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Comparative Biochemistry and Physiol

The activity of antioxidant defence enzymes in the mussel

Mytilus galloprovincialis from the Adriatic Sea

Slavica S. Borkovic a, Jelena S. Saponjic a, Sladjan Z. Pavlovic a, Duxko P. Blagojevic a,

Slavixa M. Miloxevic a, Tijana B. Kova*evic a, Ratko M. Radoji*ic b, Mihajlo B. Spasic a,

Radoslav V. Zikic c, Zorica S. Sai*ic a,*

a Department of Physiology, Institute for Biological Research ‘‘Sinixa Stankovic’’, Bulevar despota Stefana 142, 11060 Belgrade, Serbia and Montenegrob Institute of Biochemistry and Physiology, Faculty of Biology, University of Belgrade, Studentski Trg 16, 11000 Belgrade, Serbia and Montenegroc Institute of Biology and Ecology, Faculty of Sciences, University of Kragujevac, Radoja Domanovica 12, Kragujevac, Serbia and Montenegro

Received 16 November 2004; received in revised form 2 August 2005; accepted 2 August 2005

Available online 15 September 2005

Abstract

The activity of the antioxidant defence enzymes 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-S-

transferase (GST, EC 2.5.1.18) in whole mussels (Mytilus galloprovincialis) were studied. The mussels were collected in winter and in spring

at two localities in the Adriatic Sea: Bar Port and Tivat Bay. Our results show that the activities of SOD, GSH-Px and GST were seasonally

dependent with higher activities in winter. GR activity was also higher in winter, but only in mussels from Bar Port. In mussels from Tivat

Bay, GR activity was lower in winter compared to spring. In addition, a decrease in CAT activity in mussels from Bar Port compared to those

from Tivat Bay was found. It can be concluded that seasonal variations should be incorporated into interpretation of biomonitoring studies in

mussels.

D 2005 Published by Elsevier Inc.

Keywords: Adriatic Sea; Superoxide dismutase; Catalase; Glutathione peroxidase; Glutathione reductase; Glutathione-S-transferase; Mussels; Mytilus

galloprovincialis

1. Introduction

All aerobic organisms during their respiratory activity

continuously produce reactive oxygen species (ROS) (Perez-

Campo et al., 1993; Halliwell and Gutteridge, 1999). The

important sites of ROS production are mitochondria

(Boveris and Cadenas, 1982; Chance et al., 1979; Turrens

et al., 1985), microsomes (Staats et al., 1988), peroxisomes

(Dhaunsi et al., 1992) and the cytosol (Shaw and Jayatilleke,

1990). ROS induce many cellular disturbances, such as

depolymerization of polysacharides and nucleic acids,

oxidation of protein sulphydryl groups and peroxidation of

1532-0456/$ - see front matter D 2005 Published by Elsevier Inc.

doi:10.1016/j.cbpc.2005.08.001

* Corresponding author. Tel.: +381 11 2078 325; fax: +381 11 2761 433.

E-mail address: zorica.saicic@ibiss.bg.ac.yu (Z.S. Sai*ic).

fatty acids (Stohs et al., 2000). According to the classical

concept of antioxidant defence, this system includes

enzymatic (superoxide dismutase—SOD, catalase—CAT,

glutathione peroxidase—GSH-Px, glutathione reductase—

GR and glutathione-S-transferase—GST) and nonenzymatic

components (Cadenas, 1989). This view may be modified

by the notion of complex antioxidant defence (Niki, 2000)

and/or the concept of gradual antioxidant defence (Van der

Oost et al., 2003). According to the Environmental Risk

Assessment (ERA), the components of antioxidant defence

are functionally divided into biotransformation phase II

components (for instance, GST and reduced/oxidized

glutathione) and oxidative stress parameters (SOD, CAT,

GSH-Px and GR) (Van der Oost et al., 2003).

Bivalve mollusks such as Mytilus galloprovincialis are

commonly used as bioindicators in ERA. These mussels

ogy, Part C 141 (2005) 366 – 374

S.S. Borkovic et al. / Comparative Biochemistry and Physiology, Part C 141 (2005) 366–374 367

are inter-tidal filter-feeding invertebrates known to accu-

mulate high levels of trace metals and organic com-

pounds in their tissues, providing a time-integrated

indication of environmental contamination with observ-

able cellular and physiological responses (Lau and Wong,

2003). The responses in mussels make them good

bioindicators for environmental monitoring (Livingstone,

1993). They have a number of properties which make

them useful sentinels for chemical pollution: they have a

wide geographical distribution, are easy to collect and are

abundant in estuarine waters, which are submitted to high

contamination levels (Manduzio et al., 2004). Moreover,

mussels are sedentary, euryhaline and normally the

dominant species in their habitats (Sheehan and Power,

1999).

