenized in cold 1.15% KC1 with a glass-Teflon homogenizer to make up a 10% homogenate (w/v). Estimation of lipid peroxides and glutathione in liver and brain The degree of lipid peroxidation was assessed by two different methods. (al The level of malondialdehyde was measured by the thiobarbituric acid test according to the method of Ohkawa et al. [16]. The breakdown product of 1,1,3,3-tetraethoxypropane was used as standard. (b1Diene conjugate formation was determined spectrophotometrically at 233 nm, according to Buege and Aust [17], using the original 10% homogenate. The approximate amounts of hydroperoxides were calculated using a molar extinction coefficient of 2.52 x lo4 M-l. Liver and brain glutathione levels were measured with 5,5’-dithiobis-(2-nitrobenzoatel at 412 nm according to Ellman [18]. The protein concentrations of liver and brain homogenates were determined by the method of Lowry et al. [19]. Table I.
Statistical analysis The significance of observed differences between ethanol-treated and control groups were evaluated by Student’s t-test. Results A single dose of ethanol (5 g/kg, i.p.1 led to a significant increase in lipid peroxide levels when assessed by malondialdehyde (MDA) and diene conjugate assays in both liver and brain homogenates in rats (Table Il. In the liver, both MDA and diene conjugate levels increased progressively and the highest value for lipid peroxidation was observed 4 h after ethanol administration. Hepatic diene conjugate levels returned to normal values earlier than MDA levels. In the brain, the MDA levels increased by 18.6O/b, 43.6%, and 35.3% after 1, 2 and 4 h, respectively. A significant increase in brain diene conjugates was detected at 2 h. Diene conjugate levels were increased by 28.6% and 20.7% after 2 and 4 h, respectively. Both the MDA and diene conjugate levels in the brain returned to normal levels after 6 h. Table II shows liver and brain glutathione (GSH) levels following acute ethanol intoxication. The liver GSH levels decreased progressively and the lowest value for GSH levels was observed after 4 h (37.4% decrease vs. control). Brain GSH levels were also decreased 17.8%
Effect of acute ethanol administration on lipid peroxide levels in rat liver and brain (Mean f S.D., 7t = 8). Liver
Brain
Malondialdehyde,
Diene conjugates**
Malondialdehyde
Diene conjugates
Controls
512 f
13.0 + 2.23
1114 f 179
14.0 f 2.19
Ethanol (hours) 1 2 4 6 9 12 24
648 702 900 713 600 540 520
14.4 16.0 19.4 16.9 14.0 13.6 13.2
1321 1600 1508 1130 1200 1170 1100
14.9 18.0 16.9 13.6 13.7 14.0 13.8
59
f 78b f 144b + 111’ f 99 f 80d f 72 f 67
f 2.69 k 2.55” rf:2.66” + 2.66b -e 2.57 f 2.70 f 2.50
‘P < 0.001; ‘P < 0.01; ‘P < 0.02; dP< 0.05 as compared to controls. *pmol/mg protein; **nmol hydroperoxidelmg protein.
-c f f f f f f
119’ 237” 224b 191 170 160 147
f f 2 + + + f
3.38 2.98b 2.78’ 3.44 3.00 2.50 2.17
Table II. Effect of acute ethanol administration on glutathione levels in rat liver and brain (Mean f S.D., n = 8).
Controls Ethanol (hours) 1 2 4 6 9 12 24
Liver glutathione’
Brain glutathione*
28.1 zt 2.48
15.7 f 1.24
25.8 23.2 17.6 20.4 26.2 27.5 26.7
f 1.68d f 1.70a f 2.17’ zt 1.361 f 2.00 f 2.10 zt 2.20
14.8 12.9 13.6 14.6 14.7 15.1 15.5
f f zt + f f +
1.55 2.65’ 1.95d 2.33 2.44 1.92 1.28
.P < 0.001; hP < 0.01; =P < 0.02; dP < 0.05 as compared to controls. *nmol/mg protein.
