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Effect of thyroidectomy and adrenalectomy on changes in liver glutathione and malonaldehyde levels after acute ethanol injection
Free Radical Biology & Medicine, Vol. 14, pp. 655-660, 1993 Primed in the USA. All rights reserved.
0891-5849/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd.
Brief Communication EFFECT OF THYROIDECTOMY AND ADRENALECTOMY ON CHANGES IN LIVER GLUTATHIONE AND MALONALDEHYDE LEVELS AFTER ACUTE ETHANOL INJECTION
J. P. TEARE,S. M. GREENFIELD,J. S. MARWAY,*V. R. PREEDY,*N. A. PUNCHARD,T. J. PETERS,* and R. P. H . THOMPSON Gastrointestinal Laboratory, The Rayne Institute, St Thomas' Hospital, London SE1 7EH, England; and *Department of Clinical Biochemistry, King's College Hospital, London SE5 9RS, England (Received 19 October 1992; Revised 1 December 1992; Accepted 17 December 1992)
Abstract--At low concentrations ethanol is metabolized largely by alcohol dehydrogenase to acetaldehyde, while at higher concentrations a microsomal ethanol oxidising system (MEOS) is involved, namely cytochrome P450 liE 1, which also probably generates free radical species. In hyperthyroidism hepatic glutathione stores are depleted and net superoxide anion production occurs. In contrast, in hypothyroidism hepatic glutathione may be increased and thus renders the liver less sensitive to alcohol generated free radical production. Steroid hormones inhibit lipid peroxidation. Sixty male Wistar rats either underwent thyroidectomy, adrenalectomy, or sham procedures. Twenty control animals were pair fed with thyroidectomized animals, whilst another twenty fed ad libitum. An intraperitoneal injection of alcohol (75 mmol/kg) was given 2.5 h prior to sacrifice to half the animals in each group, the remainder receiving saline. The total hepatic glutathione contents of the pair fed and the ad libitum groups were not different, but were significantly increased by thyroidectomy (p = <0.001 ). This effect was significantly reduced by alcohol (p < 0.01). The sham procedures and dietary restrictions had no effect. The ethanol alone reduced total hepatic glutathione, but this only reached statistical significance in the thyroidectomized and sham-adrenalectomized groups. Hepatic malonaidehyde (MDA) levels were significantly reduced in the thyroidectomy group but alcohol had no effect on them. We conclude that hypothyroidism increased hepatic glutathione status, presumably by reducing radical production by enzyme systems, which would otherwise consume this important scavenger. Long term exposure to ethanol with induction of MEOS is probably required for it to generate toxic levels of free radical species. Keywords--Alcohol, Malonaldehyde, Glutathione, Thyroidectomy, Adrenalectomy, Liver, Free radicals
INTRODUCTION
The latter is induced by alcohol and can generate toxic radical metabolites in addition to acetaldehyde.2 The liver is effectively protected against oxidative stresses by intracellular glutathione 9 via several enzymatic and non-enzymatically catalyzed reactions. ~3 These include reduction of hydroperoxides by glutathione transferase and handling ofglutathione conjugates by glutamyl transferases. ~4 In both man 9 and primates ~schronic alcohol use results in depressed hepatic reduced glutathione. Adrenal hormones may inhibit lipid peroxidation both in vitro ~6and in vivo,17-19achieved through inhibition ofcytochrome P450 hydroxylations and superoxide anion p r o d u c t i o n . 2°,21 Hyperthyroid states in man lead to the production of superoxide anion and thiobarbituric acid reactive s u b s t a n c e s . 22 We therefore studied the effect of an acute dose of
The exact mechanism of the hepatic toxicity of ethanol is unknown, and although the overall risks of liver damage rise with increasing daily consumption, many 'heavy' abusers seem to avoid physical damage from alcohol. It was initially proposed by Di Luzio and Hartman ~that hepatic lipid peroxidation was generated by free radical production following alcohol ingestion, and subsequent experimental evidence has supported t h i s v i e w . 2-12 Radical production may occur either as a result of acetaldehyde4,~° formed through the alcohol dehydrogenase, or through cytochrome P450II E 1, part of the microsomal ethanol oxidising system. Address correspondence to: Dr. J. P. Teare. 655
656
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ethanol on hepatic glutathione status and the levels of the product of lipid peroxidation, malonaldehyde. In addition we studied the added effect of decreasing either adrenal or thyroid hormone levels, since both these hormone systems have a role in the regulation of radical production. METHODS
Male Wistar rats were obtained from Interfauna UK Ltd (Huntingdon, UK) and maintained in 12 hr light and dark cycles. Two weeks prior to the study, groups of 24 rats were either thyroidectomized, adrenalectomized, or sham operated. The thyroidectomized animals had the smallest food intakes, and so the other operated animals had their intakes restricted to this group. A group of 24 control animals were pair fed to this group, whist another group was allowed to feed ad libitum. All rats were allowed free access to tap water except the adrenalectomized rats, which were given 0.9% Sodium Chloride as drinking water ad libitum. Two and a half h prior to sacrifice, half the animals in each group were injected with ethanol (75 mmol/kg body weight intraperitoneally) the rest with an equivalent volume of saline. Animals were sacririced over a 6 h time period following the normal overnight feeding period, to control for the effects of fasting on hepatic glutathione.
