Centre for Toxicology, Department of Pharmacology,
1 Department of Pharmaceutical and Biological Chemistry, The School of Pharmacy, University of London, 2939 Brunswick Square, London WC1N 1AX and
2 Biochemical Toxicology, Department of Pharmacy, King's College London, Manresa Road, London SW3 6LX, UK
Received 1 October 1998; in revised form 11 December 1998; accepted 5 January 1999
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ABSTRACT |
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INTRODUCTION |
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Chronic alcohol administration leads to proliferation of the smooth endoplasmic reticulum. This ultrastructural alteration is associated with an enhanced activity of the MEOS. MEOS involves a specific alcohol-inducible form of cytochrome P450 (CYP2E1). CYP2E1 not only catalyses the metabolism of alcohol, but also activates a number of xenobiotics to hepatotoxic and carcinogenic metabolites (Farinati et al., 1989). Alcoholics tend to display tolerance to alcohol as a result of CYP2E1 induction (Lieber, 1997
).
Recent reports have indicated that changes in methionine metabolism or methylation in the liver may have an important role in alcohol toxicity. Methionine has to be converted to S-adenosylmethionine (SAM) in order to be utilized for functions such as phosphatidylcholine synthesis. SAM also provides a source of cysteine, via the trans-sulphuration pathway, for reduced glutathione (GSH) production, a major hepatoprotective agent against liver injury, including lipid peroxidation. The usefulness of SAM administration in repleting GSH levels has been demonstrated in the baboon (Lieber et al., 1990) and in clinical studies (see Fig. 1
) (Vendemiale et al., 1989
). Methionine may be deficient in alcohol-treated rats, as a result of methionine synthase inhibition (Kerai et al., 1998
). Therefore, methionine supplementation has been considered as a treatment for alcoholic liver injury (Finkelstein and Martin, 1986
).
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There is significant evidence to suggest that taurine has protective properties both as an endogenous compound and when administered therapeutically (Alvarez and Story, 1983; Nakashima et al., 1983). Recent studies have demonstrated the protective effects of taurine against hepatic steatosis and lipid peroxidation, when co-administered with alcohol for 28 days (Kerai et al., 1998
). The aim of the current study was to show whether the administration of taurine for 2 days following alcohol treatment for 28 days to rats, would reverse the pathological and biochemical lesions induced by alcohol (for example hepatic steatosis and lipid peroxidation). The role of the modulation of CYP2E1 activity and methionine synthase activity in the toxicity was investigated further.
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MATERIALS AND METHODS |
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Animals
Female SpragueDawley rats (125150 g) were obtained from Charles River (UK) and acclimatized for 7 days after delivery. Animals were housed in communal cages, fed a rat and mouse maintenance cube diet (691 diet; Quest Nutrition Ltd, Wingham, Kent, UK) and water ad libitum. During experiments, animals were housed in individual metabolism cages designed to separate and collect faeces and urine (Techmate Ltd, Milton Keynes, UK), and given powdered diet (691 diet, Quest Nutrition Ltd, Wingham, Kent, UK) and water ad libitum prior to introduction of the liquid diet. Lighting was controlled to give a regular 12 h light12 h dark cycle (lights on at 08.00); room temperature was maintained at 21 ± 1°C. Urine samples (24 h) were collected over ice and diluted to 25 ml with UHQ water, centri-fuged (2000 r.p.m., 10 min, 4°C) to remove hair and food debris and stored (80°C) in aliquots for later analysis. The liquid diet intake and general condition of the animals were monitored daily and rats were weighed twice a week. Animals were used under the British Home Office regulations.
Liquid diet technique of alcohol administration
Due to the tendency of animals given alcohol to reduce their solid food consumption, animals were given a liquid diet. Chronic alcohol feeding was achieved by incorporation of alcohol in a nutritionally adequate totally liquid diet obtained from Dyets Inc. (Pennsylvania, PA, USA). The liquid diet provided 1 kcal/ml, of which 35% of total calories were derived from fat, 47% from carbohydrates and 18% from protein. Alcohol-treated animals were given diet where maltosedextrin was isocalorically replaced by alcohol. The alcohol provided 36% of the calories. Animals were started on the diet at a body weight of 125150 g and alcohol was introduced progressively as 30 g/l of the liquid diet for 2 days, 40 g/l for the subsequent 2 days followed by the final formula containing 50 g/l (Lieber and DeCarli, 1989; Kerai et al., 1998
).