and 13.4Orbafter 2 and 4 h of ethanol treatment, respectively. Chronic ethanol treatment resulted in increased MDA and GSH levels in the liver, whereas hepatic diene conjugate levels remained unchanged (Table III). However,
Effect of chronic ethanol administration on Table III. lipid peroxide and glutathione levels in rat liver and brain (Mean f S.D.). Controls (n = 10)
Ethanol (n = 10)
Liver
Malondialdehyde 490 zt 72.5 (pmollmg protein) Diene conjugates 11.8 k 1.62 (nmol hydroperoxidel mg protein) Glutathione 28.7 f 3.62 (nmollmg protein) Brain Malondialdehyde 1165 + 167 tpmollmg protein) Diene conjugates 13.9 f 1.96 (nmol hydroperoxidel mg protein) Glutathione 16.0 A 1.75 (nmollmg protein) aP < 0.05 as compared to controls.
568 +
85.2’
13.2 f
2.70
32.8 +
3.80’
1210 f 213 14.2 *
3.85
15.6 k
2.70
there was no significant change in brain MDA, diene conjugate and GSH levels after chronic ethanol treatment. Discussion
The increase in brain lipid peroxidation after acute ethanol treatment was first reported by Nordmann et al. [14] and Rouach et al. [15] who examined lipid peroxidation in cerebellum, the most sensitive site of the brain open to oxidative attacks. They detected that the rate of lipid peroxide formation was increased significantly 2 and 4 h after ethanol administration (2.3 g/kg, i.p.1. They also observed decreases in vitamin E and vitamin C levels 4 h after intoxication. In our study, rats received a single i.p. injection of 5 g/kg ethanol and the examinations were carried out in whole brain minus cerebellum. According to our results lipid peroxidation was stimulated in brain homogenates of ethanol-loaded rats as evaluated by two different assays. Malondialdehyde levels were shown to increase 1, 2 and 4 h after ethanol treatment whereas increases in diene conjugates were observed 2 and 4 h after intoxication (Table I). In addition, glutathione levels were found to decrease 2 and 4 h after 5 g/kg ethanol treatment. The decrease found in glutathione levels in brain homogenates after acute ethanol treatment is a new finding. These results, parallel with those of Nordmann et al. [la] and Rouach et al. [15], demonstrated that lipid peroxidation in brain is stimulated by acute ethanol loading in rats. However+ the chronic ethanol treatment for 6 weeks (5 g/kg, p.0.) did not cause any change in the brain lipid peroxide and glutathione levels. These results are in accordance with those we obtained when rats were given water containing 20% ethanol (v/v) for 2 months [Ill. On the other hand, it is known that brain glutathione levels [20] and superoxide dismutase activity [lo] were decreased in chronic ethanol intoxication in rats. Therefore, as it was the case in acute ethanol intoxication, we expected an increase in lipid peroxide levels in brain homogenates after chronic ethanol treatment,
230
but we could not observe such an increase. Thus, further studies need to be carried out. References
10 11 12 13
M.U. Dianzani, Alcohol Alcoholism, 20 (1985) 161. R. Nordmann, C. Rib&e and H. Rouach, M/S-Med. Sci.. 6 (1988) 336. N. KoGak-Toker et al., IRCS Med. Sci., ll(1983) 915. Y. Kera et al., Res. Commun. Chem. Pathol. Pharmacol., 47 (1985) 203. M. Uysal et al., Biochem. Med., 34 (1985) 370. T. Suematsu et al., Alcoholism: Clin. Exp. Res., 5 (1981) 427. S. Shaw, K.P. Rubin and C.S. Lieber, Dig. Dis. Sci., 28 (1983) 585. M. Uysal et al., Drug Alcohol Depend., 18 (1986) 385. H.H.H. Oei et al., Res. Commun. Chem. Pathol. Pharmacol., 51(1986) 195.
14 15 16 17 18 19 20
M. Ledig, J.-R. M’Paria and P. Mandel, Neurochem. Res., 6 (1981) 385. M. Uysal et al., Drug Alcohol Depend., 18 (1986) 73. N.R. Di Luzio and A.D. Hartmann, Fed. Proc., 26 (1967) 1436. F.P. Corongiu, M. Lai and A. Milia, Biochem. J., 212 (1983) 625. R. Nordmann et al., Bull. Acad. Natle Med., 169 (1985) 1201. H. Rouach et al., Bioelectrochem. Bioenerg., 18 (1987) 211. H. Ohkawa. N. Ohishi and K. Yagi, Anal. Biochem., 95 (1979) 351. J.A. Buege and S.D. Aust, Methods Enzymol., 52 (1978) 302. G. Ellman, Arch. Biochem. Biophys., 82 (1959) 70. O.H. Lowry et al., J. Biol. Chem., 193 (1951) 265. C. Guerri and S. Grisolia, Pharmacoi. Bioehem. Behav., 13 (Suppl. 1) (1980) 53.