Preparation of samples Hepatic giutathione analysis: within four min of sacrifice, the liver was removed and 1.0 gram liver homogenised in five volumes of 1% picric acid. The homogenate was then centrifuged at 2500 g for 15 min and the supernatant stored at -70°C for less than four weeks until later analysis according to our reported adaptation 23 of the method of Griffith.24 Hepatic malonaldehyde concentration was measured in an aliquot of liver homogenized in phosphate buffered saline (pH 7.4) and stored at -70°C for less than four weeks until analysis, by high performance liquid chromatography (HPLC) by the method of Esterbauer, z5 This is more specific than the measurement of thiobarbituric acid reactive material26 which also registers decomposition products that arise during the heating stage of the r e a c t i o n 27 a s well as nonlipid derived products. Malonaldehyde was separated from the whole homogenate by precipitation with an equal volume of acetonitrile and centrifuging the mixture, after thoroughly mixing, at 3000 g for five min. The clear supernatant was then injected (via a Rheodyne injection valve with a 20 ul loop) onto an $5 Spherisorb-NH2
column, connected to a Shimadzu LC 6A pump and SPD 6A UV detector set at 270 nm. The column was eluted with acetonitrile/0.03 M Tris at pH 7.0 (1:9 v/v) and the absorbance read at 270 nm and compared to that for MDA standards which were prepared from 1,1,3,3,tetra-methoxy propane (TMP) by hydrolysing the TMP for 1 h at 50°C and then calculating the molar extinction coefficient.
Statistical analysis As the groups were paired, multiple paired t-tests were performed. To avoid the defects in the use of multiple t-tests an overall test based on the analysis of variance was first performed; if this indicated differences, modified t-tests were performed. Significance was taken at the 5% level. RESULTS
Hepatic glutathione content There was a difference between the total hepatic glutathione content of pair fed and the ad libitum groups suggesting that restricting the intakes to that of the thyroidectomized animals impaired hepatic glutathione synthesis (p < 0.005) (Fig. 1). In the thyroidectomized animals injected with saline the level of total glutathione was significantly (p < 0.001) increased compared to the pair fed controls, and this effect was significantly (p < 0.01) reduced by acute administration of alcohol (Fig. 1). This was not an effect of the surgical procedure, as the sham operated animals were not different to the pair fed controls. Results expressed in the figures are for total glutathione, as there was no difference in the ratio of oxidised:reduced glutathione in any of the groups studied. Adrenalectomy had no effect on the mean total glutathione levels compared to pair fed controls, and alcohol injection in these animals made no difference. The sham procedure also had no effect.
Effect of alcohol Hepatic total glutathione levels were reduced following alcohol infusion, but only in the thyroidectomized (< 0.01), and sham adrenalectomized (< 0.01) did this reach statistical significance (Fig. 1).