As mussels are thermocomforming organisms, they must

deal with oscillations in the environmental temperature and

their metabolic rate and consequently with oscillations in the

levels of ROS (Wilhelm Filho et al., 1993). As a

consequence, ROS generation, oxidation rates and antiox-

idant status are most likely related to the ambient temper-

ature and the metabolic activity (Wilhelm Filho et al., 2000),

as we have previously shown for fish (Pavlovic et al., 2004)

and rodents (Spasic et al., 1993).

Fig. 1. The geographical position of Bar Po

An important factor influencing musselVs biochemistry

and physiology is seasonality. Depending on the availability

of nutrients, reproductive status, growth rate related with

season and other factors, the activity of antioxidant defence

enzymes and other biomarkers fluctuate significantly

throughout the year (Sheehan and Power, 1999). Seasonal

variations in antioxidant defences were observed in tissues

of horse mussels (Modiolus modiolus) (Lesser and Kruse,

2004), blue mussels (Mytilus edulis) (Manduzio et al., 2004)

and in the digestive gland of brown mussels (Perna perna)

(Wilhelm Filho et al., 2001).

It is also known that several classes of pollutants are

capable of enhancing the formation of ROS and thereby

provoke oxidative stress. Some of these pollutants include

polychlorinated biphenyls (PCBs), polycyclic aromatic

hydrocarbons (PAHs), phenols and heavy metals (Van der

Oost et al., 2003). Many studies have shown positive

correlations between levels of antioxidant defences and the

presence of xenobiotics (Orbea et al., 2002). Oxidative

stress is also a highly seasonal phenomenon in bivalve

mollusks (Lesser and Kruse, 2004). The oxidative stress

modulation by environmental pollutants is a factor which

might complicate the interpretation of biomonitoring studies

(Sheehan and Power, 1999).

rt and Trivat Bay in the Adriatic Sea.

Table 1

Values of salinity (�), temperature (-C) and oxygen concentration (mg/L)

at 0.5 m depth in Bar Port and Tivat Bay on the day of mussel collection

during winter and spring

Bar Port Tivat Bay

winter spring winter spring

Temperature (-C) 8.6 19.9 12.0 20.1

O2 (mg/L) 9.6 7.6 8.2 7.8

Salinity (�) 32.2 36.5 33.0 34.3

S.S. Borkovic et al. / Comparative Biochemistry and Physiology, Part C 141 (2005) 366–374368

The studied areas of Bar Port and Tivat Bay were

selected because both receive extensive industrial and

urban waste water discharges. These areas have similar

climates, and the lowest mean water temperature occurs in

February and highest in August. The mean water

temperature in February is 12.5 -C and in May is 20.4

-C. Tivat Bay is characterized by a higher inflow of

120

2

4

6

8

10

12

14

******

Winter

(A)

ANOVA comparison: (S): p<0.001 (L): N.S. SxL: N.S.

Spring

Bar PortTivat Bay

450

100

200

300

400

*** #

***

ANOVA comparison: (S): p<0.001(L): p<0.01SxL: N.S.

(B)

SpringWinter

Bar Port Tivat Bay

U/g

wet

mas

sU

/mg

prot

ein

Fig. 2. The specific (A) and total (B) activities of SOD in mussels from Bar

Port (n =20) and Tivat Bay (n =13) in winter and spring. Results are

expressed as meanTSE. The statistical significance was analyzed by two-

way ANOVA test with season (S) and locality (L) as factors. N.S.=not

significant. A minimal confidence interval of 5% ( p <0.05) was considered.

freshwater than Bar Port. The mean sea depth in Bar Port

is 10 m and Tivat bay is 25 m. The bottoms of the

biotopes are covered with thick stratum of fine terri-

genous mud containing particles of detritus. The sea

currents in both localities are very irregular, in the

summer they are slight, while in winter they are very

strong (Stjep*evic, 1974).