Elsevier Scientific Publishers
23 (19891227 - 230 Ireland Ltd.
227
Ethanol-induced changes in lipid peroxidation and glutathione content in rat brain
Miijdat Uysal, Giildal Kutalp, Gi.il ozdemirler Department
of Biochemistry,
Istanbul Faculty of Medicine,
University
and Giilqin Aykaq of Istanbul, Cap% Istanbul (Turkey)
(Received November 24th. 19881
The effect of acute and chronic ethanol administration on brain lipid peroxide and glutathione levels was investigated in rats. Acute ethanol administration (5 g/kg, i.p.1 led to an increase in lipid peroxide levels and a decrease in glutathione levels in whole brain homogenates without cerebellum. However, there was no change in brain lipid peroxide and glutathione levels of rats chronically treated with ethanol. Key words: ethanol; lipid peroxidation;
glutathione:
brain; liver; rat
Introduction It is now generally accepted that lipid peroxides play an important role in the pathogenesis of ethanol-induced hepatotoxicity [1,2]. Most reports indicate that lipid peroxidation is stimulated in ethanol-treated animals [3- 51 and human chronic alcoholics [6- 81. It has been suggested that the changes in lipid peroxidation are not restricted to the liver, but occur also in extrahepatic tissues [4,5,9]. Acute and chronic ethanol treatment in rats have been reported to decrease the brain superoxide dismutase activity [lo]. Therefore, it has been proposed that this may account for an accumulation of cytotoxic superoxide radicals, which are known to initiate lipid peroxidation. However, in our previous study [ll], chronic ethanol treatment was shown to be inefficient in inducing changes in lipid peroxide levels in rat whole brain homogenates. It has also been reported that acute ethanol administration had no effect on brain lipid peroxide levels [12,13]. Furthermore, it has been demonstrated [14,15] that after an acute ethanol load, lipid peroxide 0 1989 Elsevier Scientific Publishers 0376-8716/89/$03.50 Printed and Published in Ireland
levels increased and vitamin E and vitamin C levels decreased in rat cerebellum. It was also proposed that lipid peroxidation may be one of the mechanisms responsible for some of the toxic effects of ethanol on the central nervous system [2,15]. Because of these contradictory results, the present study was designed to investigate the effect of acute ethanol administration on brain lipid peroxidation as well as glutathione levels in rats. In addition, we studied rat brain lipid peroxidation in a chronic model different from that used in our previous study. Materials
and methods
Male Wistar rats (NO- 200 gl were given a single dose of ethanol (5 g/kg, i.p., as a 50% solution in saline) and the control group saline only. The animals were killed 1,2,4,6,9, 12 and 24 h after treatment. Both the saline- and ethanoltreated animals had a fasting period 14 - 16 h prior to decapitation. For chronic treatment, ethanol (5 g/kg per day as 50% solution in saline) was given to WisIreland Ltd.
228
tar rats orally for 6 weeks. The control group received the same volume of saline solution. Both groups were fed on a standard diet ad libiturn. Chronically ethanol-treated rats and controls were decapitated following an overnight fasting. Livers and whole brains, except the cerebellum, were rapidly dissected, washed and homogenized in cold 1.15% KC1 with a glass-Teflon homogenizer to make up a 10% homogenate (w/v). Estimation of lipid peroxides and glutathione in liver and brain The degree of lipid peroxidation was assessed by two different methods. (al The level of malondialdehyde was measured by the thiobarbituric acid test according to the method of Ohkawa et al. [16]. The breakdown product of 1,1,3,3-tetraethoxypropane was used as standard. (b1Diene conjugate formation was determined spectrophotometrically at 233 nm, according to Buege and Aust [17], using the original 10% homogenate. The approximate amounts of hydroperoxides were calculated using a molar extinction coefficient of 2.52 x lo4 M-l. Liver and brain glutathione levels were measured with 5,5’-dithiobis-(2-nitrobenzoatel at 412 nm according to Ellman [18]. The protein concentrations of liver and brain homogenates were determined by the method of Lowry et al. [19]. Table I.
