protein adduct species in muscle and liver of rats following acute

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protein adduct species in muscle and liver of rats following acute
Alcohol & Alcoholism Vol. 40, No. 6, pp. 485–493, 2005
Advance Access publication 30 August 2005
doi:10.1093/alcalc/agh196
PROTEIN ADDUCT SPECIES IN MUSCLE AND LIVER OF RATS FOLLOWING
ACUTE ETHANOL ADMINISTRATION
VINOOD B. PATEL1*, SIMON WORRALL2y, PETER W. EMERY3 and VICTOR R. PREEDY3
1
Department of Biomedical Sciences, University of Westminster, 115 New Cavendish Street, London W1W 6UW, UK, 2Alcohol Research Unit,
Department of Biochemistry and Molecular Biology, The University of Queensland, Brisbane, Queensland, QLD 4072, Australia and
3
Nutrition, Food and Health Research Centre, King’s College London, 150 Stamford Street, London SE1 9NN, UK
(Received 2 March 2005; first review notified 29 March 2005; in revised form 15 July 2005; accepted 26 July 2005; advance access publication 30 August 2005)
Abstract — Aims: Previous immunohistochemical studies have shown that the post-translational formation of aldehyde–protein
adducts may be an important process in the aetiology of alcohol-induced muscle disease. However, other studies have shown that in
a variety of tissues, alcohol induces the formation of various other adduct species, including hybrid acetaldehyde–malondialdehyde–
protein adducts and adducts with free radicals themselves, e.g. hydroxyethyl radical (HER)–protein adducts. Furthermore,
acetaldehyde–protein adducts may be formed in reducing or non-reducing environments resulting in distinct molecular entities, each
with unique features of stability and immunogenicity. Some in vitro studies have also suggested that unreduced adducts may be converted to reduced adducts in situ. Our objective was to test the hypothesis that in muscle a variety of different adduct species are formed
after acute alcohol exposure and that unreduced adducts predominate. Methods: Rabbit polyclonal antibodies were raised against unreduced and reduced aldehydes and the HER–protein adducts. These were used to assay different adduct species in soleus (type I fibrepredominant) and plantaris (type II fibre-predominant) muscles and liver in four groups of rats administered acutely with either [A]
saline (control); [B] cyanamide (an aldehyde dehydrogenase inhibitor); [C] ethanol; [D] cyanamide+ethanol. Results: Amounts of unreduced acetaldehyde and malondialdehyde adducts were increased in both muscles of alcohol-dosed rats. However there was no increase
in the amounts of reduced acetaldehyde adducts, as detected by both the rabbit polyclonal antibody and the RT1.1 mouse monoclonal
antibody. Furthermore, there was no detectable increase in malondialdehyde–acetaldehyde and HER–protein adducts. Similar results
were obtained in the liver. Conclusions: Adducts formed in skeletal muscle and liver of rats exposed acutely to ethanol are mainly unreduced acetaldehyde and malondialdehyde species.
INTRODUCTION
et al., 2000). Immunohistochemical studies have shown that
treatment of rats with ethanol and cyanamide + ethanol
increases the amount of malondialdehyde- and acetaldehydederived protein adducts in skeletal muscle (Niemela et al.,
2002). Greater adduct scores were observed in a type II
fibre-predominant muscle (represented by the plantaris) than
a type I fibre-predominant muscle (represented by the soleus)
(Niemela et al., 2002).
From the aforementioned study, a number of issues have
been raised. The first pertains to the fact that the immunohistochemical analysis, although productive for identifying
the cellular location of the adducts provided limited information on the type of adduct formed. For example, a number of
protein adduct species have been identified elsewhere including hybrid malondialdehyde–acetaldehyde (Worrall et al.,
2000a; Thiele et al., 2001) and hydroxyethyl radical (HER)–
protein adducts (Iimuro et al., 1996; Worrall et al., 2000a).
The formation of malondialdehyde–protein adduct species
suggests that increased lipid peroxidation occurs in alcoholexposed muscle (Singh et al., 2001; Worrall et al., 2001).
If malondialdehyde—acetaldehyde (MAA)–protein adducts
were found it would suggest that ethanol oxidation and lipid
peroxidation occur simultaneously within the same microenvironment for a prolonged time (Thiele et al., 2001). Secondly, two types of acetaldehyde–protein adducts have been
identified, depending on whether they are formed under reducing or non-reducing conditions (Worrall et al., 1994, 2000b).
It has been suggested that antibodies raised against unreduced
adducts may actually recognize reduced adducts (Klassen
et al., 1999). This may arise as unreduced acetaldehyde–
protein epitopes are reconfigured or transformed to their
reduced counterpart epitopes during the raising of the antibodies, and a similar process may be responsible for the
appearance of circulating antibodies to reduced-acetaldehyde
Alcohol-induced muscle disease is one of the most prevalent
skeletal muscle disorders in the western hemisphere affecting between 40–60% of all chronic alcohol misusers
(Estruch et al., 1993; Fernandez-Sola et al., 1995; Sacanella
et al., 1995; Preedy et al., 2001c). This disease is characterized
by selective atrophy of type II fibres (glycolytic, fasttwitch, anaerobic) whereas the type I (oxidative, slow
twitch, aerobic) fibres are relatively protected (Preedy et al.,
2001b, c, d).
