GLUTATHIONE AS А SUITABLE BIOMARKER IN HEPATOPANCREAS, GILLS AND MUSCLE OF THREE FRESHWATER CRAYFISH SPECIES

We determined the contents of total glutathione (tGSH), reduced glutathione (GSH), and oxidized glutathione (GSSG) and values of the glutathione redox index (GSH RI) in hepatopancreas, gills, and muscle of three freshwater crayfish species: noble crayfish (Astacus astacus) from the Southern Morava River, stone crayfish (Austropotamobius torrentium) from the Kra�kovačka River, and spinycheek crayfish (Orconectes limosus) from the Danube River. The obtained data show strong tissue and species specifity of investigated parameters: tGSH, GSH, GSSG, and GSH RI in the hepatopancreas, gills, and muscle of the indicated crayfish species. Our work represents the first study of its kind and showed that the investigated parameters can be considered suitable biomarkers of the cellular glutathione redox status in of freshwater crayfish species.


INTRODUCTION
Glutathione (GSH) is the most important water-soluble low-molecular antioxidant.It is widely distributed in all living organisms and is found largely inside cells, where it reaches relatively high concentrations (0.1-10 mM) (D e n e k e and F a n b u r g , 1989).In contrast, values of GSH content in the plasma are usually in the low micromolar range, but they may vary extensively.The given antioxidant accounts for 90% of intracellular nonprotein thiols and it is therefore the most important intracellular reducing agent.GSH is a three-peptide (�-�-glutamyl-�-three-peptide (�-�-glutamyl-�cysteinyl-glycine) present in the free or bound form in the cytosol, nucleus, and mitochondria (S e n et al., 1992).There are ).There are two forms: reduced glutathione (GSH) and its oxidized dimeric form, glutathione disulfide (GSSG).Mitochondria contain 15 to 20% of total cellular GSH, which is also present in the total cellular GSH, which is also present in the nucleus and endoplasmic reticulum (B r i b i v a et al., 1993; Vo e h r i n g e r et al., 1998).).
Numerous physiological functions have been physiological functions have been attributed to GSH: 1) scavenging of hydrogen per-oxide and other peroxides and free radicals; 2) pres-preservation of SH groups in a reduced state in proteins, of SH groups in a reduced state in proteins, enzymes, and some other molecules; 3) detoxification of foreign compounds by their con�ugation with foreign compounds by their con�ugation with compounds by their con�ugation with compounds by their con�ugation with their con�ugation with GSH; 4) acting as a co-enzyme for some enzymes (e.g., glyoxalase); 5) translocation of amino acids translocation of amino acids of amino acids across cell membranes; and 6) catalysis of disulfide catalysis of disulfide exchange reactions (M e i s t e r , 1985).In general, GSH plays a key role in cellular defenсe and serves as a reservoir for the amino acid cysteine.GSH depletion leads to cell death and has been documented in many degenerative conditions.Mitochondrial GSH depletion may be the ultimate factor determining vulnerability to oxidant attack.
Cellular GSH contents reflect a steady-state balance between its synthesis and loss.Generation of loss.Generation of GSH occurs as a result of de novo synthesis from particular amino acids, as well as due to glutathione reductase (GR)-mediated regeneration of GSH from GSSG.In some conditions (such as stress or the presence of xenobiotics), GSH is transformed into GSSG.An elevated content of GSSG can be regarded as oxidative stress marker, while the GSH/GSSG  , 2006).The gills and hepatopancreas were chosen for the present work because gills are the respiratory organs and exposed to ambient oxygen, while the hepatopancreas is responsible for regulation of overall body metabolism (M u r i a n a et al., 1993).Also, the hepatopancreas is the organ most involved with the detoxification of xenobiotics.Muscle is important because of its role as a consumer.
The aim of this study was to investigate the contents of total glutathione (tGSH), reduced glutathione (GSH), and oxidized glutathione (GSSG) in the hepatopancreas, gills, and muscle of three freshwater crayfish species: noble crayfish (Astacus astacus), stone crayfish (Austropotamobius torrentium), and spinycheek crayfish (Orconectes limosus).We also evaluated the glutathione redox index (GSH RI), which is a comprehensive marker of the mutual relationship between components of the GSH system (B e n z i et al., 1988).

Site description, sample collection, and preparation of specimens
Three freshwater crayfish species were collected: noble crayfish (Astacus astacus) from the Southern Morava River (42°30'44'' N, 21°53'42.1''E); stone crayfish (Austropotamobius torrentium) from the Kra�kovačka River (43°20'48.3''N, 21°38'23.7''E); and spinycheek crayfish (Orconectes limosus) from Danube River (44°41'31.6''N, 20°57'38.5''E).The sites are indicated in Fig. 1.The specimens (n=30, 10 of each of the all three freshwater crayfish species) were collected (specimens of both sexes with similar dimensions and similar weights were taken in order to avoid age differences) in summer (July and August, 2004) using deep nets or by hand from their natural habitats.Water temperature was 18°C in the Southern Morava River, 14°C in the Kra�kovačka River, and 19°C in the Danube River.The hepatopancreas, gills, and muscle of each crayfish species were immediately dissected and frozen in liquid nitrogen (at the site of collection) and then stored at -80°C before further biochemical analyses.All chemicals were from Sigma (St.�ouis, MO, U. S. A.).