Preparation of diet
As vitamin A degrades when mixed with other dry ingredients, vitamins and minerals were incorporated into the liquid diet at the time of preparation using a kitchen-type blender. The diet was kept refrigerated, in the dark and used within 1 week of preparation.
Pair-feeding
The alcohol-fed animals were allowed liquid diet consumption ad libitum and their daily intake was monitored. The control animals were then given the same amount of control liquid diet during the following 24 h feeding period. This pair-feeding process was repeated every 24 h. The technique of daily pair-feeding was adopted to assure a strict caloric intake in both alcohol-treated animals and their individual pair-fed controls (Lieber and DeCarli, 1989).
Study design
Rats (n = 12) were treated with alcohol which was administered in the liquid diet for 28 days. Pair-fed control rats (n = 12) were also provided with the same liquid diet, but without alcohol. After 28 days, alcohol administration was stopped and alcohol pre-treated animals (n = 6) and pair-fed controls (n = 6) received the control liquid diet. The remaining alcohol pre-treated animals (n = 6) and pair-fed controls (n = 6) received the control liquid diet with 3% taurine added. After 2 days of taurine treatment, animals were killed and blood and tissue removed for analysis and microsomes prepared from the liver.
Post-mortem procedure
Animals were exsanguinated from the abdominal aorta under anaesthesia [Hypnorm: Hypnovel: water, 1:1:2, 3.33 ml/kg intraperitoneally (i.p.)] and blood samples were collected into Microtainers® (Becton Dickinson & Co., Rutherford, NJ, USA) for the separation of serum. After standing at room temperature for 45 min, the Microtainers® were centrifuged (13 000 r.p.m., 45 s, MSE minifuge) and stored at 80°C. Serum was analysed for serum enzymes and biochemical parameters using appropriate kits (Boehringer Mannheim GmbH Diagnostica, Mannheim, Germany) with a centrifugal IL Monarch 2000 (Instrumentation Laboratory, UK, Ltd). The liver was removed, weighed and ~200 mg taken from the right lobe and immediately homogenized in trichloroacetic acid (TCA, 10% w/v, 4 ml, 4°C), frozen in liquid nitrogen and stored at 80°C for subsequent analysis of ATP. Approximately 200 mg of liver were also taken from the right lobe and immediately homogenized in sulphosalicylic acid (0.2 M, 2 ml, 4°C), frozen in liquid nitrogen and stored at 80°C for subsequent analysis of taurine, total non-protein sulphydryls (TNPSH) and oxidized glutathione (GSSG).
Biochemical determinations
Taurine.
A high performance liquid chromatographic method with fluorimetric detection was used for the determination of taurine in urine, serum and liver tissues, essentially by the method of Waterfield (1994). Taurine was derivatized with o-phthaldehyde/2-mercaptoethanol prior to injection onto a C18 column. Isocratic elution of the adduct was carried out using NaH2PO4 (0.05 M, pH 5.4) in methanol and water (43:57 v/v). Homoserine was used as an internal standard to facilitate the standardization and quantification of samples. Analysis was completed in 6 min with homoserine and taurine eluting after 3 and 4 min, respectively.
Triglycerides. Hepatic content of triglyceride was determined by a modified method of Butler et al. (1962). Briefly, phospholipids were separated from triglycerides by adsorption on a synthetic Zeolite. The triglycerides were then extracted into chloroform, hydrolysed and measured as esterified glycerol with non-esterified samples used as individual blanks.
Lipid peroxidation. Lipid peroxidation, measured as malondialdehyde (MDA) production in liver samples, was determined by the method of Sawicki et al. (1963) employing MDA as standard.
ATP.
ATP content of liver samples was determined by luciferase-linked bioluminescence in TCA extracts of liver samples using a firefly lantern extract (Jenner and Timbrell, 1994).
Total non-protein sulphydryls (TNPSH). Liver TNPSH were measured by the method of Ellman (1959) as a measure of liver GSH, which constitutes most (>95%) of the liver TNPSH (DeMaster and Redfern, 1987).
GSSG. Hepatic oxidized glutathione (GSSG) was determined by the method of Griffith (1980) using 2-vinylpyridine to mask GSH.