The aim of this study was to compare the activity of

antioxidant defence enzymes in mussels between two

different localities, one with intensive industrial pollution

(Bar Port), and second with intensive anthropogenic

pollution (Tivat Bay) in two seasons. The activity of

antioxidant defence enzymes, superoxide dismutase, cata-

lase, glutathione peroxidase and glutathione reductase and

the activity of glutathione-S-transferase was measured in

musselMytilus galloprovincialis collected from the Bar Port

and Tivat Bay from the Adriatic Sea in winter and late

spring.

890

2

4

6

8

10

12

*

#

(A)

ANOVA comparison: (S): p<0.05 (L): p<0.01 SxL: N.S.

SpringWinter

Bar Port Tivat Bay

11 12

µmol

H2O

2/m

in/g

wet

mas

s

50

100

150

200

250

300

#

#

ANOVA comparison: (S): N.S.(L): p<0.01SxL: N.S.

(B)

SpringWinter

Bar Port Tivat Bay

µmol

H2O

2/m

in/m

g pr

otei

n

0

Fig. 3. The specific (A) and total (B) activities of CAT in mussels from

Bar Port (n =20) and Tivat Bay (n =13) in winter and spring. The number

of samples and type of statistical analysis was identical to that indicated in

Fig. 2.

29 300

5

10

15

20

25

30

*

*

ANOVA comparison: (S): N.S.(L): N.S.SxL: p<0.05

SpringWinter

(A)

Bar Port Tivat Bay

600

700

800

ANOVA comparison: (S): N.S.(L): N.S.SxL: p<0.05

(B)

Bar Port Tivat Bay

t mas

snm

ol N

AD

PH/m

in/m

g pr

otei

n

S.S. Borkovic et al. / Comparative Biochemistry and Physiology, Part C 141 (2005) 366–374 369

2. Materials and methods

2.1. Site description

Mussels (Mytilus galloprovincialis) were collected in

winter (February) and late spring (May) at two localities:

Bar Port (42- 05V 58� N and 19- 05V 40� E) and Tivat Bay

(42- 24V 16� N and 18- 43V 23� E) (Fig. 1).

The two sites were chosen to compare the activity of

antioxidant defence enzymes between periods of lower

metabolic activity (winter) and higher metabolic activity

(spring). At the time of sampling environmental parameters

including water temperature, oxygen concentrations and

salinity were measured (Table 1).

2.2. Sample collection and preparation

At the Bar Port, 20 (10 in winter and 10 in spring)

specimens were collected, whereas in Tivat Bay 13 (7 in

winter and 6 in spring) specimens were collected. Mussels

from Bar Port were larger (4.5–5 cm shell length) than

15 160

2

4

6

8

10

12

******

ANOVA comparison: (S): p<0.001(L): N.S.SxL: N.S.

(A)

SpringWinter

Bar Port Tivat Bay

190

100

200

300

400

******

(B)

SpringWinter

ANOVA comparison: (S): p<0.001(L): N.S.SxL: N.S.

Bar Port Tivat Bay

nmol

NA

DPH

/min

/mg

prot

ein

nmol

NA

DPH

/min

/g w

et m

ass

Fig. 4. The specific (A) and total (B) activities of GSH-Px in mussels from

Bar Port (n =20) and Tivat Bay (n =13) in winter and spring.

32 330

100

200

300

400

500*

SpringWinter

nmol

NA

DPH

/min

/g w

e

Fig. 5. The specific (A) and total (B) activities of GR in mussels from Bar

Port (n =20) and Tivat Bay (n =13) in winter and spring.

those from Tivat Bay (2.5–3 cm). The latter has a higher

inflow of freshwater that enables higher organic production

and abundant appearance of young mussels (Stjep*evic,

1974). After collecting, mussels were frozen in liquid

nitrogen and then stored at �70 -C. In each group, total

soft tissue from the whole body was dissected from all

collected mussels. The tissues were ground and homogen-

ized in 5 vol. (Lionetto et al., 2003) of 25 mmol/L sucrose

containing 10 mmol/L Tris–HCl, pH 7.5 at 4 -C with an

Ultra-Turrax homogenizer (Janke and Kunkel, IKA-Werk,

Staufen, Germany) (Rossi et al., 1983). The homogenates

were sonicated for 30s at 10 kHz on ice to release enzymes

(Takada et al., 1982) and sonicates were then centrifuged at

4 -C at 100,000 g for 90 min. The resulting supernatants

were used for biochemical analyses.

2.3. Biochemical analyses

Total protein concentration in the supernatant was

determined according to the method of Lowry et al.