Although the causative agent is known, the steps between
alcohol exposure and development of biochemical and functional abnormalities in muscle have not been fully elucidated.
However, there is increasing evidence that the development of
alcohol-related diseases may involve the formation of protein
adducts, which are post-translationally modified proteins
formed by covalent linkage of reactive nucleophiles (e.g. aldehydes) to parent proteins (Worrall et al., 1993; Niemela et al.,
1999; Thiele et al., 2001; Worrall and Thiele, 2001). Protein
adducts may perturb tissue biochemistry via neoantigen
formation and concomitant antibody generation (Rolla et al.,
2000; Worrall and Thiele, 2001), by rendering affected
proteins more susceptible to proteolysis (Nicholls et al.,
1994) and/or by inactivating the affected proteins (Chen
*Author to whom correspondence should be addressed at: Department of
Biomedical Sciences, University of Westminster, 115 New Cavendish Street,
London W1W 6UW UK. Tel: +44 020 7911 5000 (ext. 3543); Fax: +44 020
7911 5087; E-mail: [email protected]
yAuthor to whom reprint requests should be addressed at: Alcohol Research
Unit, Department of Biochemistry and Molecular Biology, The University
of Queensland, Brisbane, Queensland, QLD 4072, Australia. Tel: +61 7
3365 4626; Fax: +61 7 3365 4699; E-mail: [email protected]
485
The Author 2005. Published by Oxford University Press on behalf of the Medical Council on Alcohol. All rights reserved
486
V. B. PATEL et al.
protein adducts in alcohol-treated animals and patients
(Klassen et al., 1999). However, the extent to which
reduced-acetaldehyde protein adducts are formed in alcoholexposed muscle has not been investigated.
To address these issues we measured a variety of protein
adducts in alcohol-exposed muscle using a combination of
polyclonal and monoclonal antibodies. We compared skeletal
muscles with a predominance of either type I fibres (i.e.
soleus) or type II fibres (i.e. plantaris). In order to test the
effects of elevated levels of acetaldehyde animals were pretreated with cyanamide, an aldehyde dehydrogenase inhibitor.
For comparative purposes, protein–aldehyde adducts were
measured in liver.
METHODS
Animals
Male Wistar rats were obtained from Charles River (Margate,
Kent, UK) at 60 g body weight and fed ad libitum a commercial pelleted diet and housed in an air-conditioned (20–25 C),
humidified (40–60%) animal house with a 12 h light/12 h dark
cycle starting at 08:00 h.
After 1 week of acclimatization, when they weighed
110–120 g body weight, the animals were divided into
four groups: [A] saline + saline, [B] cyanamide + saline, [C]
saline + ethanol, and [D] cyanamide + ethanol.
The dosage used was 75 mmol/kg body weight for alcohol,
0.5 mmol/kg body weight cyanamide, and controls were injected with identical volume of 0.15 mol/l NaCl. The experimental procedure involved i.p. injection (0.5 ml/100 g body
weight) ‘pretreatment’ with either saline or cyanamide, followed 30 min later by i.p. injection (1 ml/100 g body weight)
‘treatment’ with either saline or ethanol. Animals were killed
by decapitation 2.5 h after the alcohol/saline treatment. The
soleus (type I fibre-predominant) and plantaris (type II fibrepredominant) muscles were quickly dissected, and a portion
of the liver was also dissected from each rat. Tissues were
immediately frozen in liquid nitrogen (–196 C) and then
stored at –70 C until analysis.
Generation of modified proteins for antisera production
The conditions and verification of adduct formation were
defined using radiolabelled [14C] acetaldehyde and [3H] lysine
(Tuma et al., 1987). Human plasma protein was modified as
described below and used to generate antisera reactive with
each type of modification. Once the modification reactions
had been carried out the modified proteins were used to raise
polyclonal antibodies in rabbits. The antibodies raised against
each type of modification were then tested for crossreactivity
against unmodified proteins and each type of adduct. These
experiments showed there was little or no crossreactivity
against the adducts but, as expected, some reactivity against
the original carrier protein. This was removed by incubation
of small amounts of antiserum with immobilised carrier protein prior to use. At the end of the incubations, the reaction
mixtures were extensively dialysed against buffer for 12 h
at 4 C before being used as immunogens or in ELISAs. Modified proteins were stored in aliquots at –80 C for <1 month
before use.
Acetaldehyde-modified proteins
Acetaldehyde-modified proteins were produced by incubating
protein (10 mg/ml) with 1 mmol/l acetaldehyde in phosphatebuffered saline (PBS), pH 7.4. Unreduced-acetaldehyde protein adducts were generated during a 5 h incubation at 37 C.