Microsomal analysis. Microsomes were prepared from livers, essentially as described by Lake (1987). Total cytochrome P450 content of liver samples was determined by the method of Omura and Sato (1964). 4-Nitrophenol hydroxylase (NPOH) activity was determined by the modified method of Prough et al. (1978). 4-Nitrophenol is a substrate for the alcohol-inducible CYP2E1. The method relies on the formation of 4-nitrocatechol, which can be detected spectrophotometrically after total ionization under alkaline conditions. The protein content of microsomes was determined by the method of Lowry et al. (1951) using bovine serum albumin as standard.
Homocysteine and cysteine. A high performance liquid chromatographic method with fluorimetric detection was used for determination of total homocysteine and cysteine (oxidized and reduced) in urine and serum, according to Fortin and Genest (1995). Homocysteine and cysteine were reduced by 10% tri-n-butylphosphine in dimethylformamide then derivatized with SBD-F (ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate) at 60°C for 1 h (stable for 1 week at 4°C), prior to injection onto a C18 column. Isocratic elution of the adduct was carried out using sodium acetate (0.1 M), acetic acid (0.1 M) and 2% methanol, pH 4.0. N-Acetylcysteine was used as an internal standard to facilitate the standardization and quantification of samples. Analysis was completed in 14 min with cysteine, homocysteine and N-acetylcysteine eluting after 2.5, 3.5 and 6 min, respectively.
Methionine synthase. Methionine synthase was measured in the liver cytosol essentially as described by Nicolaou et al. (1997). Assay mixtures (total volume 300 µl) contained 50 mM potassium phosphate buffer pH 7.2, 400 µM (dl)-homocysteine, 35 µM SAM, 236 µM MTHF (2658 dpm/nmol), 60 µM hydroxycobalamin, 25 mM dl-dithiothreitol and the enzyme source. Incubations were performed in light-protected stoppered serum vials under nitrogen. Reaction mixtures were pre-incubated for 5 min (at 37°C), prior to the initiation of the reaction by the addition of homocysteine through a syringe. Incubations (at 37°C) were performed for 45 min. The enzyme reaction was terminated by the addition of 400 µl of ice-cold water, and solutions immediately passed through a 0.5x5.0 cm column of Bio-Rad AG1-X8 resin. [14C]Methionine was eluted with 2 ml of water, collected and quantified by scintillation spectrometry. Protein concentrations were determined with the Bio-Rad protein assay based on the method of Bradford (1976) with bovine serum albumin as standard.
Acetaldehyde. Acetaldehyde in the liver and serum was determined by the method of McCloskey and Mahaney (1981).
Histology
Tissues were fixed in 10.5% (v/v) phosphate-buffered formalin (pH 7.2) and embedded in paraffin wax. Sections (4 µm) were cut and stained with Mayer's haematoxylin and eosin. Frozen liver sections from fixed tissues were cut (10 µm) and stained for lipid with Oil Red O in triethylphosphate with Mayer's haematoxylin as counter stain.
Statistical analysis
Statistical evaluation of data was performed by Duncan's multiple range test to make comparisons between groups. Values quoted are means ± SEM of six animals. The level of significance was set at 0.05.
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RESULTS |
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Histological analysis, hepatic and serum triglyceride and lipid peroxidation
Histological examination of liver tissue showed that animals treated with alcohol (Fig. 3c) had developed marked steatosis, compared to pair-fed controls (Fig. 3a
). The extent of fat accumulation in animals given alcohol followed by taurine (Fig. 3d
) was distinctly less than in animals treated with alcohol alone, and also less than in taurine-treated animals (Fig. 3b
). No method of scoring fat accumulation was used, as triglycerides were also measured biochemically. The hepatocytes in the livers from animals treated with alcohol followed by taurine appeared swollen and vacuolated.
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Hepatic ATP, GSH, GSSG and microsomal analysis
No difference was detected in the levels of ATP (data not shown) or GSSG between groups after 28 days of alcohol administration (Table 1). However, alcohol and alcohol followed by taurine treatments significantly raised levels of GSH, compared to levels in pair-fed controls, and animals treated with alcohol followed by taurine had significantly lower levels of GSH compared to animals treated with alcohol alone (Table 1
). There was no change in 4-nitrophenol hydroxylase activity 2 days after withdrawing alcohol from the diet in any of the treatment groups (Table 1
). Total cytochrome P450 was significantly raised by alcohol treatment and raised significantly more when alcohol was followed by taurine treatment (Table 1
).