(1951) and expressed in mg/g wet mass. The activity of

Table 2

Pearson correlation coefficients between the specific activity of antioxidant

defence enzymes in the musselMytilus galloprovincialis in Bar Port (below

diagonal) and in Tivat Bay (above diagonal) during winter

SOD CAT GSH-Px GST GR

SOD 0.64 0.97 0.66 0.06

CAT 0.23 0.80 �0.16 �0.73

GSH-Px 0.49 �0.32 0.47 �0.17

GST 0.37 0.01 �0.01 0.79

GR 0.78* �0.38 0.66 0.22

(* p <0.05).

S.S. Borkovic et al. / Comparative Biochemistry and Physiology, Part C 141 (2005) 366–374370

antioxidant defence enzymes was measured simultaneously

in triplicate for each mussel 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), based on the capacity

of SOD to inhibit the autooxidation of epinephrine to

adrenochrome, and expressed as U/mg of protein, as well as

U/g wet mass. CAT activity was evaluated by the rate of

hydrogen peroxide (H2O2) decomposition and expressed as

Amol H2O2/min/mg protein, and as Amol H2O2/min/g wet

mass (Beutler, 1982). The activity of GSH-Px was

determined following the oxidation of nicotinamide adenine

dinucleotide phosphate (NADPH) as a substrate with t-butyl

hydroperoxide (Tamura et al., 1982) and expressed in nmol

NADPH/min/mg protein, as well as nmol NADPH/min/g

wet mass. The activity of GR was measured as described by

Glatzle et al. (1974), and expressed as nmol NADPH/min/

mg protein, as well as nmol NADPH/min/g wet mass. GST

activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was

determined by the method of Habig et al. (1974), and

expressed as nmol GSH/min/mg protein and as nmol GSH/

22 230

10

20

30

40

50

60

******

ANOVA comparison: (S): p<0.001(L): N.S.SxL: N.S.

(A)

SpringWinter

Bar Port Tivat Bay

25 260

200

400

600

800

1000

1200

1400

1600

******

ANOVA comparison: (S): p<0.001 (L): N.S. SxL: N.S.

SpringWinter

(B) Bar Port Tivat Bay

nmol

GSH

/min

/g w

et m

ass

nmol

GSH

/min

/mg

prot

ein

Fig. 6. The specific (A) and total (B) activities of GST in mussels from Bar

Port (n =20) and Tivat Bay (n =13) in winter and spring.

min/g wet mass. All chemicals were products of Sigma-

Aldrich (St. Louis, MO, USA).

Salinity, temperature, and oxygen concentration of the

sea water were measured with a WTW (Wissenschaftlich-

Technische Werkstatten, Weilheim, Germany) multilab

system.

2.4. Statistical analyses

Data are expressed as the MeanTSE (Standard Error).

Statistical significance and other statistical tests (descriptive

statistics, analyses of variance-ANOVA, correlation analy-

ses) were established by protocols described in Hinkle et al.

(1994) and Manley (1986).

3. Results

The protein concentration of the soft part of the mussels

from Bar Port in winter was 33.84T6.91 mg/g wet mass,

and in spring 36.74T14.26 mg/g wet mass. In mussels from

Tivat Bay the protein concentration in winter was

36.48T2.26 mg/g wet mass, and in spring 27.10T5.87mg/g wet mass. No statistical significance was found

between the protein concentration in the mussels from both

localities and from both seasons.

A two-way ANOVA test considering the effects of both

the season and locality showed that season had a statistically

significant influence on specific and total SOD activities

( p <0.001), (Fig. 2A and B). These activities were higher in

winter than in spring in both localities.

As presented in Fig. 3A and B, specific and total

activities of CAT were significantly lower in mussels from

Table 3

Pearson correlation coefficients between the total activity of antioxidant

defence enzymes in the musselMytilus galloprovincialis in Bar Port (below

diagonal) and in Tivat Bay (above diagonal) during winter

SOD CAT GSH-Px GST GR

SOD 0.37 0.93 0.38 �0.03

CAT �0.53 0.69 �0.72 �0.94

GSH-Px 0.01 �0.53 0.02 �0.39

GST �0.06 0.15 �0.05 0.91

GR 0.52 �0.93** 0.37 0.05

(** p <0.01).