Reduced-acetaldehyde protein adducts were produced by a
1 h incubation at 37 C followed by the addition of 1 ml of
40% (w/v) sodium cyanoborohydride per 10 ml of reaction
mixture. A further 30 min incubation at 37 C was then carried
out to allow the reduction of Schiff bases.
HER-modified proteins
HER-modified proteins were prepared by incubating 10 mg/ml
protein with 50 mmol/l ethanol, 100 mmol/l ferrous ammonium sulphate, 200 mmol/l EDTA and 100 mmol/l hydrogen
peroxide, in 50 mmol/l sodium phosphate buffer (pH 7.4) at
37 C for 30 min (Moncada et al., 1994).
Malondialdehyde-modified proteins
Malondialdehyde-modified proteins were produced by incubating (10 mg/ml protein) with 1 mmol/l malondialdehyde
for 12 h at 37 C in PBS, pH 7.4. Malondialdehyde was
produced immediately before use by the hydrolysis of the
dimethylacetal with 1 mol/l HCl for 30 min at 37 C. A portion
of the acidic solution was then diluted with water and adjusted
to pH 7.4 with 1 mol/l NaOH to prepare neutral malondialdehyde solution for use in the modification reactions.
Malondialdehyde–acetaldehyde modified proteins
It has been shown previously that a mixture of malondialdehyde (MDA) and acetaldehyde react with protein to produce
malondialdehyde–acetaldehyde–protein adducts which have
been termed MAA adducts. Fluorescence was used to measure
the formation of MAA adducts and the number of amino
groups was measured to determine the level of adduct formation (Tuma et al., 1996; Xu et al., 1997). Protein, 10 mg/ml
in 100 mmol/l sodium phosphate (pH 7.4), was incubated
with 1 mmol/l acetaldehyde and 1 mmol/l malondialdehyde
for 3 days at 37 C.
Generation of anti-protein adduct antisera
New Zealand white rabbits (8 months old) were immunized
with modified human plasma protein in three sets of multisite injections as described previously (Worrall et al., 1989).
After the final booster injection, the rabbits were exsanguinated by cardiac puncture under Nembutal anaesthesia. Blood
was collected using heparinized needles and tubes and the
resulting plasma was stored at –80 C. To decrease binding to
unmodified proteins, antisera were purified as described previously (Worrall et al., 1989; Nicholls et al., 1994). Briefly, 1 ml
of rabbit antiplasma and 1 ml of Buffer A (20 mmol/l Tris–
HCl, pH 8.0, containing 28 mmol/l NaCl) were mixed and
loaded onto a Biogel P-6 DG desalting column (Biorad).
The eluate was collected as a single fraction and applied to a
DEAE-Affigel blue column (Biorad) pre-equilibrated with
Buffer A. The column was eluted with Buffer A and 2 ml fractions collected. Those containing the highest protein concentrations were pooled and applied to an immunoadsorption
column for 4 h at 4 C. The eluate from the immunadsorption
PROTEIN ADDUCT SPECIES IN MUSCLE
column was collected as a single fraction and stored at 4 C for
<1 week prior to use in the ELISAs described below. The
immunoadsorption column was made by reacting 2 ml of rat
plasma (from a control rat) with 10 ml of Affigel-10 activated
ester gel (Biorad) suspended in coupling buffer (40 mmol/l
HEPES, pH 7.5, containing 160 mmol/l CaCl2) for 4 h at
room temperature. The gel was poured into a column and
washed with 20 bed-volumes of coupling buffer to remove
unbound protein. The antibodies raised against each type of
modification were then tested for crossreactivity against
unmodified proteins and each type of adduct. These experiments showed there was little or no crossreactivity against
the adducts but, as expected, some reactivity against the
original carrier protein. This was removed by incubation of
small amounts of antiserum with immobilized carrier protein
prior to use.
RT1.1 antibody was a generous gift from Professor Dean
Tuma and Associate Professor Geoff Thiele (Department of
Veteran’s Affairs Alcohol Research Centre and the University
of Nebraska Medical Centre, Omaha, Nebraska). This antibody only recognizes epitopes generated by incubating proteins with acetaldehyde in the presence of excess sodium
cyanoborohydride (a specific reducing agent for Schiff bases)
(Worrall et al., 1994). The archetypal modification formed
under these circumstances is N-ethyllysine (i.e. a lysine residue ethylated on the side chain e-amino group) and will not
react against unreduced proteins (Thiele et al., 1994). In
general when the proposed modification is unknown it is
difficult to state with certain the specificity of the antibody.
However, aldehydes can react with proteins containing arginine, histidine, lysine, and tryptophan residues (Esterbauer
et al., 1991).
Detection of modified proteins in tissue
homogenates by ELISA
Indirect ELISA detected the presence of modified proteins in
muscle and liver homogenates, with all samples being analysed in quadruplicate. Briefly, tissues were homogenized
in TBS [1:10 (w/v)] using an Ultra-Tarrax homogenizer. Samples were then diluted to 100 ng protein/ml in TBS and 100 ml
was added to each well of a 96-well plate. After 2 h at 4 C, the
plates were washed with Tris-buffered saline (TBS), pH 7.4.