Serum and urinary homocysteine and cysteine and hepatic methionine synthase
From day 2 of treatment (30 g alcohol/l), urinary homocysteine levels were raised significantly in animals treated with alcohol and alcohol followed by taurine, compared to the pair-fed controls (Fig. 5). The higher levels of urinary homocysteine were maintained in these animals up to day 14 of alcohol treatment. Animals treated with taurine for 2 days had slightly but significantly higher levels of homocysteine and cysteine in the urine. However, alcohol did not affect urinary cysteine levels (data not shown). Although there was no effect of alcohol on levels of serum cysteine, alcohol and alcohol followed by taurine treatment significantly raised levels of serum homocysteine (Table 1
). Serum homocysteine levels were significantly higher in animals treated with alcohol followed by taurine than animals treated with alcohol alone. Animals treated with alcohol and alcohol followed by taurine had hepatic methionine synthase activities which were significantly inhibited, compared to pair-fed controls (Table 1
). Taurine-treated animals alone had significantly lower methionine synthase activity compared to the non-alcohol-treated group.
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DISCUSSION |
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Despite strict isocaloric pair-feeding, alcohol-fed animals did not gain as much weight as their pair-fed controls, although they received diets with the same energy content (Lieber and DeCarli, 1989). This may be due to the oxidation of alcohol without phosphorylation by MEOS. When alcohol is oxidized to acetaldehyde via the ADH pathway, NADH is generated. However, the oxidation of alcohol via MEOS utilizes NADPH resulting in energy wastage as heat which may explain the slower weight gain of the rats fed the alcohol-containing liquid diets, despite their similar calorific intakes and their increase in weight following alcohol withdrawl. The slower weight gain of the animals in this study compared to those reported by some authors (see, e.g., Lindros and Järveläinen, 1998) could be attributed to strain and/or sex differences of the rats used, or the conditions of housing, such as the room temperature.
The administration of alcohol for 28 days to rats, with or without 2 days of taurine treatment, caused a significant increase in total and relative liver and kidney weights. The increase in the liver weight could not be accounted for by the accumulation of triglycerides as this was <2% of the total increase in liver weight measured in animals treated with alcohol alone. The lack of triglyceride accumulation in animals subsequently treated with taurine also suggests that an additional factor, which is currently unknown, was contributing to the increase in liver weight, although the appearance of the liver suggested that there was oedema of the tissue. The increased total and relative kidney weights suggests an effect of alcohol alone on the kidneys, as previously noted (Kerai et al., 1998).
Alcohol may have caused slight bile duct damage and mild cholestasis, as serum alkaline phosphatase was significantly raised by alcohol although it was raised significantly more when alcohol treatment was followed by taurine. The raised serum cholesterol levels also suggests slight cholestasis (Evans, 1996). The lack of raised serum bilirubin levels suggests that any bile duct injury was slight or that cholestasis was not complete. Hepatic and serum taurine levels were also raised in animals given taurine for 2 days, but the levels were markedly higher in animals given chronic alcohol treatment followed by taurine.
Although taurine is known to increase bile flow (Masuda and Horisaka, 1986), the greater accumulation of liver taurine and serum alkaline phosphatase in alcohol-treated rats post-treated with taurine suggests that taurine may have exacerbated the cholestasis caused by alcohol. The slightly raised level of serum AST, which was greater in animals given taurine following alcohol treatment, may indicate a worsening of the parenchymal injury. However, the data are inconclusive as ALT and GDH were also moderately raised. However, it is not known what effect alcohol treatment may have had on the sphincter of Oddi which controls the release of bile, although it is thought to reduce the motility of the sphincter (Cullen et al., 1997
). An increase in bile volume coupled with a reduction in ability to release bile into the duodenum could increase back pressure and may account for the apparent increase in liver taurine and serum markers of hepatic injury such as alkaline phosphatase. Thus, alcohol treatment caused cholestasis, resulting in bile duct damage (raised alkaline phosphatase), raised serum cholesterol and reduced elimination of liver taurine effects which may have been enhanced with taurine treatment.