Table 5

Pearson correlation coefficients between the total activity of antioxidant

defence enzymes in the musselMytilus galloprovincialis in Bar Port (below

diagonal) and in Tivat Bay (above diagonal) in spring

SOD CAT GSH-Px GST GR

SOD 0.09 0.17 �0.001 �0.04

CAT �0.30 0.45 0.59 0.15

GSH-Px 0.13 �0.01 0.80** 0.25

GST 0.32 �0.14 0.53 0.17

GR 0.06 �0.47 0.22 0.22

(** p <0.01).

S.S. Borkovic et al. / Comparative Biochemistry and Physiology, Part C 141 (2005) 366–374 371

Bar Port than from Tivat Bay in both seasons ( p <0.01). A

two-way ANOVA test considering the effects of both the

season and locality showed that the season had a statistically

significant influence only on the specific activity of CAT

( p <0.05), which was lower in winter than in spring in

mussels sampled at Tivat Bay.

The specific and total activities of GSH-Px (Fig. 4A and

B) were significantly higher in winter in both localities

( p <0.001). However, there were no significant differences

in the specific and total GSH-Px activities between Bar Port

and Tivat Bay in both seasons.

GR activity was higher in winter compared to spring in

mussels collected from Bar Port (Fig. 5A and B), while in

mussels from Tivat Bay, only the specific GR activity was

lower in winter compared to spring ( p <0.05).

As shown in Fig. 6A and B, the specific and total

activities of the phase II biotransformation enzyme GST

were significantly higher in winter than in spring in both

localities ( p <0.001), and no statistically significant differ-

ences between both localities were observed in both

seasons. Correlation analyses are statistical tools to explore

functional connections between antioxidant components on

a statistical base (Blagojevic et al., 1998; Pavlovic et al.,

2004; Jovanovic-Galovic et al., 2004; Speers-Roesch and

Ballantyne, 2005). Degree of correlation is expressed as

Pearson correlation coefficient (determines the extent to

which values of the two variables are ‘‘proportional’’ to each

other) and its statistical significance based on the assump-

tion that the distribution of the residual values (i.e., the

deviations from the regression line) for the dependent

variable follows the normal distribution, and that the

variability of the residual values is the same for all values

of the independent variable ( p <0.05 is considered as

significant). The results from such analyses from data

presented in this present paper are in Tables 2–5. We

concluded that there were no significant correlations in

winter in mussels from Tivat Bay, contrary to mussels from

Bar Port. In mussels from Bar Port collected during winter, a

positive correlation between SOD and GR and a negative

one between CAT and GST activities were detected. In

spring, several correlations were noticed in mussels from

Tivat Bay (positive between GST and GSH-Px, CAT and

GST, SOD and GR, as well as CAT and GST), and negative

between SOD and GR in mussels from Bar Port. This

Table 4

Pearson correlation coefficients between the specific activity of antioxidant

defence enzymes in the musselMytilus galloprovincialis in Bar Port (below

diagonal) and in Tivat Bay (above diagonal) in spring

SOD CAT GSH-Px GST GR

SOD 0.50 0.12 0.41 0.77**

CAT 0.38 0.51 0.68* 0.44

GSH-Px 0.12 0.47 0.71* 0.42

GST 0.61 0.73* 0.41 0.59

GR �0.71* 0.10 0.32 �0.19

(** p <0.01, * p <0.05).

suggests that basic homeostatic relationships between the

antioxidant defence enzymes in mussels from Bar Port were

different than those from Tivat Bay, depending on the period

they were collected.