Non-specific binding sites were blocked by incubation with
200 ml of saturated casein solution [2.0% (w/v), pH 7.5–8.0]
for 1 h at 4 C. Purified rabbit anti-protein adduct antiplasma
(100 ml) was then added to each well and incubated for 4 h
at 4 C. The plate was washed with TBS-casein [TBS containing 0.5% (w/v) casein] and 100 ml of biotinylated antirabbit immunoglubulin antibody added. After 2 h at 4 C, the
plates were again washed with TBS-casein and 100 ml of
streptavidin-alkaline phosphatase complex added. After 1 h
at 37 C, the plates were again washed with TBS. The final
step was to add 100 ml of p-nitrophenyl phosphate (1 mg/ml)
in diethanolamine buffer (10 mmol/l diethanolamine,
0.5 mmol/l MgCl2, pH 9.5) and incubate at 37 C. The absorbance of each well at 405 nm was measured using a Titertek
multiscan plate reader after 1–3 h incubation depending on
the tissue and the type of protein adduct assayed. Appropriate
negative and positive controls were carried out for each
analysis (Worrall et al., 1989, 2000a, b, 2001; Nicholls et al.,
1994). The data displayed refers to assay conditions designed
487
to produce optimum optical densities (OD) and are shown as
OD units (at 405 nm) per 10 ng protein.
Statistics
All data are expressed as the mean ± SEM (n = 7–8). The
significance of differences between treatments was assessed
using two-way ANOVA. Significance was indicated when
P < 0.05. Statistics where P > 0.05 are not displayed.
RESULTS
Unreduced-acetaldehyde–protein adducts
In the type I fibre-predominant soleus muscle (Fig. 1) alcohol
treatment caused a highly significant increase in the levels
of unreduced acetaldehyde adducts. There was also a highly
significant interaction with cyanamide pretreatment, which
potentiated the effect of alcohol treatment. Alcohol treatment
also caused a highly significant increase in the levels of unreduced acetaldehyde adducts in the type II fibre-predominant
plantaris muscle (Fig. 2), and cyanamide pretreatment also
caused a significant increase in levels of unreduced acetaldehyde adducts that was independent of alcohol treatment.
In the liver (Fig. 3) alcohol treatment again caused a highly
significant increase, which was potentiated by cyanamide
pretreatment.
Reduced-acetaldehyde protein adducts
Use of both the rabbit polyclonal and the mouse monoclonal
RT1.1 antibody showed that there was no measurable increase
in levels of reduced-acetaldehyde adducts and in particular
N-ethylated lysine residues, in the soleus, plantaris, or liver
in response to cyanamide, ethanol, or cyanamide + ethanol
(Figs 1–3).
Malondialdehyde–protein adducts
In both the soleus and the plantaris muscles, malondialdehyde–
protein adduct scores increased in response to both cyanamide
and ethanol with no significant interaction (Figs 1 and 2). In
the liver, malondialdehyde–protein adducts also increased
in response to both cyanamide and ethanol, although in this
case there was some interaction as cyanamide appeared to
have a much greater effect in combination with alcohol than
on its own (Fig. 3).
HER– and MAA adducts
There was no measurable increase in these adducts in the
soleus, plantaris, or liver in any of the experimental conditions
(Figs 1–3).
Differences between the soleus and plantaris
Because soleus and plantaris were taken from the same animals and assayed under the same analytical conditions direct
comparisons between these two muscles were made. The calculated ratio of the amounts of adducts in the two muscles for
each animal were for plantaris/soleus 1.69 (95% CI 1.17–2.20;
n = 30) for unreduced-acetaldehyde adducts and 1.65 (95% CI
1.45–1.84) for MDA adducts. Thus in both cases the average
level of adducts was greater in the plantaris than the soleus.
However, when the ratios were subjected to analysis of
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V. B. PATEL et al.
Fig. 1. Protein adduct species in soleus of rats subjected to ethanol and cyanamide treatments. Data are OD units per 10 ng protein, shown as mean ± SEM of
n = 7–8 observations in each group.
variance there were no significant effects of either alcohol
or cyanamide and no significant interactions. It appears that
neither muscle was significantly more sensitive to either
alcohol or cyanamide treatment in terms of adduct formation.