Hepatic malonaldehyde levels In the saline group, thyroidectomy significantly (p < 0.03) decreased hepatic MDA production, compared to pair fed controls. Adrenalectomy had no effect and nor did the sham procedures (Fig. 2), as there
Changes in liver after acute ethanol injection
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0 Tx
sham Tx
Adx
sham Adx
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Ad lib
Fig. 1. Total Hepatic glutathione levels following adrenalectomy (Adx) and thyroidectomy (Tx). *p < 0.005 versus pair fed saline controls; **p < 0.001 versus pair fed saline controls; +p < 0.01 versus saline control. Results expressed as mean _+ SEM.
was data variability. Alcohol had no effect in any of the groups studied (Fig. 2). DISCUSSION
At low concentrations most alcohol is metabolised by cytosolic alcohol dehydrogenase to acetaldehyde, while another microsomal ethanol oxidising system (MEOS), which is linked to cytochrome P450, requires an higher ethanol concentration for maximal activity and is induced by chronic alcohol administration. 2 Induction of the P450 system also increases pro-
duction of acetaldehyde, which may cause liver injury by forming adducts with proteins and stimulating the formation of antibodies, 3 but also inactivates enzymes, damages cell membranes, 4 depletes glutathione, and induces free radical damage and hepatic collagen synthesis.4 Acetaldehyde causes lipid peroxidation by free radical generation in isolated perfused livers, 5 in rat liver microsome systems6 and in v i v o . 7 By binding to cysteine or glutathione, it depresses hepatic glutathione levels and reduces the scavenging of radicals by this tripeptide. Glutathione levels are normally lowest in centrilobular hepatocytes where the
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sham Tx
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sham Adx
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Ad lib
Fig. 2. Hepatic malonaldehyde levels following adrenalectomy and thyroidectomy. *p < 0.03 versus pair fed saline control. Results expressed as mean _+ SEM.
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J.P. TEARE et aL
earliest lesions in alcoholic liver disease (ALD) are seen, and reduction of hepatic glutathione levels increases hepatic damage after alcohol ingestion.8 Liver biopsy specimens from chronic alcohol abusers have reduced glutathione levels, suggesting that this important radical scavenger has been depleted, 9-H and increased levels of by-products of radical formation as measured by diene conjugates.9,~2 Increased formation of hydroxyl radicals, 28-32 superoxide anion, 33 thiobarbituric acid reactive substances, 34and metabolites that adduct to electron spin t r a p s 35-37 have also been demonstrated. Steroid hormones are known to inhibit lipid peroxidation in vitro 16and similarly in vivo. 17-19Corticosteroids are known to affect the activity of a number of enzymes, including aryl hydrocarbon hydroxylase, a cytochrome P450 related enzyme/7 Cytochrome P450 hydroxylations are known to produce superoxide anion, and are influenced by steroid hormones, 2°'2~cortisol decreasing the production in some biological systems19 but not in others. 37 The greatest production is seen however in the adrenalectomized animal 16'18and is reversed by cortisol administration. However in this study adrenalectomized rats had normal hepatic glutathione and MDA levels suggesting that there was no excess of free radical production. Adrenalectomy with an associated reduction of antioxidant corticosteroid hormones should reduce glutathione levels and increase lipid peroxides.~6-18 However, this was not seen in our model, and could be explained by alterations in the activities of relative hydroxylase and oxidase enzyme systems.38Hydroxylation systems tend to produce superoxide anion, 2° and a lack of steroid hormones would result in a decrease in the activity of this enzyme. We have shown a rise in hepatic glutathione levels following thyroidectomy, which could be due to either increased production, reduced sinusoidal ettlux, or reduced utilization of this important free radical scavenger. This would be in keeping with previous r e p o r t s 22'39-46 where markers of lipid peroxidation were elevated in hyperthyroid states. Hyperthyroidism in man and rats increases the production of superoxide anion and thiobarbituric acid reactive substances, 22 an effect that can be abolished by antithyroid agents such as propylthiouracil.22'4° This phenomenon seems to be mediated by enhanced oxidative capacity of liver microsomes41 with increased activity of NADPH cytochrome P450 reductase42'44 and NADPH oxidase44'45 enzymes whose activity promotes superoxide production. 