Alcohol treatment increased liver GSH, as previously found (Kawase et al., 1989; Kerai et al., 1998
). GSH levels may have been raised as a result of: (a) rebound synthesis of GSH; (b) conversion of homocysteine (which was raised, possibly due to reduced methionine synthase activity) to GSH, or (c) mild cholestasis (Yan et al., 1993
). Hepatic levels of GSSG, a marker of oxidative stress, were not significantly changed. There was no apparent effect of alcohol on hepatic levels of ATP.
Hepatic steatosis is the most common pathological change induced by alcohol and is also one of the earliest pathological manifestations of alcoholic liver disease. In the present study, there was a significant accumulation of hepatic triglycerides 2 days following the withdrawal of alcohol. This suggests that levels were probably elevated to a greater extent before the 2-day recovery period. Treatment of animals with taurine for 2 days reversed hepatic steatosis to below control values, as assessed biochemically and histologically. Serum triglycerides were raised by alcohol treatment (as shown previously; Kerai et al., 1998), but this increase was significantly greater in animals treated with alcohol, followed by taurine. This suggests that triglycerides were secreted from the liver (Yan et al., 1993) at an increased rate in animals treated with taurine following alcohol withdrawal. It is likely that this increase in triglyceride efflux was responsible for the reduced hepatic triglyceride levels in animals treated with taurine. The mechanism of this reduction in hepatic triglycerides is currently being investigated in vitro in isolated hepatocytes. Thus, taurine not only protects against alcohol-induced hepatic steatosis when co-administered for 28 days with alcohol (Kerai et al., 1998
) but can also reverse hepatic steatosis once it has developed.
Alcohol metabolism is associated with the generation of reactive oxygen species. The polyunsaturated fatty acids that are abundant in cell membranes are susceptible to oxidative damage by free radicals, with consequent lipoperoxide formation, which can lead to the degeneration of membrane phospholipids. The crucial role of lipid peroxidation in the pathogenesis of alcoholic liver injury can be illustrated by the use of antioxidants (Lieber et al., 1994). There is evidence to suggest that taurine may protect against free radical damage (Banks et al., 1991
; Huxtable, 1992
). Taurine has been shown to suppress lipid peroxidation in the liver of carbon tetrachloride-intoxicated rats (Nakashima et al., 1983
) and rabbit spermatozoa (Alvarez and Story, 1983). In the present study, alcohol administration for 28 days caused significant lipid peroxidation (as determined by MDA production) even though measurements were made 2 days following alcohol withdrawal. The levels were reversed by treatment with taurine for 2 days, although the ratio of MDA:liver triglycerides was higher than in the other treatments. There was a slight correlation between individual liver triglyceride and MDA levels (r2 = 0.2297, P = 0.02, data not shown). Chronic alcohol treatment of rats raises serum bile acids, which are toxic (Kerai et al., 1998
). Taurine, however, is known to increase bile flow (Yan et al., 1993
; Seabra and Timbrell, 1997
). Thus, an attractive hypothesis to explain the loss of MDA products of lipid peroxidation could be increased rate of removal of bile acids and lipid peroxides by increasing bile flow. There is also evidence that taurine results in the synthesis of phospholipids with a higher proportion of saturated fatty acids and a lower proportion of both polyunsaturated and monounsaturated fatty acids (Yan et al., 1993
). Lower levels of unsaturated fatty acids would be expected to result in fewer lipoperoxides being formed, which could have contributed to the lower levels of MDA found in taurine-treated animals.
In our previous study, it was shown that co-administering taurine with alcohol resulted in almost complete inactivation of the alcohol metabolizing cytochrome P450 isoform, CYP2E1 (Kerai et al., 1998). However, in the present study, CYP2E1 activity appeared to be unchanged after 2 days of taurine treatment following alcohol withdrawal, which probably reflects the short half-life of this enzyme (
6 h) (Roberts et al., 1994
). Protein measurements will need to be made in future studies to verify these biochemical observations, as 4-nitrophenol hydroxylation is not specific for CYP2E1. However, the total hepatic cytochrome P450 in these animals was actually greater than in animals given alcohol alone, although it is not known which isoenzymes contributed to this increase. There was also no apparent elevation of serum bile acids 2 days following alcohol withdrawal and after taurine treatment, although there was evidence of cholestasis (raised alkaline phosphatase). Thus, by the time cytochrome P450 measurements were made, any inhibition of CYP2E1 by taurine-conjugated bile acids at an earlier time point may have been reversed. As the toxicity of alcohol appeared to be reversed by taurine, without the apparent inhibition of CYP2E1 activity, inactivation of CYP2E1 may not be the main mechanism for the protective effects of taurine seen in the present study.
