GLUTATHIONE DEPENDENT ENZYME ACTIVITIES IN THE FOOT OF THREE FRESHWATER MUSSEL SPECIES IN THE SAVA RIVER, SERBIA

We investigated activities of glutathione peroxidase (GSH-Px), glutathione reductase (GR), and the phase II biotransformation enzyme glutathione-S-transferase (GST) in the foot of three freshwater mussel species: Unio pictorum (Up), Unio tumidus (Ut), and Sinanodonta woodiana (Sw) from the Sava River. Specific and total GSH-Px activity was lower in Sw than in Up and Ut. Total GR activity was higher in Up than in Sw. Specific GST activity was higher in Up than in Ut. Total GST activity was higher in Up than in Ut and Sw. Electrophoretic analysis of proteins shows species specifities between the investigated mussel species. Our study represents the first comprehensive report of the investigated glutathione-dependent enzyme activities in the foot of three freshwater mussel species from the Sava River, Serbia.


INTRODUCTION
Activity of the antioxidant defense system can be increased or inhibited under chemical stress and antioxidant parameters therefore represent biomarkers of interest (Doyotteet al., 1997).Studies on aquatic species have been carried out mainly on marine species (Livingstoneet al., 1990;Di Giulioet al., 1993) and little is known about the response of the antioxidant defense system to pollutants in freshwater species.Bivalve mollusks are commonly used as bioindicators in the assessment of environmental quality because they are widely distributed, live in direct contact with the substrate, and have a high capacity for bioaccumulation.A number of antioxidant defense mechanisms are present in bivalve mollusks, including low-molecular-weight compounds and specially adapted enzymes (Winston, 1991).Use of the biotransformation enzymes involved in cellular detoxification as biomarkers of exposure to xenobiotics has been intensively studied in mollusks (Pe-terset al., 1999;Moreiraet al., 2001).
Glutathione peroxidase (GSH-Px), glutathione reductase (GR), and glutathione-S-transferase (GST) are glutathione-dependent enzymes because they use glutathione (GSH) as a cofactor.The first two enzymes are antioxidant defense enzymes, while GST is a phase II biotransformation enzyme.The ubiquitous tripeptide GSH (L-γ-glutamyl-cysteinyl-glycine), the most abundant soluble cellular thiol, is involved in processes essential for synthesis and degradation of proteins, formation of deoxyribonucleotides, regulation of enzymes, and protection of cells against reactive oxygen species (Meisterand Anderson, 1983).Glutathione exists in two forms: reduced glutathione (GSH) and oxidized glutathione (GSSG).The former is present usually in high concentrations in tissues and is one of the most important endogenous antioxidants.On the other hand, GSSG is toxic and can be used as an indication of oxidative stress (Parris and Kidd, 1997).
Glutathione peroxidase binds GSH with high affinity and oxidizes it to GSSG.This enzyme can detoxify hydrogen peroxide and organic hydroperoxides which are formed during tissue oxidative stress.Glutathione reductase reduces GSSG and is thus at the base of the regeneration of GSH necessary to the operation of GSH-Px, GST, and many other enzymes of the cell (Winston and Di Giulio, 1991).The phase II biotransformation enzyme GST catalyzes the conjugation of GSH to a wide variety of xenobiotics with an electrophilic site, yielding xenobiotics more water soluble and facilitating their excretion (Mannervikand Danielson, 1988).
The aim of our study was to determine activites of the glutathione dependent enzymes GSH-Px (EC 1.11.1.9),GR (EC 1.6.4.2), and the phase II biotransformation enzyme GST (EC 2.5.1.18)in the foot of three freshwater mussel species: Unio pictorum (Painter's mussel), Unio tumidus (swollen river mussel),and Sinanodonta woodiana (Chinese pond mussel) from the Sava River near the city of Šabac.The first two species are native to Serbian freshwaters, but S. woodiana is an invasive species and originates from Eastern Asia (Paunović et al., 2006).We also measured the concentration of total proteins and considered the gross patternof mussel proteins using electrophoresis.
The homogenates were sonicated for 30 s at 10 kHz on ice to release enzymes and then were centrifuged at 4°C at 100000 x g for 90 min (Takadaet al. 1982).The resulting supernatants were used for biochemical analyses.

Biochemical analyses
Activities of glutathione-dependent enzymes were measured simultaneously in triplicate for each mussel using a Shimadzu UV-160 spectrophotometer and a temperature-controlled cuvette holder.The activity of GSH-Px was determined following oxidation of nicotinamide adenine dinucleotide phosphate (NADPH) as a substrate with t-butyl hydroperoxide (Tamuraet al., 1982) and expresed in nmol of NADPH/min/mg of protein, as well in nmol of NADPH/min/g of wet mass.Glutathione reductase activity was measured as described by Glatzleet al. (1974) and expresed in nmol of NADPH/min/mg of protein and in nmol of NADPH/min/g of wet mass.Activity of GST toward 1chloro-2,4-dinitrobenzene (CDNB) was assayed by the method of Habiget al. (1974) and expresed as nmol of GSH/min/mg of protein and as nmol of GSH/min/g of wet mass.The activities of glutathione-dependent enzymes were expressed as specific (U/mg of protein) and total (U/g of wet mass) activities as described by Barja De Quiroga et al. (1988).Total protein concentration in the supernatant was determined according to the method of Lowryet al. (1951) and expressed in Fig. 1.Site of specimen collection from the Sava River near the city of Šabac (44° 46' 17. 2'' N and 19° 42' 16. 1'' E at an altitude 70 m above sealevel).mg/mL.Protein electrophoretic profiles were examined by the standard method of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), (Laemmli, 1970).All chemicals were products of Sigma-Aldrich (St. Louis, MO, USA).