DISCUSSION
In these studies, we sought to investigate the nature of the
protein adducts formed in skeletal muscle as a consequence
of alcohol exposure. The experimental design also allowed us
to assess the effects of markedly raising circulating acetaldehyde levels, by administering cyanamide to some of the
rats that were subsequently dosed with ethanol (Hillbom
et al., 1983; Pron’ko et al., 1999; Adachi et al., 2001). Levels
of acetaldehyde in the blood of cyanamide plus ethanol treated
rats at the end of 2.5 h are 500-fold higher than levels achieved
with ethanol alone (Adachi et al., 2001). Thus the failure to
detect increased levels of reduced-acetaldehyde adducts with
both the rabbit polyclonal and mouse monoclonal (RT1.1)
PROTEIN ADDUCT SPECIES IN MUSCLE
489
Plantaris
0.4
P
0.05
<0.001
NS
0.3
0.2
0.1
0
RTL1-protein adducts (OD)
[A]
[B]
[C]
[D]
Saline Cyanamide Saline Cyanamide
+Saline
+Saline
+Ethanol +Ethanol
0.2
2 way ANOVA
Treatments
Cyanamide
Ethanol
Interaction
0.15
0.1
P
NS
NS
NS
0.05
2 way ANOVA
0.2
Reduced acetaldehydeprotein adducts (OD)
Treatments
Cyanamide
Ethanol
Interaction
0.5
0.15
P
NS
NS
NS
Treatments
Cyanamide
Ethanol
Interaction
0.1
0.05
0
[A]
[B]
[C]
[D]
Saline Cyanamide
Saline Cyanamide
+Ethanol +Ethanol
+Saline
+Saline
Hydroxyethyl radicalprotein adducts (OD)
Unreduced acetaldehydeprotein adducts (OD)
2 way ANOVA
0
0.5
0.4
0.3
2 way ANOVA
P
NS
NS
NS
Treatments
Cyanamide
Ethanol
Interaction
0.2
0.1
0
[C]
[A]
[B]
[D]
Saline Cyanamide Saline Cyanamide
+Saline
+Saline
+Ethanol +Ethanol
[C]
[A]
[B]
[D]
Saline Cyanamide
Saline Cyanamide
+Saline
+Saline
+Ethanol +Ethanol
0.75
Treatments
Cyanamide
Ethanol
Interaction
P
<0.001
<0.001
NS
0.5
0.25
0
[A]
[B]
[C]
[D]
Saline Cyanamide
Saline Cyanamide
+Saline
+Saline
+Ethanol +Ethanol
Malondialdehyde-acetaldehyde
protein adducts (OD)
Malondialdehyde-protein
adducts (OD)
2 way ANOVA
0.1
2 way ANOVA
0.08
0.06
Treatments
Cyanamide
Ethanol
Interaction
P
NS
NS
NS
0.04
0.02
0
[A]
[B]
Saline Cyanamide
+Saline
+Saline
[C]
[D]
Saline Cyanamide
+Ethanol +Ethanol
Fig. 2. Protein adduct species in plantaris of rats subjected to ethanol and cyanamide treatments.
antibodies in the present series of data must reflect the fact that
these adducts are not formed acutely rather than the possibility
that acetaldehyde levels were not sufficiently elevated.
Low levels of acetaldehyde adducts were seen in all tissues
of untreated rats, in line with previous observations in the
liver (Nicholls et al., 1994). These adducts may arise from
endogenous acetaldehyde generation by intestinal bacteria
(Salaspuro, 1997; Seitz and Poschl, 1997) or serine hydroxymethyltransferase activity which catalyses the conversion
of threonine to glycine (Nicholls et al., 1994). Rat skeletal
muscle also contains aldehyde dehydrogenase (Nilsson, 1988;
Stewart et al., 1996), so it would not be unreasonable to expect
490
V. B. PATEL et al.
Fig. 3. Protein adduct species in liver of rats subjected to ethanol and cyanamide treatments.
to see small increases in acetaldehyde adducts in control rats
injected with cyanamide alone. However, the main effect
of cyanamide on acetaldehyde adducts was to potentiate the
effect of alcohol, which was particularly apparent in the soleus
and the liver, where the interaction term in the analysis of
variance was highly significant.
Cyanamide also had an effect on MDA adduct levels in both
muscles, and this effect was independent of the effect of ethanol. We have recently found based on immunohistochemical
staining a similar effect of cyanamide on MDA adduct levels
in the gastrocnemius muscle, which has a mixed fibretype composition (Niemela O., Parkkila S., Emery P. W. and
PROTEIN ADDUCT SPECIES IN MUSCLE
Preedy V. R., unpublished data). These data suggest that cyanamide imposes oxidative stress in muscle, as has previously
been shown in the pancreas (Altomare et al., 1996) and liver
(Kera et al., 1988; Kato et al., 1990; Vendemiale et al., 1998).
Reduced-acetaldehyde adducts
To investigate the nature of the adducted protein species
formed in muscle of alcohol-dosed rats we employed an array
of rabbit polyclonal antibodies and one mouse monoclonal
antibody. The purpose of using both a mono- and polyclonalantibody reacting against reduced-acetaldehyde adducts
relates to the phenomena of epitope interconversion. It has
been observed that when unreduced protein adducts are used
to raise antibodies, antibodies reacting against both reducedand unreduced-acetaldehyde protein adducts are detected
(Klassen et al., 1999). Use of reduced-acetaldehyde protein
adducts, however, raises antibodies that react only with the
reduced adduct species (Klassen et al., 1999). The conversion
of unreduced-acetaldehyde protein adducts to reduced species
may be mediated by macrophages via a mechanism that has
yet to be elucidated (Klassen et al., 1999). Nevertheless, the
presence of antibodies reacting against reduced-acetaldehyde
adducts in man (Worrall et al., 1990; Klassen et al., 1999)
and experimental animals (Worrall et al., 2000a) has been
used to suggest that reduced protein adducts are formed as a
consequence of alcohol exposure.