Homocysteine is a sulphydryl-containing amino acid that is formed by the demethylation of methionine and is normally metabolized to cysteine, or re-methylated to methionine via methionine synthase (see Fig. 1). Reduced levels of methionine are likely to result in reduced levels of SAM. SAM is used in the methylation of phosphatidyl ethanolamine to phosphatidylcholine which is used in the transportation of triglycerides out of cells. Thus, fatty liver can result from methionine deficiency. The administration of alcohol to rats in vivo has been reported to inhibit methionine synthase activity (Barak et al., 1991
; Kerai et al., 1998
) but not in vitro (Sherif et al., 1993
). Although in-vitro studies have failed to show a direct effect of ethanol on methionine synthase activity (0.5570 mM ethanol), acetaldehyde (in contrast to ethanol or acetate) was found to inhibit methionine synthase activity in a time-dependent manner (Kenyon et al., 1998
). This suggests that acetaldehyde, from alcohol metabolism may be responsible for reduced methionine synthase activity in vivo. Although acetaldehyde was undetectable in the liver 2 days after alcohol withdrawal in the present study, the inhibition was shown to be irreversible in vitro (Kenyon et al., 1998
) and therefore, possibly, in vivo as well. However, the apparent increase in triglyceride transport from the liver following taurine administration cannot be attributed to an increase in the activity of this enzyme, as taurine treatment failed to restore methionine synthase activity. It is not known whether taurine can increase the use of betaine as the methyl donor for SAM formation, which could have enabled triglyceride transport to be restored.
The effect of taurine as a protective agent may not be specific. Indeed a recent report (Yin et al., 1998) has shown that glycine (2% in liquid diet) enhances recovery from alcohol-induced liver injury. Although glycine is not a sulphur-containing amino acid, both of these amino acids are part of the methionine/trans-sulphuration pathway. Other sulphur-containing amino acids may also have beneficial effects: methionine through its conversion to S-adenosyl-methionine (which may help reverse alcoholic liver damage) and cysteine as a precursor for glutathione, although this is relatively toxic and has poor bioavailability. All of these would ultimately be metabolized to taurine. As far as we are aware, this is the first time that taurine has been shown to reverse alcohol-induced fatty liver and lipid peroxidation when given for 2 days following chronic alcohol administration to rats. The protective effects of taurine may be attributed to enhanced triglyceride secretion from the liver, which may also indirectly have reduced the lipid peroxidation. This is currently under investigation.
Unlike rats, humans have a limited capacity to synthesize taurine and rely more on dietary intake to maintain tissue levels. In view of this, the use of taurine as a dietary supplement, following chronic alcohol consumption, has the potential to be used as a promising therapeutic agent in the treatment of alcoholic liver disease.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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REFERENCES |
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Banks, M. A., Porter, D. W., Martin, W. G. and Castranova, V. (1991) Ozone induced lipid peroxidation and membrane leakage in isolated rat alveolar macrophages: protective effects of taurine. Journal of Nutritional Biochemistry 2, 308313.[ISI]
Barak, A. J., Beckenhauer, H. C. and Tuma, D. J. (1991) Hepatic transmethylation and blood alcohol levels. Alcohol and Alcoholism 26, 125128.[ISI][Medline]
Bradford, M. M. (1976) A rapid and sensitive method for quantification of microgram quantities of protein utilising the principle of protein-dye binding. Analytical Biochemistry 72, 248254.[ISI][Medline]
Butler, W. M., Maling, H. M., Horning, H. G. and Brodie, B. B. (1962) The direct determination of liver triglycerides. Journal of Lipid Research 2, 9596.[ISI]
Chesney, R. W. (1985) Taurine: its biological role and clinical implications. Advances in Paediatrics 32, 142.