Statistical analyses
Data are given as the mean ± SE (standard error).Statistical significance between the species was analyzed using the unpaired Student t-test considering at a level of p<0.05 to be significant (Hoel, 1966).

RESULTS
Total protein concentration of the mussels is shown in Table 1.The obtained results demonstrate significantly lower total protein concentration in S. woodiana compared to the other two species, U. pictorum and U. tumidus (p<0.005).Data on glutathione dependent antioxidant enzyme activities are given in Figs.2-4.Specific and total GSH-Px activity was significantly lower in S. woodiana than in U. pictorum and U. tumidus (p<0.005)(Figs.2A and 2B).As presented in Fig. 3B, total GR activity was considerably higher in U. pictorum than in S. woodiana (p<0.005).Specific GST activity was significantly higher in U. pictorum than in U. tumidus (p<0.005) (Fig. 4A).Also, total GST activity was considerably higher in U. pictorum than in U. tumidus and S. woodiana (p<0.005) (Fig. 4B).Electrophoretic analysis of proteins shows species specifities between the investigated freshwater mussel species (Fig. 5).

DISCUSSION
Different animals in aquatic ecosystems have developed various ways of protection from changing environmental conditions and pollution (Adamset al., 2000).Useful as indicators in biomonitoring investigations are the activities of antioxidant defense enzymes and biotransformation enzymes.Activity of the antioxidant defense system can be increased or inhibited under chemical stress.These two kinds of response depend on the intensity and duration of the stress applied and on susceptibility of the exposed living species.Induction of the antioxidant defense system can be considered an adaptation of species to their environment, whereas inhibition may lead to antioxidant-mediated toxicities (Winstonand Di Giulio, 1991).The responses in mussels make  them good bioindicators for environmental monitoring (Livingstone, 1993).A great number of biomarker studies have been performed on bivalves for the following reasons: their wide distribution, direct contact with the substrate, great tolerance to a huge variety of environmental conditions, and significant bioconcentration of environmental toxicants due to high filtration activity.
The results obtained in our study show that total and specific GSH-Px activities were significantly lower in S. woodiana compared to the other investigated freshwater species, U. pictorum and U. tumidus.Cossuet al. (2000) observed the antioxidant defense system in the freshwater bivalve U. tumidus transplanted from a control site to four different contaminated areas and reported that the most susceptible antioxidant parameters were se-lenium-dependent GSH-Px (Se GSH-Px) and GR activities, which decreased.Decline of those parameters indicates that the mussels were under oxidative stress as a result of exposure to prooxidant chemicals.Géretet al. ( 2002) also detected a reduction in the levels of SeGSH-Px and total GSH-Px activities in Ruditapes decussates exposed to copper after one day of exposure.The enzyme SeGSH-Px is considered an efficient protective agent against lipid peroxidation (Winstonand Di Giulio, 1991).On the other hand, in some studies where the induction of SeGSH-Px was recorded, this increase was not sufficient to prevent oxidative damage (Di Giulioet al., 1993). Jinget al. (2006) maintained that GSH-Px may be potentially useful as a biomarker in biotesting of metalpollution.

Up Ut Sw
In our experiments, we found that total GR activity was higher in U. pictorum than in S. woodiana.Glutathione reductase has a crucial role in cellular antioxidant protection because of its ability to regenerate GSH.Previous reports indicated that GR activity decreased during exposure of the bivalve Anadara granosa to mercury (Patelet al., 1990). Doyotteet al. (1997) measured GR activity in the gills and digestive gland of U. tumidus after exposure to copper and/or thiram and observed a reduction of its activity.The same authors also found that GR activity is a more sensitive parameter than activity of SeGSH-Px.Specific GST activity was significantly higher in U. pictorum than in U. tumidus, while total GST activity was considerably higher in U. pictorum than in U. tumidus and S. woodiana.As a phase II biotransformation enzyme, GST has been used as a biomarker of organic industrial effluents (Sheehanet al., 1995).In addition, GST has been used as a biomarker of exposure to anthropogenic organics (Fitzpatricket al., 1997). Di Giulioet al. (1993) reported induction of some GST isoenzymes by substrates such as polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenols (PCBs).Lenartovaet al. (1997) also reported induction of GST, but after exposure to metals.This enzyme is the most sensitive biomarker of the influence of environmental pollution on the organism and its activity has been shown to increase in the whole organism or particular organs (gills, digestive gland) as a function of the xenobiotic concentration (Stienet al., 1998).Some authors found that the activity of GST in mussels was significantly higher in winter, which is in accordance with higher sensitivity to oxidative stress during this period (Van der Oostet al., 2003;Borković et al., 2005).
Total protein concentration of the mussels was significantly lower in S. woodiana than in U. pictorum and U. tumidus.Electrophoretic analysis of proteins also shows species specifities between the investigated freshwater mussel species.
In conclusion, our present study represents the first comprehensive report on activities of the investigated glutathione-dependent enzymes (GSH-Px, GR, and the phase II biotransformation enzyme GST) in the foot of three freshwater mussel species (Unio pictorum,Unio tumidus and Sinanodonta woodiana) from the Sava River.It is important to note that the presented experiments were the first ones ever performed on the non-indigenous species Sinanodonta woodiana from the Serbian part of the Sava River.The parameters used in our work can be useful biomarkers for estimation of the effects of environmental contamination on freshwater invertebrates.