46 These effects are seen with nano-molar concentrations of thyroid hormones present in clinical hyperthyroid states in man. 4° This cellular response is associated
with glutathione depletion, cytochrome P450 loss and decreased concentration of membrane fatty a c i d s . 47 The thyroidectomized state might therefore reverse these effects, with a reduction in markers of lipid peroxidation and subsequent elevation of tissue levels of glutathione, which is a potent free radical scavenger. In general there was a reduction in hepatic glutathione following the alcohol injection, though this was only significant in two of the groups. This effect has been noticed before, and is presumably partly due to utilization of the radical scavenger in mopping up the small amounts of peroxides produced by the cytochrome P450 II E1 in its preinduced status and those peroxides forced from the metabolism of acetaldehyde.4 The lack of difference in the ratios of oxidised (GSSG) to reduced glutathione (GSH) that we have shown in the groups studied could be due to the close regulation of oxidised glutathione within cell. Intracellular GSH depletion occurs largely due to effiux across sinusoidal and biliary canalicular membranes. 48 Maintenance of the cellular redox state depends upon the rapid and efficient conversion of GSSG back to its reduced state or to mixed protein disulphides.49 These reactions are catalysed by GSH peroxidase and reductase systems, and are dependent on the NADP+/NADPH redox pair. That we have not seen a rise in hepatic GSSG highlights the efficiency of these enzyme systems in preserving intracellular GSH. The decrease in total glutathione in the sham adrenalectomised group is difficult to explain, but may represent a minor variation in nutritional status at the time of sacrifice, despite attempts to control for this by sacrificing all animals over a short time period following overnight feeding, since fasting rapidly depletes hepatic glutathione. 5° Significant production of peroxide radicals detectable as malonaldehyde was not seen in this model, and this supports the hypothesis that long term exposure to ethanol, with induction of the cytochrome P450 IIE 1, is necessary for free radical production in response to alcohol ingestion. Acknowledgement - - We are grateful to the Trustees of St Thomas'
Hospital for their continuing support.
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hyperthyroid patients: Suppression by propyl thioumcil treatment. Free Rad. Res. Comms. 5:1-10; 1988. 23. Teare, J. P.; Punchard, N. A.; Poweil, J. J.; Lumb, P. J.; Mitchell, W. D.; Thompson, R. P. H. An automated spectrophotometric method for the determination of oxidised and reduced glutathione in the liver, Clin. Chem. 1993; in press. 24. Grifiith, D. W. Glutathione and glutathione disulphide. In: Bergmeyer, H. U., ed. Methods of enzymatic analysis. Vol III. Berlin, Germany: VCH Publishers; 1983:521-529. 25. Esterbauer, H.; Lang, J.; Zadravec, S.; Slater, T. F. Detection of malonaldehyde by high performance liquid chromatography. In: Packer, L., ed. Methods in enzymology. Vol. 105. Orlando: Academic Press; 1984:319-328. 26. Stocks, J.; Offerman, E. L.; Modell, C. B.; Dormandy, T. L. The susceptibility to autooxidation of human red cell lipids in health and disease. B. J. Haematol. 23:713-724; 1972. 27. Slater, T. F. Free radical mechanisms in tissue injury. London: Pion, 1972. 28. Fink, R.; Clemens, M. R.; Dormandy, T. L. et al. Increased free radical activity in alcoholics. Lancet 2:291-294; 1985. 29. Cederbaum, A. R. Microsomal generation of hydroxyl radicals: Its role in microsomal ethanol oxidising system (MEOS) activity and its requirement for iron. Ann-N-Y-Acad. Sci. 492:3549; 1987. 