Cullen, J. J., Ledlow, A., Murray, J. A. and Conklin, J. L. (1997) The effect of ethanol on sphincter of Oddi motility in vitro. Journal of Surgical Research 67, 5861.[ISI][Medline]
De Master, E. G. and Redfern, B. (1987) High performance liquid chromatography of hepatic thiols with electrochemical detection. In Methods of Enzymology, Vol. 143, Jakoby, W. B. and Griffith, O. W. eds, pp. 110114. Academic Press, New York.
Ellman, G. L. (1959) Tissue sulphydryl groups. Archives of Biochemistry and Biophysics 82, 7077.[ISI][Medline]
Emudianughe, T. S., Caldwell, J. and Smith, R. L. (1983) The utilisation of exogenous taurine for the conjugation of xenobiotic acids in the ferret. Xenobiotica 13, 133 138.
Evans, G. O. (1996) Lipids. In Animal Clinical Chemistry: A Primer for Toxicologists, Evans, G.O. ed., pp. 185 177. Taylor & Francis, London.
Farinati, F., Lieber, C. S. and Garro, A. J. (1989) Effects of chronic ethanol consumption on carcinogen activating and detoxifying systems in rat upper alimentary tract tissue. Alcoholism: Clinical and Experimental Research 13, 357360.[ISI][Medline]
Finkelstein, J. D. and Martin, J. J. (1986) Methionine metabolism in mammals. Adaptation to methionine excess. Journal of Biological Chemistry 261, 15821587.
Fortin, L. -J. and Genest, J. (1995) Measurement of homocysteine in the prediction of arteriosclerosis. Clinical Biochemistry 28, 155162.[ISI][Medline]
Griffith, O. W. (1980) Determination of glutathione and glutathione disulphide using glutathione reductase and 2-vinylpyridine. Analytical Biochemistry 106, 207212.[ISI][Medline]
Hoffman, A. F. (1976) The enterohepatic circulation of bile acids in man. Advances in Internal Medicine 21, 501534.[Medline]
Huxtable, R. J. (1992) Physiological actions of taurine. Physiology Reviews 7, 101163.
Jenner, A. M. and Timbrell, J. A. (1994) Effect of acute and repeated exposure to low doses of hydrazine on hepatic microsomal enzymes and biochemical parameters in vivo. Archives of Toxicology 68, 240 245.[ISI][Medline]
Kawase, T., Kato, S. and Lieber, C. S. (1989) Lipid peroxidation and antioxidant defence systems in rat liver after chronic ethanol feeding. Hepatology 10, 815 821.
Kenyon, S. H., Nicolaou, A. and Gibbons, W. A. (1998) The effect of ethanol and its metabolites upon methionine synthase activity in vitro. Alcohol 15, 305309.[ISI][Medline]
Kerai, M. D. J., Waterfield, C. J., Kenyon, S. H., Asker, D. S. and Timbrell, J. A. (1998) Taurine: Protective properties against ethanol-induced hepatic steatosis and lipid peroxidation during chronic ethanol consumption in rats. Amino Acids 15, 5376.[ISI][Medline]
Lake, B. G. (1987) Investigations and characterisation of microsomal fractions for studies of xenobiotic metabolism. In Biochemical Toxicology: A Practical Approach, Snell, K. and Mullock, B. eds, pp. 183215. IRL Press, Oxford.
Lieber, C. S. (1993) Biochemical factors in alcoholic liver disease. Seminars in Liver Disease, 13, 136153.[ISI][Medline]
Lieber, C. S. (1997) Cytochrome P-4502E1: its physiological and pathological role. Physiological Reviews 77, 517544.
Lieber, C. S. and DeCarli, L. M. (1989) Liquid diet technique of ethanol administration: 1989 update. Alcohol and Alcoholism 24, 197211.[ISI][Medline]
Lieber, C. S., Casini, A., DeCarli, L.M., Kim, C., Lowe, N., Sasaki, R. and Leo, M.A. (1990) S-adenosyl-l-methionine attenuates alcohol-induced liver injury in the baboon. Hepatology 11, 165172.[ISI][Medline]
Lieber, C. S., Robins, S. J., Li, J., DeCarli, L. M., Mak, K. M., Faulo, J. M. and Leo, M. A. (1994) Phosphatidylcholine protects against fibrosis and cirrhosis in the baboon. Gastroenterology 106, 152159.[ISI][Medline]
Lindros, K. O. and Järveläinen, H. A. (1998) A new oral low-carbohydrate alcohol liquid diet producing liver lesions: a preliminary account. Alcohol and Alcoholism 33, 347353.[Abstract]
Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 263, 265275.