Statistical analysis The significance of observed differences between ethanol-treated and control groups were evaluated by Student’s t-test. Results A single dose of ethanol (5 g/kg, i.p.1 led to a significant increase in lipid peroxide levels when assessed by malondialdehyde (MDA) and diene conjugate assays in both liver and brain homogenates in rats (Table Il. In the liver, both MDA and diene conjugate levels increased progressively and the highest value for lipid peroxidation was observed 4 h after ethanol administration. Hepatic diene conjugate levels returned to normal values earlier than MDA levels. In the brain, the MDA levels increased by 18.6O/b, 43.6%, and 35.3% after 1, 2 and 4 h, respectively. A significant increase in brain diene conjugates was detected at 2 h. Diene conjugate levels were increased by 28.6% and 20.7% after 2 and 4 h, respectively. Both the MDA and diene conjugate levels in the brain returned to normal levels after 6 h. Table II shows liver and brain glutathione (GSH) levels following acute ethanol intoxication. The liver GSH levels decreased progressively and the lowest value for GSH levels was observed after 4 h (37.4% decrease vs. control). Brain GSH levels were also decreased 17.8%
Effect of acute ethanol administration on lipid peroxide levels in rat liver and brain (Mean f S.D., 7t = 8). Liver
Brain
Malondialdehyde,
Diene conjugates**
Malondialdehyde
Diene conjugates
Controls
512 f
13.0 + 2.23
1114 f 179
14.0 f 2.19
Ethanol (hours) 1 2 4 6 9 12 24
648 702 900 713 600 540 520
14.4 16.0 19.4 16.9 14.0 13.6 13.2
1321 1600 1508 1130 1200 1170 1100
14.9 18.0 16.9 13.6 13.7 14.0 13.8
59
f 78b f 144b + 111’ f 99 f 80d f 72 f 67
f 2.69 k 2.55” rf:2.66” + 2.66b -e 2.57 f 2.70 f 2.50
‘P < 0.001; ‘P < 0.01; ‘P < 0.02; dP< 0.05 as compared to controls. *pmol/mg protein; **nmol hydroperoxidelmg protein.
-c f f f f f f
119’ 237” 224b 191 170 160 147
f f 2 + + + f
3.38 2.98b 2.78’ 3.44 3.00 2.50 2.17
Table II. Effect of acute ethanol administration on glutathione levels in rat liver and brain (Mean f S.D., n = 8).
Controls Ethanol (hours) 1 2 4 6 9 12 24
Liver glutathione’
Brain glutathione*
28.1 zt 2.48
15.7 f 1.24
25.8 23.2 17.6 20.4 26.2 27.5 26.7
f 1.68d f 1.70a f 2.17’ zt 1.361 f 2.00 f 2.10 zt 2.20
14.8 12.9 13.6 14.6 14.7 15.1 15.5
f f zt + f f +
1.55 2.65’ 1.95d 2.33 2.44 1.92 1.28
.P < 0.001; hP < 0.01; =P < 0.02; dP < 0.05 as compared to controls. *nmol/mg protein.
and 13.4Orbafter 2 and 4 h of ethanol treatment, respectively. Chronic ethanol treatment resulted in increased MDA and GSH levels in the liver, whereas hepatic diene conjugate levels remained unchanged (Table III). However,
Effect of chronic ethanol administration on Table III. lipid peroxide and glutathione levels in rat liver and brain (Mean f S.D.). Controls (n = 10)
Ethanol (n = 10)
Liver
Malondialdehyde 490 zt 72.5 (pmollmg protein) Diene conjugates 11.8 k 1.62 (nmol hydroperoxidel mg protein) Glutathione 28.7 f 3.62 (nmollmg protein) Brain Malondialdehyde 1165 + 167 tpmollmg protein) Diene conjugates 13.9 f 1.96 (nmol hydroperoxidel mg protein) Glutathione 16.0 A 1.75 (nmollmg protein) aP < 0.05 as compared to controls.