4. Discussion

The seasonal pattern of antioxidant defence enzymes

found in M. galloprovincialis appears to be closely

correlated with the seasonal variations of temperature and

the reproductive cycle. The activity of antioxidant enzymes

and antioxidant compounds (vitamin E and reduced

glutathione) are known to be under extensive seasonal

control (Sheehan and Power, 1999). Lesser and Kruse

(2004) have shown that the mussel Modiolus modiolus

seasonally compensates for decreases in temperature by

increasing the concentration of rate-limiting metabolic

enzymes while maintaining the same level of antioxidant

protection in summer and winter. The increase in temper-

ature is followed by an increase in oxygen consumption

and by an increase in ROS generation. The second season-

related determining factor in the level of various antiox-

idants in mussels seems to be the metabolic particularity of

the stages of their reproductive cycle. The above results

and conclusions reinforce the importance of seasonality on

the antioxidant status of Bivalvia in relation to the

interpretation of biomonitoring data (Wilhelm Filho et

al., 2001). In our experiments both populations of mussels

expressed differences in the antioxidant defence enzymes

regarding winter and spring (data from correlation analysis,

Tables 2–5). Antioxidant defence enzymes are constitutive

in nature and susceptible to variations due to intrinsic

biological processes and cycles and ontogenic processes,

beside the extrinsic influences, such as environmental

changes and contaminants. The interpretation of these

responses, in an environmental context, is very complex to

account for all the possible causes (Sheehan and Power,

1999). When organisms are employed as monitoring

subjects, particularly for routine long-term monitoring

purposes and in areas with contaminants at sub-lethal

concentrations, the intrinsic biological variations such as

size, tissue specificity, and natural variations of the

biochemical responses, such as food availability and

S.S. Borkovic et al. / Comparative Biochemistry and Physiology, Part C 141 (2005) 366–374372

environmental changes become key factors to be consid-

ered (Lau and Wong, 2003).

It is important that besides physiological and environ-

mental factors, the contribution of ontogenetic development

is also a determining factor of antioxidant levels in mussels

(Rudneva, 1999). This is in accordance with the fact that

physiological functions in mussels are influenced by size

(Sukhotin et al., 2002). Studies regarding the effect of aging

and size on the activity of CAT in mussels have given rise to

different results, as this effect appears to be specific to

species, sex, tissue and age. Several authors found a decrease

in CAT activity in the course of ageing in the mussel Mytilus

edulis (Viarengo et al., 1991), the marine shrimp Aristeus

antennatus (Mourente and Diaz-Salvago, 1999), as well as in

cephalopods Sepia officinalis and Lolliguncula brevis

(Zielinski and Portner, 2000). In contrary, Sukhotin et al.

(2002) found a weight-specific decrease in CAT activity

within the separate age classes of M. edulis. This finding

illustrates that in those studies where age-related decrease of

CAT has been reported, it might be due to the increasing size

of the ageing mussels and not due to the effect of age per se.

Our investigations showed an increase in specific and total

activity of CAT in M. galloprovincialis collected during

May, which is probably linked to the increased metabolic

activity related to the seasonal temperature elevations and

intense reproductive activity that occurs in spring. Further-

more, in the Mediterranean Sea, the winter period is

characterized by gametogenesis, whereas spring is charac-

terized by intensive egg release. Summer is characterized by

the deposition of lipids and glucose, whereas the autumn by

the beginning of gametogenesis (Stjep*evic, 1974). This is in

accordance with the findings of other authors which

postulated that changes in the reproductive cycle are

connected with the season and the rate of metabolic activity

(Robledo et al., 1995).

The activities of SOD, GSH-Px and GST showed

elevated levels in winter. It was suggested that SOD and

GSH constituted the integral components of the cellular

antioxidant defence. Namely, reduced glutathione (GSH) is

a physiologically useful scavenger if the reaction with

superoxide is prevented (Munday and Winterbourn, 1989).

Elevated SOD activity was accompanied with elevated

GSH-Px activity and positive correlation with GR activity

suggests coordinated action toward possible hydrogen

peroxide induced oxidative attack. The study of Orbea et

al. (2002) showed a higher antioxidant defence enzyme

activities in summer. Seasonal factors might affect bio-

marker responses to a greater extent than pollution

variations. Apart from natural biological cycles, the annual

cycles of climatic conditions may also induce stress in the

organisms thereby triggering antioxidant defences. Cellular

rearrangements of metabolic components to compensate

environmental fluctuations have already been found (Lesser

and Kruse, 2004). The antioxidant defence system might

regulate its activity by rearrangements of components in

spite of changing its activity (Blagojevic et al., 1998). The

recomposition of antioxidant defence enzymes regarding

season was observed by correlation analyses in the present

work. Davenport (1983) stated that the asymmetrical pattern

of ctenidial ciliary activity and other physiological

responses depended on the water temperature and salinity.

The low temperature could reduce the metabolic activities,

and hence lower enzymatic activities in general. One

possible explanation for the higher antioxidant defence

enzyme activities in the winter samples might be the higher

sensitivity to stress induced by chemicals from the sea water

in the winter season (Power and Sheehan, 1996). Salinity is

another major physical factor affecting the physiological

responses such as inhalant and exhalant siphon closure, and

shell valve closure in mussels (Davenport, 1983). The

optimal salinity for the mussel M. galloprovincialis is in the

range of 32–37� (Renzoni, 1962). In our study, the salinity

in the seasons was within the optimal range (in winter 32–

33� and in spring 34–36.5�), which enabled mussels to

maintain normal filtration activity (Table 1). It is known that

the production of phytoplankton, which is the main energy

source for filtering organisms such as the mussel M.

galloprovincialis increases during spring. Reproductive

activity and temperature-associated changes in patterns of

food storage and utilization is also likely to cause changes in

the hormonal and nutritional status which might also affect

the levels of enzyme activity (Orbea et al., 1999).