30. Ekstrom, G.; Cronholm, T.; Ingelman-Sundberg,M. Hydroxyl radical production and ethanol oxidation by liver microsomes isolated from ethanol treated rats. Biochems. 233:755-761; 1986. 31. Krikun, G.; Lieber, C. S.; Cederbaum, A. I. Increased microsomal oxidation of ethanol by cytochrome P-450 and hydroxyl radical-dependent pathways after chronic ethanol consumption. Biochem. Pharmacol. 33:3306-3309; 1984. 32. Cederbaum, A. I.; Cohen, G. Microsomal oxidant radical production and ethanol oxidation. Methods Enzymol. 105:516522; 1984. 33. Williams, A. J.; Barry, R. E. Free radical generation by neutrophils: A potential mechanism of cellular injury in acute alcoholic hepatitis. Gut 28:1157-1161; 1987. 34. Ekstrom, G.; Ingelman-Sundberg, M. Rat liver microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P4so. Biochem. Pharmacol. 38:1313-1319; 1989. 35. Reinke, L. A.; Rau, J. M.; McCay, P. B. Possible roles of free radicals in alcoholic tissue damage, Free Rad. Res. Comm. 9:205-211; 1990. 36. Knecht, K. T.; Bradford, B. V.; Mason, R. P.; Thurman, R. G. In-vivo formation of a free radical metabolite of ethanol. Mol. Pharmacol. 38:26-30; 1990. 37. Albano, E.; Tomasi, A.; Goria-Gatti, L.; Dianzani, M. U. Spin trapping of free radical species produced during the microsomal metabolism of ethanol. Chem. Biol. Interact. 65:223234; 1988. 38. Nelson, Dh.; Ruhmann-Wennhold, A. Corticosteroids increase superoxide anion production by rat liver microsomes. J. Clin. Invest. 56:1062-1065; 1975. 39. Nord Bloom, G. D.; Coon, M. J. Hydrogen peroxide formation and stoichiometry of hydroxylastion reactions catalyzed by highly purified liver microsomal cytochrome P45o. Arch. Biochem. Biophys. 180:343-347; 1977. 40. Faure, M.; Lissi, E.; Kidela, L. A. Evaluation ofthe antioxidant properties of thyroid hormones and propyithiouracil in the brain-homogenate autooxidation system and in the free radical mediated oxidation of erythrocyte membranes. Chem. Biol. Interact. 77:173-185; 1991. 41. Marzoev, A. I.; Kozliv, A. V.; Andryuschenko, A. P.; Vladimiro, Y. A. Activation of lipid peroxidation in liver mitochondria of hyperthyroid rabbits. Bull. Exp. Biol. Med. 93:269-272; 1982. 42. De Araujo, P. S.; De Andrade-Silva, R.; Raw, I. Effects of drugs
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J.P. TEAREel al. and hormones on rat liver dimethylamin benzene reductase activity. Braz. J. Med. Biol. Res. 15:17-28; 1982. Landriscina, C.; Petragallo, V.; Morini, P.; Marcotrigiano, G. O. Lipid peroxidation in rat microsomes--stimulation of the NADPH cytochrome P450reductase dependent process in the hyperthyroid state. Biochem. Int. 17:385-393; 1988. Kato, R.; Takahashi, A. Thyroid hormone and activities of drug metabolizing enzymes and electron transport systems of rat liver microsomes. Mo. Pharmacol. 4:109-120; 1968. Fernandez, V.; Llesuy, S.; Solari, L.; et al. Chemiluminescent and respiratory response related to thyroid hormone oxidative stress. Free. Rad. Res. Comms. 5:77-84; 1988. Fong, K. L.; McCay, P. B.; Poyer, J. L.; Keele, B. B.; Misra, H. Evidence that peroxidation of lysosomal membranes is initi-
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0891-5849/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd.
Brief Communication EFFECT OF THYROIDECTOMY AND ADRENALECTOMY ON CHANGES IN LIVER GLUTATHIONE AND MALONALDEHYDE LEVELS AFTER ACUTE ETHANOL INJECTION
J. P. TEARE,S. M. GREENFIELD,J. S. MARWAY,*V. R. PREEDY,*N. A. PUNCHARD,T. J. PETERS,* and R. P. H . THOMPSON Gastrointestinal Laboratory, The Rayne Institute, St Thomas' Hospital, London SE1 7EH, England; and *Department of Clinical Biochemistry, King's College Hospital, London SE5 9RS, England (Received 19 October 1992; Revised 1 December 1992; Accepted 17 December 1992)
