Available online at www.sciencedirect.com
Comparative Biochemistry and Physiology, Part C 147 (2008) 122 – 128
www.elsevier.com/locate/cbpc
Antioxidant defence enzyme activities in hepatopancreas, gills and muscle of
Spiny cheek crayfish (Orconectes limosus) from the River Danube☆
Slavica S. Borković a , Sladjan Z. Pavlović a , Tijana B. Kovačević a , Andraš Š. Štajn b ,
Vojislav M. Petrović a , Zorica S. Saičić a,⁎
a
Department of Physiology, Institute for Biological Research “Siniša Stanković”, University of Belgrade, Bulevar despota Stefana 142, 11060 Belgrade, Serbia
b
Institute of Biology and Ecology, Faculty of Sciences, University of Kragujevac, Radoja Domanovića 12, 34000 Kragujevac, Serbia
Received 23 May 2007; received in revised form 20 August 2007; accepted 22 August 2007
Available online 28 August 2007
Abstract
The aim of our study was to determine the activity of antioxidant defence (AD) enzymes: superoxide dismutase (SOD), catalase (CAT),
glutathione peroxidase (GSH-Px), glutathione reductase (GR) and the phase II biotransformation enzyme glutathione-S-transferase (GST) in the
hepatopancreas, the gills and muscle of Spiny cheek crayfish (Orconectes limosus) from the River Danube and to compare tissue specificities of
investigated enzymes. Our results indicated that both specific and total SOD activities in the hepatopancreas were lower compared to the gills and
muscle. Total SOD activity in the gills was lower with respect to that in muscle. CAT and GSH-Px (both specific and total) activities were higher in
the hepatopancreas compared to those in the gills and muscle. In the gills the specific and total GR activities were higher than in the
hepatopancreas and muscle. The specific and total GST activities were higher in the hepatopancreas compared with the gills and muscle. Our study
represents the first comprehensive report of AD enzymes in tissues of O. limosus caught in the River Danube. The noted tissue distributions of the
investigated AD enzyme activities most likely reflected different metabolic activities and different responses to environmental conditions in the
examined tissues.
© 2007 Elsevier Inc. All rights reserved.
Keywords: Antioxidant defence enzymes; Biomonitoring; Oxidative stress; River Danube; Spiny cheek crayfish
1. Introduction
Aquatic life is constantly exposed to chemical contamination
by an increasing variety of anthropogenic activities that can
induce many different mechanisms of toxicity, each contributing to varying degrees to the final overall deleterious effect
(Correia et al., 2003). The main antioxidant defence (AD)
enzymes in all organisms are superoxide dismutase (SOD),
catalase (CAT), glutathione peroxidase (GSH-Px), glutathione
reductase (GR) and the phase II biotransformation enzyme
glutathione-S-transferase (GST). These enzymes can be induced
by reactive oxygen species (ROS) and therefore they may
☆
This article is dedicated to the memory of the Academician Prof. Dr Vojislav
M. Petrović.
⁎ Corresponding author. Tel.: +381 11 2078 325; fax: +381 11 2761 433.
E-mail address: zorica.saicic@ibiss.bg.ac.yu (Z.S. Saičić).
1532-0456/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpc.2007.08.006
represent indicators of oxidative stress (Pavlović et al., 2004).
The induction of AD enzymes may provide sensitive earlywarning signals of incipient oxidative stress conditions.