GST, one of the phase II biotransformation enzyme

systems, has been used as a biomarker of organic industrial

effluents (Sheehan et al., 1995). In addition, GST has been

used as a biomarker of exposure to anthropogenic organics

(Fitzpatrick et al., 1997). Xenobiotic chemicals may be

biotransformed according to the simplified mechanism of

route I, which can be subdivided into phases I, II and III.

Phase I is a non-synthetic alteration (oxidation, reduction or

hydrolysis) of the original foreign molecule, which can then

be conjugated in phase II and catabolised in phase III (Van

der Oost et al., 2003). GST, a phase II biotransformation

enzyme catalyzes the initial step of mercapturic acid

synthesis and a conjugation of GSH with xenobiotics and

their metabolites such as the alkyl transferase and epoxide

transferase, detoxifying PAH epoxide produced by cyt P450

(George, 1994). This enzyme is also the most sensitive

biomarker for the influence of environmental pollution on

the organism. Our results show that the activity of GST was

significantly higher in winter which is in accordance with

higher sensitivity to oxidative stress in this period (Van der

Oost et al., 2003). At the same time, no statistically

significant differences were detected between the two

localities investigated in both seasons, indicating a similar

degree of pollution. Our finding of a positive correlation

between GST and GSH-Px activities could be explained as a

coordinated expression of total GST and its peroxidase-like

isoform. At the same time, there was significant positive

correlation between specific GST and CAT activities in both

localities, suggesting a similar pattern for hydrogen per-

oxide elimination.

S.S. Borkovic et al. / Comparative Biochemistry and Physiology, Part C 141 (2005) 366–374 373

Although many compounds possess antioxidant proper-

ties, cellular antioxidant defence is provided by functional

overlapping of antioxidant enzymes and correlation

between its activities. This means, if cellular antioxidant

defence represents a physiological system, then changes

in the activity of an individual antioxidant component

should be accompanied by subsequent changes in the

activity of others. This can be explored statistically by

correlation analysis, which determinates probability if the

activity of one component is correlated with another.

Comparative analyses of correlations between antioxidant

enzymes in different species, seasons and the environment

have been performed in our previous work (Blagojevic et

al., 1998; Pavlovic et al., 2004; Jovanovic-Galovic et al.,

2004). In the recent paper similar approach was used,

and several positive correlations between measured

antioxidant components in fish were observed (Speers-

Roesch and Ballantyne, 2005) suggesting coordinated

regulation of antioxidant enzymes. It seems that anti-

oxidant components undergo recomposition together with

the changes in its activity to respond different devel-

opmental, seasonal and environmental impact during life

cycle.

Our study represents the first comprehensive report of

antioxidant defence enzyme activities in the mussel M.

galloprovincialis collected from the Montenegrine coast-

line from the Adriatic Sea. The results obtained in this

work indicate a significant influence of seasonal factors on

specific and total activities of SOD, CAT, GSH-Px and

GST and a significant influence of location on CAT

activity. Therefore, it can be concluded that seasonal

factors should be incorporated into interpretation of

mussels-based biomonitoring studies. The relationship

between oxygen consumption and ROS generation also

seems to apply to the antioxidant status of thermocom-

formers, whose oxidative metabolism depends directly on

the environmental temperature, reproductive cycle and

seasonality (Wilhelm Filho et al., 2001). This functional

independence is important for the homeostasis of thermo-

comformers, especially regarding their oxidative metabo-

lism and should also be taken into account for the

interpretation of biomonitoring studies related to aquatic

contamination.

Acknowledgements

This work was funded by the Federal Government of

Serbia and Montenegro, Project title:‘‘Bioindicators of

contamination of the Montenegrine coastline’’, as well as

by the Ministry for Science, Environmental Protection of

Republic of Serbia. The map of the Montenegrine

coastline was kindly provided by the Institute of Marine

Biology, Kotor, Serbia and Montenegro. The authors are

thankful to Dr. David R. Jones for proofreading the

manuscript.

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