Abstract--At low concentrations ethanol is metabolized largely by alcohol dehydrogenase to acetaldehyde, while at higher concentrations a microsomal ethanol oxidising system (MEOS) is involved, namely cytochrome P450 liE 1, which also probably generates free radical species. In hyperthyroidism hepatic glutathione stores are depleted and net superoxide anion production occurs. In contrast, in hypothyroidism hepatic glutathione may be increased and thus renders the liver less sensitive to alcohol generated free radical production. Steroid hormones inhibit lipid peroxidation. Sixty male Wistar rats either underwent thyroidectomy, adrenalectomy, or sham procedures. Twenty control animals were pair fed with thyroidectomized animals, whilst another twenty fed ad libitum. An intraperitoneal injection of alcohol (75 mmol/kg) was given 2.5 h prior to sacrifice to half the animals in each group, the remainder receiving saline. The total hepatic glutathione contents of the pair fed and the ad libitum groups were not different, but were significantly increased by thyroidectomy (p = <0.001 ). This effect was significantly reduced by alcohol (p < 0.01). The sham procedures and dietary restrictions had no effect. The ethanol alone reduced total hepatic glutathione, but this only reached statistical significance in the thyroidectomized and sham-adrenalectomized groups. Hepatic malonaidehyde (MDA) levels were significantly reduced in the thyroidectomy group but alcohol had no effect on them. We conclude that hypothyroidism increased hepatic glutathione status, presumably by reducing radical production by enzyme systems, which would otherwise consume this important scavenger. Long term exposure to ethanol with induction of MEOS is probably required for it to generate toxic levels of free radical species. Keywords--Alcohol, Malonaldehyde, Glutathione, Thyroidectomy, Adrenalectomy, Liver, Free radicals
INTRODUCTION
The latter is induced by alcohol and can generate toxic radical metabolites in addition to acetaldehyde.2 The liver is effectively protected against oxidative stresses by intracellular glutathione 9 via several enzymatic and non-enzymatically catalyzed reactions. ~3 These include reduction of hydroperoxides by glutathione transferase and handling ofglutathione conjugates by glutamyl transferases. ~4 In both man 9 and primates ~schronic alcohol use results in depressed hepatic reduced glutathione. Adrenal hormones may inhibit lipid peroxidation both in vitro ~6and in vivo,17-19achieved through inhibition ofcytochrome P450 hydroxylations and superoxide anion p r o d u c t i o n . 2°,21 Hyperthyroid states in man lead to the production of superoxide anion and thiobarbituric acid reactive s u b s t a n c e s . 22 We therefore studied the effect of an acute dose of
The exact mechanism of the hepatic toxicity of ethanol is unknown, and although the overall risks of liver damage rise with increasing daily consumption, many 'heavy' abusers seem to avoid physical damage from alcohol. It was initially proposed by Di Luzio and Hartman ~that hepatic lipid peroxidation was generated by free radical production following alcohol ingestion, and subsequent experimental evidence has supported t h i s v i e w . 2-12 Radical production may occur either as a result of acetaldehyde4,~° formed through the alcohol dehydrogenase, or through cytochrome P450II E 1, part of the microsomal ethanol oxidising system. Address correspondence to: Dr. J. P. Teare. 655
656
J.P. TEARE el al.
ethanol on hepatic glutathione status and the levels of the product of lipid peroxidation, malonaldehyde. In addition we studied the added effect of decreasing either adrenal or thyroid hormone levels, since both these hormone systems have a role in the regulation of radical production. METHODS
Male Wistar rats were obtained from Interfauna UK Ltd (Huntingdon, UK) and maintained in 12 hr light and dark cycles. Two weeks prior to the study, groups of 24 rats were either thyroidectomized, adrenalectomized, or sham operated. The thyroidectomized animals had the smallest food intakes, and so the other operated animals had their intakes restricted to this group. A group of 24 control animals were pair fed to this group, whist another group was allowed to feed ad libitum. All rats were allowed free access to tap water except the adrenalectomized rats, which were given 0.9% Sodium Chloride as drinking water ad libitum. Two and a half h prior to sacrifice, half the animals in each group were injected with ethanol (75 mmol/kg body weight intraperitoneally) the rest with an equivalent volume of saline. Animals were sacririced over a 6 h time period following the normal overnight feeding period, to control for the effects of fasting on hepatic glutathione.
