REGULATION OF MANGANESE SUPEROXIDE DISMUTASE (MnSOD) IN

Alcohol & Alcoholism Vol. 35, No. 2, pp. 159–163, 2000
REGULATION OF MANGANESE SUPEROXIDE DISMUTASE (MnSOD) IN
CHRONIC EXPERIMENTAL ALCOHOLISM: EFFECTS OF VITAMIN E-SUPPLEMENTED
AND -DEFICIENT DIETS
OSVALDO KOCH, STELLA FARRÉ, MARIA EMILIA DE LEO1,
PAOLA PALOZZA1, BERNARDETTA PALAZZOTTI1, SILVIA BORRELO1,
GUGLIELMO PALOMBINI1, AMERIS CRAVERO and TOMMASO GALEOTTI1*
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)
Abstract — In order to investigate the pathogenic mechanism responsible for liver injury associated with chronic alcoholism, we
studied the effects of different dietary vitamin E levels in chronically ethanol (EtOH)-fed rats on the activity and mRNA regulation of
the manganese superoxide dismutase (MnSOD) enzyme. Evidence is accumulating that intermediates of oxygen reduction may in fact
be associated with the development of alcoholic liver disease. Since low vitamin E liver content seems to potentiate EtOH-linked
oxidative stress, we studied the effect of EtOH treatment in livers from rats fed a diet deficient or supplemented with vitamin E. Chronic
EtOH feeding enhanced hepatic consumption of vitamin E in both groups of EtOH-treated animals, irrespectively of the vitamin E level
of the basal diet and the effect was observed in both the microsomal and mitochondrial fractions. Both EtOH-fed groups exhibited
increased MnSOD gene expression, while the enzyme activity was enhanced only in the vitamin E-deprived group of EtOH-treated
animals. The significant increase in manganese liver content found only in this last group could explain the rise of enzyme activity. In
fact, in the absence of a parallel increase of the prosthetic ion manganese, MnSOD mRNA induction was not accompanied by a higher
enzymatic activity. These findings support the role of oxidative alteration in the EtOH-induced chronic hepatotoxicity in which
MnSOD response might represent a primary defence mechanism against the damaging effect of oxygen radical species.
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 EtOHfed 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.
Although many theories have been advanced, the pathogenic
mechanism underlying liver injury associated with chronic
alcoholism remains unsettled. Since the pioneering report of
Di Luzio (Di Luzio, 1973), much evidence has been provided
that free radicals are usually implicated in this process (Koch
et al., 1991; Nordmann et al., 1992; Lieber, 1997). Besides
electron-spin resonance (ESR) detection (Albano et al., 1988;
Reinke et al., 1997) in liver microsomes isolated from ethanol
(EtOH)-fed rats of EtOH- and lipid-derived free radicals,
reactive oxygen species (ROS) have been reported to accumulate
in various subcellular compartments of hepatocytes after
EtOH consumption (Nordmann et al., 1992). Such a condition
of oxidative-stress is frequently associated with enhanced
lipid-peroxidation, as established by the use of non-invasive
techniques (Sies et al., 1979; Boveris et al., 1983) and with
protein (Remmer et al., 1989; Rouach et al., 1997) and DNA
(Rajasinghe et al., 1990; Kurose et al., 1997) damage. Data are
also available in the literature on the level of non-enzymatic
antioxidants in the liver of chronic EtOH-fed rats (Nordmann
et al., 1992). A previous report from our group (Koch et al.,
1994) showed that rat liver after chronic EtOH feeding
exhibited an increased manganese superoxide dismutase
(MnSOD) activity with an upregulation of the enzyme at the
mRNA level. The same study showed that chronic intake of
EtOH also led to a significant decrease in the content of
vitamin E in both the liver mitochondrial and microsomal
fractions, suggesting the occurrence of enhanced lipid
peroxidation.