AD enzymes play a crucial role in maintaining cellular
homeostasis. AD enzymes, such as SOD, CAT, GSH-Px and
GR act by detoxifying the ROS generated. Furthermore, the
phase II detoxifying enzyme GST produces a glutathione (GSH)
conjugate that appears to be the first step in the detoxification of
many toxins (Sureda et al., 2006). The enzymes mentioned
above have been proposed as biomarkers of contaminantmediated oxidative stress in a variety of marine and freshwater
organisms and their induction reflects a specific response to
pollutants (Borković et al., 2005; Cossu et al., 1997). Previous
reports have considered GSH-dependent enzymes and other AD
enzymes as markers for oxidative stress in fish (Hasspieler et al.,
1994; Pavlović et al., 2004). AD enzymes can be induced by
various environmental pro-oxidant conditions (those that induce
ROS generation, for example exposure to various types of
S.S. Borković et al. / Comparative Biochemistry and Physiology, Part C 147 (2008) 122–128
pollution). AD enzymes may also be regulated by other
endogenous/exogenous factors such as age (Arun and Subramanian, 1998), diet (Peters et al., 1994), seasonality/
reproductive cycle (Ringwood and Conners, 2000), temperature
variation (Abele et al., 1998) and hypoxia/hyperoxia (AbeleOeschger and Oeschger, 1995).
Crustaceans are frequently used as bioindicators in various
aquatic systems (Brouwer et al., 1997). They are widely
distributed in a number of different habitats including marine,
terrestrial and freshwater environments. Therefore, they are
suitable candidates for comparative studies. Some of the special
features of crustaceans, particularly their reproductive strategies, may be significant for the interpretation of data from
bioindicator studies and for the development of ecotoxicological endpoints.
The Spiny cheek crayfish (Orconectes limosus Rafinesque,
1817) is the native species in the Eastern part of the USA.
During the last century it was introduced into Europe and it has
been found in more than twenty European countries (Lodge
et al., 2000). We detected this species for the first time in the
Serbian part of the River Danube (Pavlović et al., 2006).
In this study the activities of superoxide dismutase (SOD, EC
1.15.1.1), catalase (CAT, EC 1.11.1.6), glutathione peroxidase
(GSH-Px, EC 1.11.1.9), glutathione reductase (GR, EC 1.6.4.2)
and the phase II biotransformation enzyme glutathione-Stransferase (GST, EC 2.5.1.18) were investigated in the
hepatopancreas, the gills and the muscle of Spiny cheek crayfish
(O. limosus) from the River Danube.
The main goal of our study was to establish and compare
tissue specificities between investigated AD enzymes and to
detect the variables that significantly contributed to differences
between examined tissues.
123
10 mmol/L Tris–HCl, pH 7.5 at 4 °C using an IKA-Werk
Ultra-Turrax homogenizer (Janke and Kunkel, Staufen, Germany) (Rossi et al., 1983). The homogenates were sonicated for
30 s at 10 kHz on ice to release enzymes (Takada et al., 1982)
and then centrifuged in a Beckman ultracentrifuge (90 min,
85,000 ×g, 4 °C). The resulting supernatants were used for
further biochemical analyses.
2.3. Biochemical analyses
All AD enzyme activities were measured simultaneously in
triplicate for each crayfish using a Shimadzu UV-160
spectrophotometer and a temperature controlled cuvette holder.
The activity of SOD was assayed by the epinephrine method
(Misra and Fridovich, 1972). One unit of SOD activity was
defined as the amount of protein causing 50% inhibition of the
autoxidation of adrenaline at 26 °C (Petrović et al., 1982) and
was expressed as specific activity (U/mg protein) and as total
(U/g wet mass). CAT activity was evaluated by the rate of
hydrogen peroxide (H2O2) decomposition (Clairborne, 1984).
The method is based on H2O2 degradation by the action of CAT
contained in the examined samples. In this procedure 50 mM
phosphate buffer (pH 7.0) was used and 30 mM H2O2 as
substrate. CAT activity was expressed as μmol H2O2/min/mg
2. Materials and methods
2.1. Site description and sample collection
Spiny cheek crayfish (O. limosus, Rafinesque) were caught
in the Serbian part of the River Danube, near the city of
Smederevo (in August, 2004, 1112 km from the water source,
44°41′31.6″ N, 20°57′38.5″ E) (Fig. 1).
The crayfish were collected using deep nets and by hand
from their natural habitats. We collected 10 specimens (both
sexes, 3 males and 7 females). The average length of the O.
limosus specimens were 10.76 ± 0.26 cm and their masses
averaged 43.10 ± 4.02 g. Using the age scale according to
Jarvekulg (1958), Spiny cheek crayfish were aged between 3
and 4 years. At the time of collection, the water temperature was
19 °C and the atmospheric temperature 25 °C. After collection,
the tissue samples (hepatopancreas, gills and abdominal
muscle) were immediately dissected on ice and then frozen in
liquid nitrogen before storage at − 80 °C.
