Bull Vet Inst Pulawy 53, 673-676, 2009
ACTIVITY OF ANTIOXIDANT ENZYMES
IN UTERINE TISSUES OF BITCHES WITH PYOMETRA
MAREK SZCZUBIAŁ AND ROMAN DĄBROWSKI
Department and Clinic of Animal Reproduction, Faculty of Veterinary Medicine,
University of Life Sciences, 20-612 Lublin, Poland,
[email protected]
Received for publication June 29, 2009
Abstract
The aim of the study was to determine the activities of glutathione peroxidase (GSH-Px), superoxide dismutase, and
catalase in the uterine tissues of bitches with pyometra. The uterus from 11 bitches with pyometra and from 18 healthy bitches, which
had undergone sterilisation (controls), were used. The activities of enzymes were determined in uterine tissue homogenisates using
spectrophotometric methods. Lower GSH-Px activities in the uterine tissues of bitches with pyometra indicate deteriorated capacities
of cells to protect against ROS and suggest possible involvement of oxidative stress in the aetiology and pathogenesis of pyometra.
Further studies, however, are required to thoroughly elucidate this issue.
Key words: bitches, uterus, pyometra, antioxidant enzymes.
Although reactive oxygen species (ROS) are
unavoidable intermediates during metabolism, and may
have also beneficial effects, their level and balance
between their production and degradation must be
controlled. Small amounts of ROS are necessary for
numerous vital processes in the organism (16, 29, 30).
However, their uncontrollable increased generation
results in oxidative damage to almost all cellular
components, which, in turn, leads to the injury of cell
membranes, degradation of cell structures, cell lysis, and
tissue damage (12).
Human beings and animals are equipped with
defensive mechanisms to protect them against harmful
effects of ROS by maintaining the production of ROS at
a level safe for cells (11, 13). The essential elements of
antioxidative protection include antioxidant enzymes:
glutathione peroxidase (GSH-Px), superoxide dismutase
(SOD), and catalase (CAT) (11, 13, 36).
Glutathione peroxidase (GSH-Px) is a tetramer,
whose each subunit contains one selenium atom bound
with cysteine (24). Its main function is to remove
hydrogen peroxide (34). GSH-Px catalyses the hydrogen
peroxide-glutathione reaction, which results in the
oxidation of glutathione. Moreover, GSH-Px reduces
organic peroxides, mainly lipid peroxides, to alcohols
(34). In cases of lipid peroxides, this discontinues the
process of lipid peroxidation.
In mammals, superoxide dismutase (SOD)
occurs in three distinct forms varying in a cofactor
required for their proper functioning and location (10).
The dismutase dependent on zinc and copper ions
(ZnCu-SOD) occurs in the cytoplasm; its activity
constitutes about 70% of the total SOD activity. The
manganese-dependent dismutase (Mn-SOD) occurs in
the mitochondria. The third form of SOD present in the
plasma is the extracellular superoxide dismutase (ECSOD). The mechanism of SOD-catalysed reactions
consists of the reduction and oxidation of metal ions in
the active centres. The enzyme catalyses the reaction of
dismutation of superoxide anion radicals to hydrogen
peroxides (10).
Catalase (CAT) is a tetrameter composed of
four subunits, about 60 kDa, each. In mammalian cells,
CAT occurs predominantly in peroxysomes (24).
Together with glutathione peroxidase, it prevents the
accumulation of hydrogen peroxide in the cell,
degrading it to water and molecular oxygen (34). CAT is
believed to play an essential role at high concentrations
of hydrogen peroxide. At low concentrations of
hydrogen peroxide, GSH-Px is considered more relevant
(10).
At present, ROS are implicated in the aetiology
and pathogenesis of many diseases (1, 2), including
gynaecological diseases (6, 19, 25, 27, 31). Studies in
humans have demonstrated that the mechanisms of
antioxidative protection are impaired in many uterine
diseases (6, 19, 25, 27, 31).
Pyometra is the most common gynaecological
disease observed in bitches (5, 32). Generally, it occurs
from 4 weeks to 4 months after oestrus and is
characterised by the accumulation of purulent secretion
in the enlarged uterus; in some cases, vaginal purulent
discharge (open-cervix pyometra), and general clinical
symptoms (polyuria, polydipsia, apathy, inappetite,
vomiting, and enlarged abdominal integuments) are
observed (4, 32). The aetiology of pyometra in bitches is
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complex and has not been fully elucidated. The disease
is believed to be primarily caused by hormonal
disorders, resulting in cystic proliferation of the uterine
mucosa glands; bacterial infections induced mainly by
Escherichia coli are considered the secondary cause (4).
The available literature lacks data concerning the
antioxidative status in bitches with pyometra.
The aim of the present study was to determine
the activities of antioxidative enzymes: glutathione
peroxidase, superoxide dismutase, and catalase in the
uterine tissues of bitches with pyometra.
