Departmento de Patología, Facultad de Medicina, Universidad de Buenos Aires, J. Uriburu 950, P.P. 1114 Buenos Aires, Argentina and
1 Istituto di Patologia Generale, Università Cattolica del Sacro Cuore, Largo F. Vito 1, 00168 Rome, Italy
Received 3 June 1999; in revised form 6 October 1999; accepted 20 October 1999
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ABSTRACT |
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INTRODUCTION |
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According to some reports (Kawase et al., 1989; Sadrzadeh et al., 1994
), low vitamin E liver content seems to potentiate the EtOH-linked oxidative stress; thus, it may be possible that different dietary vitamin E levels in chronically EtOH-fed rats may also modify the expression of the MnSOD mRNA. In order to evaluate this possibility, the present study explored the activity and RNA regulation of MnSOD in chronically EtOH-treated rats fed either a vitamin E-deficient or -supplemented diet.
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MATERIALS AND METHODS |
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Enzyme assay
Copperzinc superoxide dismutase (CuZnSOD) and MnSOD activities were determined in 48 h-dialysed supernatants (105 000 g) of homogenates of liver specimens, by inhibition of haematoxylin to haematein autoxidation monitored at 560 nm, at pH 7.5 and 25°C (Martin et al., 1987). MnSOD was measured in the presence of 3.0 mM cyanide.
Vitamin E evaluation
Vitamin E was extracted as indicated by Handelman et al. (1988). The samples were dissolved in methanol and 20 µl aliquots were analysed by reverse phase HPLC with fluorescence detection using a PerkinElmer 650 LC fluorescence detector, with excitation at 295 nm and emission at 340 nm. Vitamin E, as well as the internal standard, tocol, was eluted with 100% methanol on an Alltech C18 3-µm column, as described previously (Palozza et al., 1992).
Metal determination
Metal concentrations were determined by atomic absorption spectrometry using a PerkinElmer 272 spectrophotometer. Thin slices of liver sample were dried at 100°C, digested with nitric acid and then analysed for metal content.
Statistical analysis
All data are presented as the means ± SEM. We estimated differences among the different treatments by one-way analysis of variance (ANOVA), using Minitab software (Minitab, State College, PA, USA). When a significant effect was found, post-hoc comparisons of means were made using Fisher's test. Differences were considered significant at P < 0.05.
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RESULTS |
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DISCUSSION |
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In this study, it remains still unclear why vitamin E is altered differently by the same dose of EtOH depending on dietary administration of the vitamin E itself. This might be explained in different ways. It is possible that different pools of vitamin E near the surfaces or in the depths of membranes might be present in liver as a consequence of a different vitamin E intake, with consequently different susceptibility to free radical species (Palozza et al., 1992). Alternatively, free radical species may be formed during oxidative stress induced by EtOH, against which even high levels of vitamin E are not effective.
MnSOD is one of the enzymatic antioxidants whose action may be referred to primarily as the ability to change the intramitochondrial levels of ROS. In a previous report (Koch et al., 1994), we have shown that chronic EtOH feeding caused an upregulation of the enzyme at the mRNA level, with a good correlation between the transcript and the enzyme activity. The results reported here extend these observations and clearly demonstrate that the increase in the MnSOD mRNA does not correlate with microsomal or mitochondrial vitamin E levels. In this regard, rats fed with EtOH plus vitamin E have still higher hepatic levels of vitamin E than non-EtOH rats fed stock diets. Although both EtOH-fed groups exhibit increased MnSOD gene expression, a parallel increase in the enzyme activity is observed only in the EtOH-treated vitamin E-deficient animal group, which is the only one characterized by a significant increase in the manganese ion. It is possible, therefore, that lower levels of Mn, such as those found in the EtOH-treated vitamin E-supplemented group, are not sufficient to activate post-translationally higher amounts of the apoenzyme accumulated following the increased mRNA. Nanji et al. (1995) showed, in the gastric continuous EtOH infusion model, that rats treated for 1 month did not exhibit increases in the hepatic levels of MnSOD mRNA nor reduced levels when rats were fed totally liquid EtOH diets higher in unsaturated lipids, such as corn oil or fish oil. This apparent discrepancy may be explained by marked differences in the chronic EtOH models used by Nanji et al. (1995) and the one reported here.
Unlike the corresponding cytosolic enzyme CuZnSOD, which is constitutively expressed, MnSOD is easily inducible by different agents, such as cytokines, auto-oxidizable drugs and ionizing radiation. In this regard, it has been shown that chronic EtOH could enhance tumour necrosis factor (TNF) expression in rats (Nanji, et al., 1994; Perera, et al., 1995
) playing an important role in the pathogenesis of both experimental and clinical liver damage (McLain and Cohen, 1989; McLain et al., 1993). Adachi et al. (1994) have shown that Kupffer cells, the main source of TNF, are activated by chronic EtOH treatment and that their inactivation is diminished early in EtOH-linked liver injury. It is noteworthy that a study performed on MnSOD in humans (Togashi et al., 1990
) showed a clear increase in this enzyme activity in chronic alcoholics with alcoholic hepatitis or cirrhosis, compared with non-alcoholics with or without overt liver diseases.
We have proposed previously that the increase of MnSOD could be a protective mechanism built up by the genetic machinery to partially overcome EtOH-induced oxidative stress. However, in the case of chronic alcoholism, the increased activity of MnSOD may aggravate the antioxidantpro-oxidant status of hepatic mitochondria, as suggested by Fernandez-Checa et al. (1997). In fact, mitochondrial hydrogen peroxide concentration may substantially increase owing to a depletion of reduced glutathione (GSH) with the subsequent lower activity of the hydrogen peroxide-metabolizing enzyme glutathione peroxidase (GPX). The depletion of GSH results from a primary defect of hepatic mitochondria from EtOH-fed rats to transport GSH from cytosol into the mitochondrial matrix (Fernandez-Checa et al., 1987; Hirano et al., 1992
). In such a condition, an increase of MnSOD may aggravate the high steady-state concentration of hydrogen peroxide. Accumulation of hydrogen peroxide would then participate in the chemistry catalysed by transition metals that would give rise to generation of potent free radicals, such as OH., and hence mitochondrial damage.
In conclusion, in animals fed chronically with EtOH, vitamin E supplementation is unable to prevent the EtOH damage and the consequent increased MnSOD expression. We believe that EtOH consumption may induce MnSOD by ROS generation against which vitamin E, at least in part, is not protective. However, in the absence of adequate activities of GPX, the induction of MnSOD may aggravate oxidatively mediated EtOH-induced mitochondrial injury.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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REFERENCES |
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