2.2. Tissue processing
The tissues were minced and homogenized in 5 volumes
(Lionetto et al., 2003) of 25 mmol/L sucrose containing
Fig. 1. The geographical position of specimen collection on the River Danube.
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S.S. Borković et al. / Comparative Biochemistry and Physiology, Part C 147 (2008) 122–128
protein and as μmol H2O2/min/g wet mass. The activity of
GSH-Px was determined following the oxidation of nicotinamide adenine dinucleotide phosphate (NADPH) with t-butyl
hydroperoxide as a substrate (Tamura et al., 1982). This reaction
is proceeded by the action of GSH-Px contained in the samples
examined on t-butyl hydroperoxide (3 mM) as substrate in
0.5 M phosphate buffer, pH 7.0, at 37 °C. The activity of GSHPx was expressed as nmol NADPH/min/mg protein and as nmol
NADPH/min/g wet tissue. GR activity was measured using the
method of Glatzle et al. (1974). The method is based on the
capability of GR to catalyze the reduction of oxidized
glutathione (GSSG) to reduce glutathione (GSH) using
NADPH as substrate in the phosphate buffer (pH 7.4). GR
activity was expressed as nmol NADPH/min/mg protein and as
nmol NADPH/min/g wet tissue. The activity of GST towards 1chloro-2,4-dinitrobenzene (CDNB) was determined by the
method of Habig et al. (1974). The method is based on the
reaction of CDNB with the -SH group of GSH catalyzed by
GST contained in the samples. The reaction proceeded in the
presence of 1 mM GSH in phosphate buffer (pH 6.5) at 37 °C.
GST activity was expressed as nmol GSH/min/mg protein and
as nmol GSH/min/g wet tissue. All chemicals were products of
Sigma-Aldrich (St. Louis, MO, USA).
All AD enzyme activities were expressed as specific (U/mg
of protein) and as total activities (U/g wet mass) as described
previously by De Quiroga et al. (1988).
Total protein concentration was determined according to the
method of Lowry et al. (1951) using bovine serum albumin as a
reference.
significantly higher in the hepatopancreas compared to that in
both the gills and the muscle (P b 0.05). A statistical difference
between the total CAT activity in the gills and the muscle was
also found (P b 0.05).
A similar trend was obtained for both the specific (Fig. 3A) and
the total GSH-Px activity (Fig. 3B). The specific activity of GSHPx was significantly higher in the hepatopancreas when compared
to that in both the gills (P b 0.05) and the muscle (P b 0.05). A
statistical difference between specific GSH-Px activity in the gills
and the muscle was also found (P b 0.05). Total GSH-Px activity
was significantly greater in the hepatopancreas when compared to
that in both the gills and the muscle (P b 0.05).
Specific GR activity (Fig. 3A) was significantly higher in the
gills than in both the hepatopancreas and the muscle (P b 0.05).
A statistical difference between specific GR activity in the gills
and the muscle was also found (P b 0.05). Total GR activity
2.4. Statistical analyses
The data are expressed as mean ± Standard Error (S.E.). The
non-parametric Mann–Whitney U-test was used to seek
significant differences between means. A minimum significance level of P b 0.05 was accepted. Additionally, a discriminant function analysis was run to detect the variables that
significantly contribute to differences in the activities of AD
enzyme in the examined tissues. Analytical protocols described
by Darlington et al. (1973) and Dinneen and Blakesley (1973)
were followed.
3. Results
The specific and total activities of the investigated AD
enzymes in the hepatopancreas, the gills and the muscle of O.
limosus from the River Danube are illustrated in Figs. 2A and B
and 3A and B. Specific (Fig. 2A) and total (Fig. 2B) SOD
activities in the hepatopancreas were significantly lower
compared to those in the gills (P b 0.05) and the muscle
(P b 0.05). In addition, total SOD activity in the gills was
significantly lower than in the muscle (P b 0.05).