MATERIALS AND METHODS
Animals and treatment
A total of 14 male Wistar rats (232–305 g initial body
weight) was divided into two groups. One group (AD) was fed
for 15 days a semi-synthetic basal diet deficient in vitamin E
ad libitum (Table 1). The other group (AS) was fed the same
Table 1. Composition of the basal diets*
Components
Egg albumin
Corn starch
Sucrose
Cellulose
Coconut oil
Salt mixture
Oligoelements
Vitamin mixture
Vitamin E
DL-methionine
Vitamin E
deficient (g/kg)
Vitamin E
supplemented (g/kg)
420
86
240
40
70
80
16
40
—
8
420
86
238.5
40
70
80
16
40
1.5
8
*Altromin-Rieper SpA, Industria Mangimi, Italy.
*Author to whom correspondence should be addressed.
159
© 2000 Medical Council on Alcoholism
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INTRODUCTION
160
O . KOCH et al.
basal diet for a similar period but supplemented with vitamin E.
Both groups were offered a mixture of 25% EtOH–20%
sucrose as the only source of drinking fluid during the entire
period of treatment (15 days). Another two control groups
(seven animals each, CD: control — vitamin E deficient and
CS: control — vitamin E supplemented) were offered water
and their solid diets were based on the final regimens (basal
diet plus EtOH–sucrose solution) consumed by the alcohol
groups, but EtOH was replaced isocalorically by a mixture of
50% sucrose–50% corn starch. These control animals were
pair-fed isocalorically with those of the alcohol groups. The
caloric percentages of ingredients of the final regimen (basal
diet plus EtOH–sucrose solution) were as follows: Group AD:
alcohol 38%, carbohydrates 37%, fat 7%, protein 18%; Group
AS: alcohol 41%, carbohydrates 35%, fat 7%, protein 17%.
The composition of the diet of the control groups was the
same, except that the alcohol-derived calories were replaced
by carbohydrates. Animals in the alcohol groups were kept
without EtOH for 18 h before death, but were allowed free
access to drinking water. Animals from all groups were
starved of food overnight.
College, PA, USA). When a significant effect was found, posthoc comparisons of means were made using Fisher’s test.
Differences were considered significant at P < 0.05.
RESULTS
Figure 1 shows the effect of EtOH treatment on the vitamin
E content of liver microsomal (A) and mitochondrial (B)
membranes from rats fed a diet deficient or supplemented with
vitamin E. EtOH treatment reduced vitamin E content of rat
liver drastically; this effect was observed in both microsomal
and mitochondrial membranes.
MnSOD activities in all groups are indicated in Table 2.
Activity was higher only in the vitamin E-deprived group of
EtOH-treated animals, compared to the respective control
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RNA isolation and quantitative analysis
RNA was isolated essentially according to Chirgwin et al.
(1979). Total RNA was size-fractionated by formaldehyde–
agarose (1.5%) gel electrophoresis, and blotted to nylon membranes. Blots were pre-hybridized at 42°C for 5 h and
hybridized overnight to rat MnSOD cDNA probe radiolabelled by the Multiprime DNA labelling system (Amersham,
UK). Autoradiograms were obtained at –70°C using Kodak
XAR film. The intensity of autoradiograms was scanned with
a transmission densitometer.
Enzyme assay
Copper–zinc 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 Perkin–Elmer 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 Perkin–Elmer 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
Fig 1. Effect of a 15-day ethanol (EtOH) treatment on vitamin E content
of liver microsomal (A) and mitochondrial (B) membranes from rats fed
a diet deficient or supplemented with vitamin E.
Values are means ± SEM of three experiments. Values not sharing the
same letter are significantly different, P < 0.05.
Abbreviations used: CS, control vitamin E supplemented; AS, vitamin E
supplemented + EtOH; CD, control vitamin E deficient; AD, vitamin E
deficient + EtOH.
MnSOD REGULATION AND VITAMIN E DIET IN CHRONIC ALCOHOLISM
161
Table 2. Superoxide dismutase activities in livers of vitamin E-deficient
and -supplemented rats after ethanol (EtOH) treatment
Treatment
MnSOD
(µg/mg wet. wt)
CuZnSOD
(µg/mg wet. wt)
CD
AD
CS
AS
198.0 ± 0.7 (4)
229.0 ± 24 (4)*
224.3 ± 14.7 (4)
218.0 ± 18 (4)
559.5 ± 34 (4)
465.8 ± 65 (4)
558.8 ± 68 (4)
428.8 ± 51 (4)
Abbreviations used: CD, control vitamin E-deficient; AD, vitamin Edeficient + EtOH; CS control vitamin E-supplemented; AS, vitamin Esupplemented + EtOH.