In the present study no significant increase in reducedacetaldehyde adducts was identified using either the RT1.1
monoclonal or polyclonal antibody in types I or II tissues.
The same observation was made even in the presence of
supra-physiological levels of acetaldehyde in the cyanamide
plus ethanol group. However, in a previous study when the
polyclonal antibody was used for immunohistochemical analysis a slight increase (P < 0.05) was observed in reduced acetaldehyde adducts following cyanamide plus alcohol treatment
in type II tissue. This observation is in contrast to the present
study where no increased levels of reduced-acetaldehyde
adduct was identified using either the RT1.1 or polyclonal
antibody following cyanamide plus alcohol treatment. It is
possible that by using immunohistochemistry subtle localized
changes are identified whereas with ELISA these site-specific
effects are diluted out.
Unreduced-acetaldehyde adducts
Comparisons were made between different types of skeletal
muscle fibres, i.e. type I anaerobic, glycolytic, fast-twitch,
and type II aerobic, oxidative, slow twitch, as represented by
the soleus and plantaris, respectively. Such an approach of
analysing anatomically distinct skeletal muscles with different
fibre types have been used before, for example, in studies
on malnutrition (Ardawi et al., 1989), hypoxia (Bigard et al.,
2000) and exercise (Hildebrandt and Neufer, 2000) as well
as alcohol exposure (Adachi et al., 2000; Smith et al., 2000).
It is well known that alcoholic myopathy affects type II fibres
more severely than type I fibres (Preedy et al., 2001a, b, c, d).
In the present experiment there was a significant increase in
the levels of unreduced acetaldehyde adducts and this effect
was potentiated by cyanamide. However, there was no difference between the two muscles in the amounts of unreduced
adducts formed in response to alcohol treatment, thus each
491
muscle type displays equal sensitivity to alcohol dosing. This
observation is in line with our previous finding in rats treated
chronically with alcohol for 6 weeks (Worrall et al., 2001).
We therefore conclude that, after acute alcohol exposure,
unreduced adducts are preferentially formed than reduced
adducts in muscle. Similar conclusions can be applied to the
liver, where the results were similar to those in muscle. These
results also emphasize the importance of oxidative stress as a
mechanism by which alcohol may damage tissues.
MDA adducts
In a previous study using immunohistochemistry, the intensity
of staining for MDA adducts in cyanamide plus ethanol
treated animals was 1.5 and 2.5 for soleus and plantaris,
respectively, (Niemela et al., 2002) suggesting a preferential
increase in type II tissues. In the current study a similar
response was observed whereby mean absorbance values
were higher in plantaris than in soleus, but the calculated ratio
of the amount of adducts in the two muscles was not significantly different. Thus although there appears to be preferential
formation of MDA adducts in type II tissue, the evidence is not
conclusive. The formation of MDA adducts observed following acute alcohol dosing may reflect a transient phenomenon
to which animals may be able to adapt over time as following
chronic alcohol administration no statistical difference was
observed (Worrall et al., 2001).
MAA adducts
Following acute ethanol exposure MAA adducts were not
detected in the liver but are significantly elevated following
chronic alcohol administration (Worrall et al., 2001). In muscle, however, MAA levels were not altered following either
acute or chronic alcohol administration (Worrall et al., 2001)
in either types I or II tissue. This suggests that the formation
of acetaldehyde and MDA by lipid peroxidation possibly
occurs in separate cellular compartments, preventing any
interaction.
CONCLUSION
Protein adducts are formed in skeletal muscle of rats acutely
exposed to alcohol and pertain to unreduced acetaldehyde
and malondialdehyde species.
Acknowledgements — We are grateful to Professors John de Jersey,
Tom A. B. Sanders and Timothy J. Peters for encouragement and support.
REFERENCES
Adachi, J., Asano, M., Ueno, Y. et al. (2000) 7alpha- and 7betahydroperoxycholest-5-en-3beta-ol in muscle as indices of oxidative stress: response to ethanol dosage in rats. Alcoholism: Clinical
and Experimental Research 24, 675–681.
Adachi, J., Asano, M., Ueno, Y. et al. (2001) Acute effect of
ethanol on 7-hydroperoxycholesterol in muscle and liver. Lipids
36, 267–271.
Altomare, E., Grattagliano, I., Vendemiale, G. et al. (1996) Acute
ethanol administration induces oxidative changes in rat pancreatic
tissue. Gut 38, 742–746.
492
V. B. PATEL et al.
Ardawi, M. S., Majzoub, M. F., Masoud, I. M. et al. (1989) Enzymic
and metabolic adaptations in the gastrocnemius, plantaris and
soleus muscles of hypocaloric rats. Biochemical Journal 261,
219–225.
Bigard, A. X., Sanchez, H., Birot, O. et al. (2000) Myosin heavy
chain composition of skeletal muscles in young rats growing
under hypobaric hypoxia conditions. Journal of Applied
Physiology 88, 479–486.