568 +
85.2’
13.2 f
2.70
32.8 +
3.80’
1210 f 213 14.2 *
3.85
15.6 k
2.70
there was no significant change in brain MDA, diene conjugate and GSH levels after chronic ethanol treatment. Discussion
The increase in brain lipid peroxidation after acute ethanol treatment was first reported by Nordmann et al. [14] and Rouach et al. [15] who examined lipid peroxidation in cerebellum, the most sensitive site of the brain open to oxidative attacks. They detected that the rate of lipid peroxide formation was increased significantly 2 and 4 h after ethanol administration (2.3 g/kg, i.p.1. They also observed decreases in vitamin E and vitamin C levels 4 h after intoxication. In our study, rats received a single i.p. injection of 5 g/kg ethanol and the examinations were carried out in whole brain minus cerebellum. According to our results lipid peroxidation was stimulated in brain homogenates of ethanol-loaded rats as evaluated by two different assays. Malondialdehyde levels were shown to increase 1, 2 and 4 h after ethanol treatment whereas increases in diene conjugates were observed 2 and 4 h after intoxication (Table I). In addition, glutathione levels were found to decrease 2 and 4 h after 5 g/kg ethanol treatment. The decrease found in glutathione levels in brain homogenates after acute ethanol treatment is a new finding. These results, parallel with those of Nordmann et al. [la] and Rouach et al. [15], demonstrated that lipid peroxidation in brain is stimulated by acute ethanol loading in rats. However+ the chronic ethanol treatment for 6 weeks (5 g/kg, p.0.) did not cause any change in the brain lipid peroxide and glutathione levels. These results are in accordance with those we obtained when rats were given water containing 20% ethanol (v/v) for 2 months [Ill. On the other hand, it is known that brain glutathione levels [20] and superoxide dismutase activity [lo] were decreased in chronic ethanol intoxication in rats. Therefore, as it was the case in acute ethanol intoxication, we expected an increase in lipid peroxide levels in brain homogenates after chronic ethanol treatment,
230
but we could not observe such an increase. Thus, further studies need to be carried out. References
10 11 12 13
M.U. Dianzani, Alcohol Alcoholism, 20 (1985) 161. R. Nordmann, C. Rib&e and H. Rouach, M/S-Med. Sci.. 6 (1988) 336. N. KoGak-Toker et al., IRCS Med. Sci., ll(1983) 915. Y. Kera et al., Res. Commun. Chem. Pathol. Pharmacol., 47 (1985) 203. M. Uysal et al., Biochem. Med., 34 (1985) 370. T. Suematsu et al., Alcoholism: Clin. Exp. Res., 5 (1981) 427. S. Shaw, K.P. Rubin and C.S. Lieber, Dig. Dis. Sci., 28 (1983) 585. M. Uysal et al., Drug Alcohol Depend., 18 (1986) 385. H.H.H. Oei et al., Res. Commun. Chem. Pathol. Pharmacol., 51(1986) 195.
14 15 16 17 18 19 20
M. Ledig, J.-R. M’Paria and P. Mandel, Neurochem. Res., 6 (1981) 385. M. Uysal et al., Drug Alcohol Depend., 18 (1986) 73. N.R. Di Luzio and A.D. Hartmann, Fed. Proc., 26 (1967) 1436. F.P. Corongiu, M. Lai and A. Milia, Biochem. J., 212 (1983) 625. R. Nordmann et al., Bull. Acad. Natle Med., 169 (1985) 1201. H. Rouach et al., Bioelectrochem. Bioenerg., 18 (1987) 211. H. Ohkawa. N. Ohishi and K. Yagi, Anal. Biochem., 95 (1979) 351. J.A. Buege and S.D. Aust, Methods Enzymol., 52 (1978) 302. G. Ellman, Arch. Biochem. Biophys., 82 (1959) 70. O.H. Lowry et al., J. Biol. Chem., 193 (1951) 265. C. Guerri and S. Grisolia, Pharmacoi. Bioehem. Behav., 13 (Suppl. 1) (1980) 53.