*P < 0.05 vs vitamin E-deficient group. Values are means ± SEM (of
the numbers of observations on parentheses).
Fig 2. Levels of MnSOD mRNA in liver of vitamin E-deficient and
-supplemented ethanol (EtOH)-treated rats.
Values of the treated animals are means ± SEM of three experiments.
Uniformity of lane to lane loading was confirmed by inspection of the
ethidium bromide-stained gel. MnSOD mRNA levels correspond to the
sum of the intensities of the two major hybridizing bands (1.08 and 0.85 kb),
normalized to 100% from control liver. *P < 0.05 vs vitamin E-deficient group.
**P < 0.05 vs vitamin E-supplemented group.
Abbreviations used: CD, control vitamin E-deficient; AD, vitamin E
deficient + EtOH; CS, control vitamin E supplemented; AS, vitamin E
supplemented + EtOH.
Fig 3. Manganese and copper contents in livers of vitamin E-deficient
and -supplemented ethanol (EtOH)-treated rats.
Values are means ± SEM of three experiments. *P < 0.05 vs vitamin
E-deficient group.
Abbreviations used: CD, control vitamin E-deficient; AD, vitamin Edeficient + EtOH; CS = control vitamin E-supplemented; AS, vitamin Esupplemented + EtOH.
DISCUSSION
The present report clearly confirms that chronic EtOH
intake enhances hepatic consumption of vitamin E, probably
caused by an increment in the formation of ROS induced
directly or indirectly by EtOH itself. This increase in hepatic
vitamin E consumption was observed in both groups of EtOHtreated animals, irrespective of the vitamin E levels of the
basal diets. These data are in agreement with those reported in
the literature showing a reduction in hepatic vitamin E content
after chronic EtOH administration (Bjorneboe et al., 1987;
Kawase et al., 1989; Koch et al., 1994; Sadrzadeh et al., 1994;
Rouach et al., 1997). In a previous study, we reported that an
EtOH diet was able to reduce significantly the hepatic vitamin E
content in rats fed a normal control diet, containing the usual
recommended allowances of vitamin E of 0.6 mg/kg body
wt/day (National Research Council, 1995). In the present
study, this effect was observed in both the microsomal and
mitochondrial fractions. In contrast, no differences in the
content of mitochondrial or microsomal vitamin E by EtOH
were reported by Kawase et al. (1989), although these authors
found a marked reduction in levels of antioxidant in whole
liver. These controversial results may be explained by the
model of chronic alcoholism employed. Liquid EtOH diets
with high lipid contents which substantially differ from those
used in the present study were used. The mechanism by which
EtOH decreases vitamin E is still under investigation. It is
possible that EtOH causes an over-production of ROS either
directly or indirectly.
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
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group. Table 2 also shows the values of CuZnSOD activities.
As reported previously (Koch et al., 1994), EtOH fails to
produce increases in this activity, but rather slightly decreases
it, irrespective of the vitamin E content of the diets.
MnSOD mRNA was increased significantly in both groups
of EtOH-treated rats after 2 weeks of EtOH consumption
(Fig. 2). These data are similar to those found previously by
our group in EtOH-treated rats fed normal vitamin E levels
(Koch et al., 1994). It is noteworthy that the increased values
found in the EtOH plus vitamin E-supplemented group differ
from the vitamin E liver contents, which were higher than the
usual levels found in normal rats fed stock diets.
Figure 3 shows the effect of EtOH on hepatic manganese
and copper cations. The manganese content was significantly
elevated only in the EtOH-treated vitamin E-deficient group.
Copper levels were unchanged in all conditions.
162
O . KOCH et al.
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.
Acknowledgements — This study was supported by University of Buenos
Aires grants M-015 and TM26 and a 60% grant from MURST. We wish to
thank Mrs Clotilde Castellani for her excellent secretarial work.
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