Specific CAT activity (Fig. 2A) was significantly higher in
the hepatopancreas when compared to that in both the gills
(P b 0.05) and the muscle (P b 0.05). A statistical difference
between the specific CAT activity in the gills and the muscle
was also found (P b 0.05). Total CAT activity (Fig. 2B) was
Fig. 2. Specific (A) (U/mg protein) and total (B) (U/g wet mass) activities of
superoxide dismutase (SOD) and catalase (CAT) in the hepatopancreas (H), in
the gills (G) and in the muscle (M) of Spiny cheek crayfish (Orconectes limosus)
from the River Danube. The data are expressed as mean ± S.E. The nonparametric Mann–Whitney U-test was used to seek significant differences
between means. A minimum significance level of P b 0.05 was accepted.
S.S. Borković et al. / Comparative Biochemistry and Physiology, Part C 147 (2008) 122–128
125
roots, the first 2 roots explained 89.31% of the overall variance.
SOD and GR were the main factors that contributed to root 1
(the greatest contribution being the gills), while GSH-Px was
the main variable that contributed to root 2 (the greatest
contribution being the hepatopancreas).
Discriminant function analysis of total AD enzyme activities
in the hepatopancreas (A), in the gills (B) and the muscle (C) of
O. limosus is illustrated in Fig. 4B. Discriminant function
analysis yielded 3 roots, the first 2 roots explained 98.53% of
the overall variance. SOD in the liver and GR in the gills were
the main factors that contributed to root 1, while CAT in the
liver and GR in the gills were the main variables that contributed to root 2. Post-hoc statistical significance (via the
Fig. 3. Specific (A) (U/mg protein) and total (B) (U/g wet mass) activities of
glutathione peroxidase (GSH-Px), glutathione reductase (GR) and phase II
biotransformation enzyme glutathione-S-transferase (GST) in the hepatopancreas (H), in the gills (G) and in the muscle (M) of Spiny cheek crayfish
(Orconectes limosus) from the River Danube. The data are expressed as mean ±
S.E. The non-parametric Mann–Whitney U-test was seen used to seek
significant differences between means. A minimum significance level of
P b 0.05 was accepted.
(Fig. 3B) was also significantly higher in the gills compared to
that in both the hepatopancreas (P b 0.05) and the muscle
(P b 0.05).
Specific and total phase II biotransformation enzyme GST
activities (Fig. 3A and B) were significantly higher in the
hepatopancreas when compared to those in both the gills and the
muscle (P b 0.05). In addition, specific and total GST activities
were significantly higher in the gills compared to those in the
muscle (P b 0.05).
Fig. 4A illustrates discriminant function analysis of specific
AD enzyme activities in the hepatopancreas (A), in the gills
(B) and in the muscle (C) of O. limosus (the tissues belong to
independent groups). Discriminant function analysis yielded 3
Fig. 4. Discriminant function analysis of AD enzyme activities in the
hepatopancreas (H), in the gills (G) and in muscle (M) of the Spiny cheek
crayfish (Orconectes limosus) expressed as specific (U/mg protein) (A) and as
total (U/g wet mass) (B). Groups were formed by root 1 (x axis) and root
2 (y axis). Statistical significance (the Mann–Whitney U-test) was not observed
between the gills and the muscle when activities were expressed as total. The
main contributions were observed for GSH-Px (P = 0.63) and GR (P = 0.075)
activities.
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Table 1
Standardized coefficients for canonical variables
Variable
SOD
CAT
GSH-Px
GR
GST
Eigenvalue
Accum. prop.