Chen, J., Petersen, D. R., Schenker, S. et al. (2000) Formation of
malondialdehyde adducts in livers of rats exposed to ethanol:
role in ethanol-mediated inhibition of cytochrome c oxidase.
Alcoholism: Clinical and Experimental Research 24, 544–552.
Esterbauer, H., Schaur, R. J. and Zollner, H. (1991) Chemistry and
Biochemistry of 4-hydroxynonenal, malondialdehyde and related
aldehydes. Free Radical Biology and Medicine 11, 81–128.
Estruch, R., Nicolas, J. M., Villegas, E. et al. (1993) Relationship
between ethanol-related diseases and nutritional status in chronically alcoholic men. Alcohol and Alcoholism 28, 543–550.
Fernandez-Sola, J., Sacanella, E., Estruch, R. et al. (1995) Significance of type, II fiber atrophy in chronic alcoholic myopathy.
Journal of the, Neurological Sciences 130, 69–76.
Hildebrandt, A. L. and Neufer, P. D. (2000) Exercise attenuates the
fasting-induced transcriptional activation of metabolic genes in
skeletal muscle. American Journal of Physiology, Endocrinology
and Metabolism 278, E1078–E1086.
Hillbom, M. E., Sarviharju, M. S. and Lindros, K. O. (1983)
Potentiation of ethanol toxicity by cyanamide in relation to acetaldehyde accumulation. Toxicology and Applied Pharmacology
70, 133–139.
Iimuro, Y., Bradford, B. U., Gao, W. et al. (1996) Detection of alphahydroxyethyl free radical adducts in the pancreas after chronic
exposure to alcohol in the rat. Molecular Pharmacology 50,
656–661.
Kato, S., Kawase, T., Alderman, J. et al. (1990) Role of xanthine
oxidase in ethanol-induced lipid peroxidation in rats. Gastroenterology 98, 203–210.
Kera, Y., Ohbora, Y. and Komura, S. (1988) The metabolism
of acetaldehyde and not acetaldehyde itself is responsible for
in vivo ethanol-induced lipid peroxidation in rats. Biochemical
Pharmacology 37, 3633–3638.
Klassen, L. W., Jones, B. L., Sorrell, M. F. et al. (1999) Conversion
of acetaldehyde-protein adduct epitopes from a nonreduced to
a reduced phenotype by antigen processing cells. Alcoholism:
Clinical and Experimental Research 23, 657–663.
Moncada C., Torres V., Varghese G. et al. (1994) Ethanol-derived
immunoreactive species formed by free radical mechanisms.
Molecular Pharmacology 46, 786–791.
Nicholls, R. M., Fowles, L. F., Worrall, S. et al. (1994) Distribution
and turnover of acetaldehyde-modified proteins in liver and blood
of ethanol-fed rats. Alcohol and Alcoholism 29, 149–157.
Niemela, O., Parkkila, S., Pasanen, M. et al. (1999) Induction of
cytochrome P450 enzymes and generation of protein-aldehyde
adducts are associated with sex-dependent sensitivity to alcoholinduced liver disease in micropigs. Hepatology 30, 1011–1017.
Niemela, O., Parkkila, S., Koll, M. et al. (2002) Generation of protein
adducts with malondialdehyde and acetaldehyde in muscles with
predominantly type I or type II fibers in rats exposed to ethanol
and the acetaldehyde dehydrogenase inhibitor cyanamide.
American Journal of Clinical Nutrition 76, 668–674.
Nilsson, G. E. (1988) A comparative study of aldehyde dehydrogenase and alcohol dehydrogenase activities in crucian carp
and three other vertebrates: apparent adaptations to ethanol
production. Journal of Comparative Physiology B, Biochemical,
Systemic, and Environmental Physiology 158, 479–485.
Preedy, V. R., Adachi, J., Peters, T. J. et al. (2001a) Recent advances
in the pathology of alcoholic myopathy. Alcoholism: Clinical and
Experimental Research 25, 54S–59S.
Preedy, V. R., Adachi, J., Ueno, Y. et al. (2001b) Alcoholic skeletal
muscle myopathy: definitions, features, contribution of neuropathy, impact and diagnosis. European Journal of Neurology 8,
677–687.
Preedy, V. R., Paice, A., Mantle, D. et al. (2001c) Alcoholic
myopathy: Biochemical mechanisms. Drug and Alcohol
Dependence 63, 199–205.
Preedy, V. R., Peters, T. J. and Adachi, J. (2001d) Pathogenic mechanisms in alcoholic myopathy, in Alcohol in health and disease
(Agarwal DP and Seitz HK eds) pp 243–259, Marcel Dekker
Inc, New York.
Pron’ko, P. S., Kuz’mich, A. B. and Abakumov, G. Z. (1999) Effect
of alcohol intoxication and aldehyde dehydrogenase inhibitors
on lipid peroxidation in rat liver. (Russian). Ukrainskii Biokhimicheskii Zhurnal 71, 79–83.