Material and Methods
The study was performed on 29 bitches of
various breeds and mongrels. The bitches were divided
into two groups. Group I comprised bitches suspected of
suffering from pyometra (n=11), aged 5-12 years. All
dogs of this group showed polydipsia and a reduced or
complete lack of appetite (anorexia). Physical
examinations revealed apathy, enlarged abdominal
integuments, and an increased pulse and respiration rates
in most of the bitches. In all animals, morphological
observations indicated leukocytosis with a left image
shift. Abdominal ultrasound (Pie Medical Scanner 200)
and the linear head 5/7.5 MHz revealed an enlarged
uterus of diameter ranging from 2 to 4 cm with a
hypoechogenic content.
Vaginoscopy indicated congested serous
membranes of the vaginal vestibule and vagina, which
could be seen to be covered with purulent secretions in
bitches with patent pyometra (n=3). On the basis of
these findings, the bitches were assigned to the pyometra
group. Group II comprised 18 bitches, aged 2-7 years,
brought for sterilisation. On the basis of abdominal
ultrasound and the results of routine haematological and
biochemical examinations of blood and urine, all bitches
in this group were considered healthy. All bitches
included in the study were 2-10 weeks after the oestrus.
All underwent ovariohysterectomy. The surgical
procedures were carried out under general anaesthesia
using intramuscular ketamine (5% Narkamon®, Spofa,
Czech Republic), 5-15 mg/kg b.w., following
premedication with subcutaneous atropine (Atropinum
sulfuricum®, Polfa, Poland), 0.05 mg/kg b.w., and
intramuscular xylazine (Rometar 2%®, Spofa, Czech
Republic), 2 mg/kg b.w.
The fragments of the entire wall of the uterine
body, about 2x2 cm in size, were collected immediately
after surgery, washed with 0.9% NaCl, frozen at -76ºC,
and kept until laboratory tests. In pyometra cases, the
samples of pus were collected from the uterine. The pus
was cultured on the agar medium with 5% ram blood.
After a 24 h incubation at 37oC under oxygen
conditions, the microorganisms were initially identified
by evaluating the morphology of colonies and
microscopic examination of Gram-stained specimens.
The isolated bacterial strains were finally identified
using commercial API tests (API-Staph, and API-20E,
BioMérieux).
The activities of enzymes were determined in
homogenisates of uterine tissue and expressed as units
per milligram of protein. The samples of uterine tissue
were homogenised in the Ultra Turrax T25 apparatus
(Ikawerk, Janke and Kunkel Inc., Germany). The
enzyme activities were measured spectrophotometrically
using Ultrospec 2000 (Pharmacia, Sweden). The activity
of GSH-Px was determined using the Paglia and
Valentine method (26), the activity of SOD - using the
Sun and Zigman method (33) while that of CAT
according to Cohen (8). The protein content in
supernatants was measured according to Lowry et al.
(17).
The data were statistically analysed by
calculating the mean, standard deviation, and
significance of differences between both groups using
the Statistica 5-0. The level of significance was set at the
P<0.05.
Results
The bacteriological tests showed the presence
of bacterial flora in nine cases. In seven (63.64%)
samples E. coli and in two (18.18%) Staphylococcus
aureus strains were found. No growth was observed in
two (18.18%) cases.
The results of glutathione peroxidase,
superoxide dismutase, and catalase determinations are
presented in Table 1. In cases of pyometra, the activity
of GSH-Px in affected uterine tissues was statistically
significantly lower (P<0.05) compared to that in healthy
tissues, i.e. 3.49 and 5.87 U/mg of protein, respectively.
The activity of SOD in affected uterine tissues was on
average 6.28 U/mg of protein and did not statistically
differ from that in healthy uterus (6.19 U/mg of protein).
The activity of CAT in the affected and healthy uterine
samples was similar, 108.02 and 105.25 U/mg of
protein, respectively.
Table 1
Activities of enzymes in uterine tissues from pyometra and healthy bitches
Parameter
Bitches with pyometra
Healthy bitches
n=11
n=18
GSH-Px (U/mg protein)
3.49*
5.87
SOD (U/mg protein)
6.28
6.19
CAT (U/mg protein)
108.02
105.25
*
- P<0.05
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Discussion
One of the key mechanisms responsible for
tissue damage is oxidative stress caused by
overproduction of ROS (15). Once the growth of these
metabolites of molecular oxygen is uncontrollable, they
are likely to cause oxidative injury to all main
constituents of the cell and are involved in the aetiology
of numerous diseases (1, 2, 12). The main event related
to ROS production is an inflammatory process (2, 3). In
pyometra, the inflammation develops in the uterine
mucosa and is the protective reaction of the organism,
yet simultaneously leads to tissue damage. At the
inflammation site, the activating factors such as
complement fragments, opsonised bacteria, viruses,
immunoglobulins, and chemotactic peptides, induce
rapid consumption of oxygen in the phagocytic cells,
called the “respiratory burst” and production of ROS
used by phagocytes to destroy the absorbed
microorganisms (35). However, the lack of proper
control of ROS production by antioxidative mechanisms
results in their overproduction and damage to the
phagocytic cells, as well as adjacent tissue cells. The in
vitro studies have demonstrated that ROS released by
activated neutrophils and macrophages may be toxic for
various somatic cells such as erythrocytes, epithelial
cells, fibroblasts, or platelets (13).