U/mg protein
U/g wet mass
Root 1
Root 2
Root 1
Root 2
0.66426
−0.50527
−0.28674
0.56229
−0.73797
29.08552
0.89306
− 0.272806
− 0.167069
0.219863
− 0.936818
− 0.534738
3.482852
1.000000
0.40250
− 0.75387
− 0.14156
0.31230
− 0.97530
42.20336
0.98527
− 0.678085
0.295131
− 0.623009
0.591255
0.073334
0.630967
1.000000
SOD—superoxide dismutase; CAT—catalase; GSH-Px—glutathione peroxidase; GR—glutathione reductase; phase II biotransformation enzyme GST—
glutathione-S-transferase; Accum. prop. = accumulated proportion.
Mann–Whitney U-test) was not observed (for the total enzyme
activities) between the gills and the muscle.
The main non-significant contributions were observed for
GSH-Px (P = 0.63) and GR (P = 0.075) activities.
Standardized coefficients for the canonical variables are
presented in Table 1.
4. Discussion
Our experiments were designed and executed to develop the
general knowledge concerning the main AD enzymes in
freshwater crayfish with a view to use them as potential future
biomarkers of river pollution.
A number of previous studies have considered ROS
generation, AD enzymes and free radical scavenger responses
and oxidative damage in molluscs (Labieniec and Gabryelak,
2007; Almeida et al., 2005; Livingstone, 2001). In addition,
some studies have reported the activity of different enzymes in
crustacean species (Daphnia magna) with respect to the
environmental pollutants (Jemec et al., 2007). However, only
a few reports have focused on other crustacean species and to
our knowledge no previous data about AD enzyme activities in
Spiny cheek crayfish have been published. As a consequence,
little is known about AD enzymes, particularly in amphipods.
The latter is of extreme interest as such organisms exhibit a
number of characteristics, such as their sensitivity to environmental disturbance, their short life cycle, and their amenability
to experimental investigation making them ideal for ecotoxicological studies (Costa and Costa, 2000).
Spiny cheek crayfish (O. limosus), an epibenthic amphipod
living above the sediment surface, possesses the key AD
enzyme activities (SOD, CAT, GSH-Px, GR and GST), similar
to a wide variety of other aquatic invertebrate species living
under aerobic conditions (including echinoderms, molluscs,
annelids and other crustaceans). A variety of contaminants
enters the aquatic environment and is taken up via sediment, the
water-column and food into the tissues of resident organisms
(Livingstone et al., 1994; Van Veld, 1990). Some studies (Pan
and Zhang, 2006) have demonstrated that the AD enzymes in
the hepatopancreas and gills of marine crab (Charybdis
japonica) can change their activities in the presence of some
toxins. Such toxins include redox-cycling compounds, polycy-
clic aromatic compounds (PAHs) (benzene and PAH oxidation
products), halogenated hydrocarbons (bromobenzene, dibromomethane and polychlorobiphenyls), lindane, dioxins, pentachlorophenol and a variety of metals (Al, As, Cd, Cr, Hg, Ni and
Va). The same general scenario of contaminant-stimulated
oxidative damage as seen in mammals targets aquatic organisms, although much less is known, in the latter, particularly
with regard to in vivo consequences and the relationship
between oxidative damage and disease (Livingstone, 2001;
Livingstone et al., 2001). Studies in fish and aquatic
invertebrates have largely been performed using gills as the
major organs of biotransformation and respiration. The
biotransformation organs include the liver of fish, the pyloric
caeca of echinoderms, the hepatopancreas of crustaceans and
the digestive gland of molluscs. Such studies have been the
subject of a number of review articles (Kelly et al., 1998;
Livingstone, 2001; Livingstone et al., 2001; Winston and Di
Giulio, 1991).