Masuda, M. and Horisaka, K. (1986) Effect of taurine and homotaurine on bile acid metabolism in dietary hyperlipidemic rats. Journal of Pharmacobiodynamics 9, 934940.
McCloskey, L. P. and Mahaney, P. (1981) An enzymatic assay for acetaldehyde in grape juice and wine. American Journal of Enology and Viticulture 32, 159 162.
Nakashima, T., Takino, T. and Kuriyama, K. (1983) Therapeutic and prophylactic effects of taurine administration on experimental liver injury. In Sulphur Amino Acids: Biochemical and Clinical Aspects, Kuriyama, K., Huxtable, R.J. and Iwata, H. eds, pp. 449459. Alan R. Liss, New York.
Nicolaou, A., Waterfield, C. J., Kenyon, S. H. and Gibbons, W. A. (1997) The inactivation of methionine synthase in isolated rat hepatocytes by sodium nitroprusside. European Journal of Biochemistry 244, 876882.[Abstract]
Omura, T. and Sato, R. (1964) The carbon monoxide binding pigment of liver microsomes. Evidence of its haemoprotein value. Journal of Biological Chemistry 239, 23702378.
Prough, R. A., Burke, M. D. and Mayer, R. T. (1978) In Methods in Enzymology, Vol. 52, Fleischer, S. and Packer, L. eds, pp. 372377. Academic Press, New York.
Roberts, B. J., Shoaf, S. E., Jeong, K. S. and Song, B. J. (1994) Induction of CYP2E1 in liver, kidney, brain and intestine during chronic ethanol administration and withdrawal: evidence that CYP2E1 possesses a rapid phase half-life of 6 h or less. Biochemical and Biophysical Research Communications 205, 10641071.[ISI][Medline]
Sawicki, E., Stanley, T. W. and Johnson, H. (1963) Comparison of spectrophotometric and spectrophotofluorometric methods for the determination of malonaldehyde. Analytical Chemistry 35, 199205.[ISI]
Seabra, V. and Timbrell, J. A. (1997) Modulation of taurine levels in the rat liver alters methylene dianiline hepatotoxicity. Toxicology 122, 193204.[ISI][Medline]
Sherif, F., Gomes, C. and Oreland, L. (1993) Methionine synthase and methionine adenosyltransferase activities in rat brain after ethanol treatment. Pharmacology and Toxicology 73, 287290.[ISI][Medline]
Skare, K. L., Schnoes, H. K. and DeLuca, H. F. (1982) Biliary metabolites of all-trans-retinoic acid in the rat: Isolation and identification of a novel polar metabolite. Biochemistry 21, 33083317.[ISI][Medline]
Vendemiale, G., Altomare, E., Trizzio, T., Le Grazzie, C., DiPadova, C., Salerno, T., Carrieri, V. and Albano, O. (1989) Effects of oral S-adenosyl-l-methionine on hepatic glutathione in patients with liver disease. Scandinavian Journal of Gastroenterology 24, 407415.[ISI][Medline]
Vendemiale, G., Grattagliano, I., Signorile, A. and Altomare, E. (1998) Ethanol-induced changes of intracellular thiol compartmentation and protein redox status in the rat liver: effect of tauroursodeoxycholate. Journal of Hepatology 28, 4653.[ISI][Medline]
Waterfield, C. J. (1994) Determination of taurine in biological samples and isolated hepatocytes by high performance liquid chromatography with fluorimetric detection. Journal of Chromatography 657, 3745.
Yan, C. C., Bravo, E. and Cantafora, A. (1993) Effect of taurine levels on liver lipid metabolism: an in vivo study in the rat. Proceedings of the Society of Experimental Biology and Medicine 202, 8896.
Yin, M., Ikejima, K., Arteel, G. E., Seabra, V., Bradford, B. U., Kono, H., Rusyn, I. and Thurman, R. G. (1998) Glycine accelerates recovery from alcohol-induced liver injury. Journal of Pharmacology and Experimental Therapeutics 286, 10141019.