Rolla, R., Vay, D., Mottaran, E. et al. (2000) Detection of circulating
antibodies against malondialdehyde-acetaldehyde adducts in
patients with alcohol-induced liver disease. Hepatology 31,
878–884.
Sacanella, E., Fernandez-Sola, J., Cofan, M. et al. (1995) Chronic
alcoholic myopathy: diagnostic clues and relationship with other
ethanol-related diseases. Quarterly Journal of Medicine 88,
811–817.
Salaspuro, M. (1997) Microbial metabolism of ethanol and
acetaldehyde and clinical consequences. Addiction Biology 2,
35–46.
Seitz, H. and Poschl, G. (1997) Alcohol and gastrointestinal cancer:
Pathogenic mechanisms. Addiction Biology 2, 19–33.
Singh, R., Leuratti, C., Josyula, S. et al. (2001) Lobe-specific
increases in malondialdehyde DNA adduct formation in the
livers of mice following infection with Helicobacter hepaticus.
Carcinogenesis. 22, 1281–1287.
Smith, C., Stamm, S. C., Riggs, J. E. et al. (2000) Ethanol-mediated
CYP1A1/2 induction in rat skeletal muscle tissue. Experimental
and Molecular Pathology 69, 223–232.
Stewart, M. J., Malek, K and Crabb, D. W. (1996) Distribution
of messenger RNAs for aldehyde dehydrogenase 1, aldehyde
dehydrogenase 2, and aldehyde dehydrogenase 5 in human
tissues. Journal of Investigative Medicine 44, 42–46.
Thiele, G. M., Wegter, K. M., Sorrell, M. F. et al. (1994) Specificity
to N-ethyl lysine of a monoclonal antibody to acetaldehydemodified proteins prepared under reducing conditions. Biochemical Pharmacology 48, 183–189.
Thiele, G. M., Worrall, S., Tuma, D. J. et al. (2001) The chemistry
and biological effects of malondialdehyde-acetaldehyde adducts.
Alcoholism: Clinical and Experimental Research 25, 218S–224S.
Tuma, D. J., Newman, B. S., Donohue, T. M. Jr et al. (1987) Covalent
binding of acetaldehyde to proteins: participation of lysine
residues. Alcoholism: Clinical and Experimental Research 11,
579–584.
Tuma, D. J., Thiele, G. M., Xu, D. et al. (1996) Acetaldehyde and
malondialdehyde react together to generate distinct protein
adducts in the liver during long-term ethanol administration.
Hepatology 23, 872–880.
Vendemiale, G., Grattagliano, I., Signorile, A. et al. (1998) Ethanolinduced changes of intracellular thiol compartmentation and protein redox status in the rat liver: effect of tauroursodeoxycholate.
Journal of Hepatology 28, 46–53.
Worrall, S. and Thiele, G. M. (2001) Protein modification in ethanol
toxicity. Adverse Drug Reactions and Toxicological Reviews 20,
133–159.
Worrall, S., De Jersey, J., Shanley, B. C. et al. (1989) Ethanol induces
the production of antibodies to acetaldehyde-modified epitopes in
rats. Alcohol and Alcoholism 217, 217–223.
Worrall, S., De Jersey, J., Shanley, B. C. et al. (1990) Antibodies
against acetaldehyde-modified epitopes: presence in alcoholic,
non-alcoholic liver disease and control subjects. Alcohol and
Alcoholism 25, 509–517.
Worrall, S., de Jersey, J., Nicholls, R. M. et al. (1993) Acetaldehyde/
protein interactions: are they involved in the pathogenesis of
alcoholic liver disease? Digestive Diseases 11, 265–277.
Worrall, S., De Jersey, J., Shanley, B. C. et al. (1994) Antiacetaldehyde adduct antibodies generated by ethanol-fed rats
react with reduced and unreduced acetaldehyde-modified
proteins. Alcohol and Alcoholism 29, 43–50.
Worrall, S., de Jersey, J. and Wilce, P. A. (2000a) Comparison of the
formation of proteins modified by direct and indirect metabolites
of ethanol in the liver and blood of rats fed the Lieber-DeCarli
diet. Alcohol and Alcoholism 35, 164–170.
Worrall, S., Richardson, P. J. and Preedy, V. R. (2000b) Experimental
heart muscle damage in alcohol feeding is associated with
PROTEIN ADDUCT SPECIES IN MUSCLE
increased amounts of reduced- and unreduced-acetaldehyde,
and malondialdehyde-acetaldehyde protein adducts. Addiction
Biology 5, 421–427.
Worrall, S., Niemela, O., Parkkila, S. et al. (2001) Protein adducts
in type I and type II fibre predominant muscles of the
ethanol-fed rat: preferential localisation in the sarcolemmal and
493
subsarcolemmal region. European Journal of Clinical Investigation 31, 723–730.
Xu, D., Thiele, G. M., Kearley, M. L. et al. (1997) Epitope characterisation of malondialdehyde-acetaldehyde adducts using an
enzyme linked immunosorbent assay. Chemical Research in
Toxicology 10, 978–986.

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