Preparation of samples Hepatic giutathione analysis: within four min of sacrifice, the liver was removed and 1.0 gram liver homogenised in five volumes of 1% picric acid. The homogenate was then centrifuged at 2500 g for 15 min and the supernatant stored at -70°C for less than four weeks until later analysis according to our reported adaptation 23 of the method of Griffith.24 Hepatic malonaldehyde concentration was measured in an aliquot of liver homogenized in phosphate buffered saline (pH 7.4) and stored at -70°C for less than four weeks until analysis, by high performance liquid chromatography (HPLC) by the method of Esterbauer, z5 This is more specific than the measurement of thiobarbituric acid reactive material26 which also registers decomposition products that arise during the heating stage of the r e a c t i o n 27 a s well as nonlipid derived products. Malonaldehyde was separated from the whole homogenate by precipitation with an equal volume of acetonitrile and centrifuging the mixture, after thoroughly mixing, at 3000 g for five min. The clear supernatant was then injected (via a Rheodyne injection valve with a 20 ul loop) onto an $5 Spherisorb-NH2
column, connected to a Shimadzu LC 6A pump and SPD 6A UV detector set at 270 nm. The column was eluted with acetonitrile/0.03 M Tris at pH 7.0 (1:9 v/v) and the absorbance read at 270 nm and compared to that for MDA standards which were prepared from 1,1,3,3,tetra-methoxy propane (TMP) by hydrolysing the TMP for 1 h at 50°C and then calculating the molar extinction coefficient.
Statistical analysis As the groups were paired, multiple paired t-tests were performed. To avoid the defects in the use of multiple t-tests an overall test based on the analysis of variance was first performed; if this indicated differences, modified t-tests were performed. Significance was taken at the 5% level. RESULTS
Hepatic glutathione content There was a difference between the total hepatic glutathione content of pair fed and the ad libitum groups suggesting that restricting the intakes to that of the thyroidectomized animals impaired hepatic glutathione synthesis (p < 0.005) (Fig. 1). In the thyroidectomized animals injected with saline the level of total glutathione was significantly (p < 0.001) increased compared to the pair fed controls, and this effect was significantly (p < 0.01) reduced by acute administration of alcohol (Fig. 1). This was not an effect of the surgical procedure, as the sham operated animals were not different to the pair fed controls. Results expressed in the figures are for total glutathione, as there was no difference in the ratio of oxidised:reduced glutathione in any of the groups studied. Adrenalectomy had no effect on the mean total glutathione levels compared to pair fed controls, and alcohol injection in these animals made no difference. The sham procedure also had no effect.
Effect of alcohol Hepatic total glutathione levels were reduced following alcohol infusion, but only in the thyroidectomized (< 0.01), and sham adrenalectomized (< 0.01) did this reach statistical significance (Fig. 1).
Hepatic malonaldehyde levels In the saline group, thyroidectomy significantly (p < 0.03) decreased hepatic MDA production, compared to pair fed controls. Adrenalectomy had no effect and nor did the sham procedures (Fig. 2), as there
Changes in liver after acute ethanol injection
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0 Tx
sham Tx
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sham Adx
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Ad lib
Fig. 1. Total Hepatic glutathione levels following adrenalectomy (Adx) and thyroidectomy (Tx). *p < 0.005 versus pair fed saline controls; **p < 0.001 versus pair fed saline controls; +p < 0.01 versus saline control. Results expressed as mean _+ SEM.
was data variability. Alcohol had no effect in any of the groups studied (Fig. 2). DISCUSSION
At low concentrations most alcohol is metabolised by cytosolic alcohol dehydrogenase to acetaldehyde, while another microsomal ethanol oxidising system (MEOS), which is linked to cytochrome P450, requires an higher ethanol concentration for maximal activity and is induced by chronic alcohol administration. 2 Induction of the P450 system also increases pro-
duction of acetaldehyde, which may cause liver injury by forming adducts with proteins and stimulating the formation of antibodies, 3 but also inactivates enzymes, damages cell membranes, 4 depletes glutathione, and induces free radical damage and hepatic collagen synthesis.4 Acetaldehyde causes lipid peroxidation by free radical generation in isolated perfused livers, 5 in rat liver microsome systems6 and in v i v o . 7 By binding to cysteine or glutathione, it depresses hepatic glutathione levels and reduces the scavenging of radicals by this tripeptide. Glutathione levels are normally lowest in centrilobular hepatocytes where the
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4
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sham Tx
Adx
sham Adx
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Fig. 2. Hepatic malonaldehyde levels following adrenalectomy and thyroidectomy. *p < 0.03 versus pair fed saline control. Results expressed as mean _+ SEM.