The excess of ROS results in the damage and
necrosis of cells through various mechanisms including
peroxidation of cellular membrane lipids, protein
denaturation, and DNA damage (7). Moreover, ROS
such as superoxide anion radical and hydrogen peroxide,
mediate in the infiltration and accumulation of
neutrophils at the inflammation site (14) and mobilise
the changes in arachidonic acid (21). ROS are also
involved in the activation of transcription factors, such
as NF-kB and AP-1. The transcription factors are
activated during inflammation in the epithelial and
inflammatory cells, in which they induce the expression
of many encoding genes, e.g. TNFα, Il-1, Il-6, nitrogen
oxide synthesis, MHC complex I antigens, and adhesive
particles (3). Furthermore, it is believed that a large
group of proteases, called metalloproteinases, is affected
by ROS, which exacerbates the tissue damage at the
inflammatory focus (35).
The essential element of the antioxidative
protection of each cell is the group of antioxidant
enzymes: GSH-Px, SOD, and CAT (11, 13). The
antioxidative action of these enzymes is to neutralise
superoxide anion radicals and hydrogen peroxide –
substrates for the production of the most toxic oxygen
metabolite – a hydroxyl radical (10, 11, 24).
Superoxide anion radical is relatively poorly
active, yet it is generated with high frequency (11). It
serves as the substrate for the formation of markedly
more toxic oxygen metabolites such as hydrogen
peroxide, hypochlorous acid, and hydroxyl radical (20).
The majority of superoxide anion radical formed
undergoes spontaneous and SOD-catalysed dismutation
to hydrogen peroxide (10).
Hydrogen peroxide may oxidise the thiol
groups and the ions of transition metals. The latter
reaction (the Fenton`s reaction) leads to the formation of
hydroxyl radical (24). Compared to superoxide anion
radical, hydrogen peroxide considerably more easily
permeates the cytoplasmatic membranes infiltrating the
extracellular space of lower capacities of anti-ROS
protection than the interior of the cell (24). It has been
demonstrated that hydrogen peroxide causes irreversible
damage to the epithelial cells (23). GSH-Px and CAT
are responsible for the neutralisation of hydrogen
peroxide formed (34).
Hydroxyl radical is the most reactive form of
oxygen and shows high toxicity at the site of its
formation. It rapidly reacts with most biological particles
(lipids, proteins, nucleic acids, carbohydrates) present at
the adjacent places of its synthesis. Due to the extremely
high speed of the reaction, the destructive effects of the
hydroxyl radical, once formed, are difficult to avoid
(24). Therefore, it is essential to prevent its formation by
removing superoxide anion radical and hydrogen
peroxide by antioxidative enzymes (11).
Antioxidative enzymes show adaptive features
enhancing their activity once the production of ROS
increases (9). Our findings showed that pyometra did not
cause significant changes in the activities of SOD and
CAT in uterine tissues; however, the activity of GSH-Px
in the affected uteruses was found to be markedly lower
compared to healthy uteruses. This is likely to indicate
impaired cellular antioxidative protection in bitches with
pyometra, as it is known that proper anti-ROS protection
of cells requires strict cooperation of all three enzymes
(28). Lower GSH-Px activity in the affected bitches
impose the risk of insufficient neutralisation of hydrogen
peroxide, the main substrate for the production of
hydroxyl radical and other toxic ROS. Hydrogen
peroxide, as the main ROS activating transcriptive
factors involved in the expression of genes encoding
various factors causing inflammation, is likely to affect
the development and course of inflammatory processes.
Moreover, lower activity of GSH-Px – involved in the
neutralisation of lipid peroxidation products – in bitches
with pyometra, indicate their deteriorated protective
capacities against the effects of lipid superoxides and
toxic aldehydes (18, 34).
It is known that oestrogens increase the activity
of GSH-Px, while progesterone does not (22). Since all
the bitches used in the study were in the luteal phase of
oestrus, the influence of sex hormones on the differences
in GSH-Px activity observed between the affected and
healthy bitches should be excluded.
In conclusion, lower GSH-Px activity in uterine
tissues from bitches with pyometra indicate reduced
abilities of cell protection against ROS and suggest
possible involvement of oxidative stress in the aetiology
and pathogenesis of pyometra. However, this issue
requires further studies.
676
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