SOD is the enzyme that detoxifies toxic superoxide anion
radicals. In eukaryotes, it exists in three different forms:
manganese containing superoxide dismutase—Mn SOD (in
mitochondria), copper zinc containing superoxide dismutase—
CuZn SOD (in cytosol), total superoxide dismutase—Tot SOD
(Mn SOD and CuZn SOD) and extracellular superoxide
dismutase—Ec SOD. SOD activity was detected in the
supernatants prepared from homogenates of the hepatopancreas,
the gills and the muscle from O. limosus. This cytosolic SOD
activity could not be inhibited by cyanide or hydrogen peroxide
indicating that the SOD activity detected did not correspond to
CuZn SOD (Brouwer et al., 1997). All organisms that use Cu
for oxygen transport may have developed an AD system that is
not Cu-dependent. However, two observations argue against
this hypothesis: the hepatopancreas of molluscs (which are
dependent on Cu-hemocyanin for oxygen transport) contains
cytosolic CuZn SOD and a recently identified high molecular
mass (130 kDa) extracellular CuZn SOD in blue crab
hemolymph (Brouwer et al., 1997). This suggests that the
lack of intracellular CuZn SOD in decapod crustaceans is not
due to hemocyanin synthesis. Brouwer et al. (1997) illustrated
that all oxygen-consuming eukaryotes had a cytosolic CuZn
SOD and that the concept of a Mn SOD residing exclusively in
the mitochondria was not applicable to a large group of
arthropods. Such organisms use two distinct forms of Mn SOD,
one in the cytosol and the other in the mitochondria (involved in
defence against potentially toxic superoxides). The functional
and regulatory properties of this novel system including its
evolutionary origin remain to be fully documented.
CAT an important AD enzyme, is a major primary
antioxidant defence component that works primarily to catalyze
the decomposition of H2O2 to H2O, sharing this function with
GSH-Px. In the presence of low H2O2 levels, organic peroxides
are the preferred substrate for GSH-Px, but at high H2O2
concentrations, they metabolised by CAT (Yu, 1994). It has
been proven that CAT plays a relatively more important role in
detoxifying invertebrates compared to vertebrates (Livingstone
et al., 1992). Specific and total CAT activities were significantly
higher in the hepatopancreas than in both the gills and in the
S.S. Borković et al. / Comparative Biochemistry and Physiology, Part C 147 (2008) 122–128
muscle. It is commonly assumed that any significant increase in
SOD must be accompanied by a comparable increase in CAT
and/or GSH-Px activities (Warner, 1994). At the same time, the
activities of GSH-Px were extremely low in prawn tissues (Arun
and Subramanian, 1998).
According to other reports, the response of this biomarker
was dependent not only on the tissue but also on the extent of
environmental pollution (Osman et al., 2007). The same trend
was obtained for both specific and total GSH-Px and GST
activities. The crustacean hepatopancreas is the site of multiple
oxidative reactions and may therefore be a site of substantial
free radical generation. In the gills of Spiny cheek crayfish
specific GR activity was significantly higher when compared
to both the hepatopancreas and the muscle. Total GR activity
was also significantly higher in the gills when compared to
both the hepatopancreas and the muscle. Our results suggest
that gills exhibit a low-threshold response to oxidative stress,
as the organ is the first tissue to come into contact with
potential water-borne contaminants. The gills' intracellular
metabolism must also be coordinated and primed in order to
provide a first line of AD.
Discriminant function analysis was used in order to detect
variables that significantly contributed to differences in AD
enzyme activities between the investigated tissues. A balanced
activity of antioxidative components is necessary for the
homeostasis of ROS and the redox state. This may be achieved
by the coordinated action of antioxidative components.
Therefore, changes in the activities of some antioxidative
component must be accompanied by correlative changes in
other AD enzymes (Pavlović et al., 2004). In the present study
we obtained differences in specific enzyme activities between
investigated tissues and in some degree overlapping in the total
enzyme activities between gills and muscle.
In conclusion, our present study represents the first
comprehensive report of AD enzyme activities in three tissues
(hepatopancreas, gills and abdominal muscle) of Spiny cheek
crayfish collected from the Serbian part of the River Danube.
The results demonstrated noticeable tissue-specific distributions
of the investigated enzyme activities. This was most likely a
reflection of different metabolic activities and different
responses to environmental conditions of the examined tissues.
Acknowledgments
This study was supported by the Ministry of Science of
Republic of Serbia, Grant No. 143035B. The authors are
thankful to Dr. Zoran Gačić for the helpful comments in the
statistical analysis and to Dr. David R. Jones for proofreading
the manuscript.
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