658
J.P. TEARE et aL
earliest lesions in alcoholic liver disease (ALD) are seen, and reduction of hepatic glutathione levels increases hepatic damage after alcohol ingestion.8 Liver biopsy specimens from chronic alcohol abusers have reduced glutathione levels, suggesting that this important radical scavenger has been depleted, 9-H and increased levels of by-products of radical formation as measured by diene conjugates.9,~2 Increased formation of hydroxyl radicals, 28-32 superoxide anion, 33 thiobarbituric acid reactive substances, 34and metabolites that adduct to electron spin t r a p s 35-37 have also been demonstrated. Steroid hormones are known to inhibit lipid peroxidation in vitro 16and similarly in vivo. 17-19Corticosteroids are known to affect the activity of a number of enzymes, including aryl hydrocarbon hydroxylase, a cytochrome P450 related enzyme/7 Cytochrome P450 hydroxylations are known to produce superoxide anion, and are influenced by steroid hormones, 2°'2~cortisol decreasing the production in some biological systems19 but not in others. 37 The greatest production is seen however in the adrenalectomized animal 16'18and is reversed by cortisol administration. However in this study adrenalectomized rats had normal hepatic glutathione and MDA levels suggesting that there was no excess of free radical production. Adrenalectomy with an associated reduction of antioxidant corticosteroid hormones should reduce glutathione levels and increase lipid peroxides.~6-18 However, this was not seen in our model, and could be explained by alterations in the activities of relative hydroxylase and oxidase enzyme systems.38Hydroxylation systems tend to produce superoxide anion, 2° and a lack of steroid hormones would result in a decrease in the activity of this enzyme. We have shown a rise in hepatic glutathione levels following thyroidectomy, which could be due to either increased production, reduced sinusoidal ettlux, or reduced utilization of this important free radical scavenger. This would be in keeping with previous r e p o r t s 22'39-46 where markers of lipid peroxidation were elevated in hyperthyroid states. Hyperthyroidism in man and rats increases the production of superoxide anion and thiobarbituric acid reactive substances, 22 an effect that can be abolished by antithyroid agents such as propylthiouracil.22'4° This phenomenon seems to be mediated by enhanced oxidative capacity of liver microsomes41 with increased activity of NADPH cytochrome P450 reductase42'44 and NADPH oxidase44'45 enzymes whose activity promotes superoxide production. 46 These effects are seen with nano-molar concentrations of thyroid hormones present in clinical hyperthyroid states in man. 4° This cellular response is associated
with glutathione depletion, cytochrome P450 loss and decreased concentration of membrane fatty a c i d s . 47 The thyroidectomized state might therefore reverse these effects, with a reduction in markers of lipid peroxidation and subsequent elevation of tissue levels of glutathione, which is a potent free radical scavenger. In general there was a reduction in hepatic glutathione following the alcohol injection, though this was only significant in two of the groups. This effect has been noticed before, and is presumably partly due to utilization of the radical scavenger in mopping up the small amounts of peroxides produced by the cytochrome P450 II E1 in its preinduced status and those peroxides forced from the metabolism of acetaldehyde.4 The lack of difference in the ratios of oxidised (GSSG) to reduced glutathione (GSH) that we have shown in the groups studied could be due to the close regulation of oxidised glutathione within cell. Intracellular GSH depletion occurs largely due to effiux across sinusoidal and biliary canalicular membranes. 48 Maintenance of the cellular redox state depends upon the rapid and efficient conversion of GSSG back to its reduced state or to mixed protein disulphides.49 These reactions are catalysed by GSH peroxidase and reductase systems, and are dependent on the NADP+/NADPH redox pair. That we have not seen a rise in hepatic GSSG highlights the efficiency of these enzyme systems in preserving intracellular GSH. The decrease in total glutathione in the sham adrenalectomised group is difficult to explain, but may represent a minor variation in nutritional status at the time of sacrifice, despite attempts to control for this by sacrificing all animals over a short time period following overnight feeding, since fasting rapidly depletes hepatic glutathione. 5° Significant production of peroxide radicals detectable as malonaldehyde was not seen in this model, and this supports the hypothesis that long term exposure to ethanol, with induction of the cytochrome P450 IIE 1, is necessary for free radical production in response to alcohol ingestion. Acknowledgement - - We are grateful to the Trustees of St Thomas'
Hospital for their continuing support.
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