Biochemical analyses
Sonicates were mixed with 10% sulfosalicylic acid (SSA) and then centrifuged at 5000 rpm for 10 min, and the resulting supernatants were stored at -80�C �C.The method is based on sequential oxidation of GSH by 5, 5´-dithiobis 2-nitrobenzoic acid (DTNB) and reduction by NADPH in the presence of GR (G r i f f i t h , 1980).The content of total GSH was measured in triplicate using a Shimadzu UV-

Statistical analysis
The data are expressed as means ± standard error (S.E.).Statistical differences between the species were analyzed using the unpaired Student ttest, a level of p<0.05 being considered significant (H o e l , 1966).

RESU�TS
The tGSH content in the hepatopancreas (Fig. 2A) of A. astacus was significantly lower than in that of A. torrentium and O. limosus (p<0.005).In gills (Fig. 2B) of A. astacus, tGSH content was significantly higher than in gills of O. limosus (p<0.05).There were no significant differences between content of tGSH in muscle of the investigated crayfish species (Fig. 2C).
Content of GSH was significantly increased in the hepatopancreas of A. torrentium in comparison with A. astacus (p<0.005) and O. limosus (p<0.05) (Fig. 3A).We also found significant differences of GSH content between A. astacus and O. limosus (p<0.01).In gills of A. astacus, content of GSH was significantly increased (Fig. 3B) in comparison with O. limosus (p<0.05).No statistically significant differences of GSH content in muscle were found between the investigated crayfish species (Fig. 3C).The GSSG content in the hepatopancreas (Fig. 4A) of O. limosus was significantly higher than in that of A. torrentium (p<0.005) and A. astacus (p<0.02).In gills of A. astacus, GSSG content was significantly higher (Fig. 4B) than in gills of O. limosus (p<0.05).Content of GSSG was significantly increased in muscle of O. limosus in comparison with A. astacus and A. torrentium (p<0.05).We also found statistical significant differences of GSSG content between A. astacus and A. torrentium (p<0.02) (Fig. 4C).
Values of the glutathione redox index in the hepatopancreas were equal in the three freshwater crayfish species, so there were no significant differences.In gills of O. limosus, GSH RI was significantly higher than the GSH RI in gills of A. astacus (p<0.05).GSH RI was significantly increased in muscle of A. astacus in relation to its values in muscle of A. torrentium (p<0.02) and O. limosus (p<0.05)(Figs 5A, 5B, and 5C).

DISCUSSION
The habitats of crustaceans are characterized by unstable physico-chemical properties, so they are exposed to daily and seasonal fluctuations in temperature and concentration of dissolved oxygen (F i l h o , 1996).Crustaceans are poikilothermic organisms, so they continuously ad�ust to environmental conditions.Because of their ecological importance, wide distribution, high availability throughout the year, sensitivity to environmental toxicants, and high capability of bioaccumulation, crustaceans are suitable as test-organisms in biomonitoring studies (S c h i l d e r m a n et al., 1999).
The results obtained in our study show the existence of tissue-specific changes of GSH content.Contents of tGSH were higher in the gills then in muscle and the hepatopancreas.These data may indicate a faster rate of GSH utilization or degradation in the former tissue, which could be responsible for the observed lower GSH content.Moreover, increase of GSH content in the gills compared with the hepatopancreas may be related to prevention of oxidative challenge in gill tissue (D a n d a p a t et al., 2000).Earlier findings also suggest that the presence of high GSH content in red cells (F i l h o , 1996) and in gills (M a r c o n and F i l h o , 1999) of fishes is associated with attenuation of oxidative stress.The highest GSSG content was detected in muscle, the lowest in the hepatopancreas.
Aquatic organisms maintain high content of GSH in tissues (T h o m a s et al., 1982), and increased content has the function of protection (T h o m a s and J u e d e s , 1992).Also, high content of GSH could be a consequence of its increased synthesis due to high cysteine accessibility, which is necessary for GSH synthesis.The content of reduced GSH in muscle was approximate equal in the three species, while in the hepatopancreas and gills interspecies differences were detected.It should be emphasized that the pattern of alteration of GSH content in tissues is completely the same as in the case of tGSH, which is understandable because reduced GSH represents 9/10 of tGSH.
Published data indicate that GSH content depends on the type of nutrition (Ta y l o r et al., 1996).Also, in tissues of some organisms, the content of GSH is seasonally dependent.In the digestive gland of the mussel Mytilus galloprovincialis, the content of GSH is lowest in winter, when there is less food and the gonads are in a state of rest (V i a r e n g o et al., 1991; P o r t e et al., 2000).GSH metabolism is regulated by several enzymes, so cellular GSH content is not constant and depends on the rates of synthesis, con�ugation, and oxidation of GSH, as well as that of GR-dependent reduction of GSSG to GSH.
It has been found that many chemical substances can alter GSH content in tissues of different organisms.In liver of Carassius auratus, a decrease of GSH content was detected as a consequence of exposure to 2, 4-dichlorophenol for 40 days (Z h a n g et al., 2004).Other organic pollutants also provoke increase of GSH content, thereby providing extra protection against cytotoxic effects of biologically active molecules.Cellular GSH content increased after treatment with subtoxic Cd concentrations (S o n et 2001a; S o n et al., 2001b).This could provide the first line of defense against the influence of toxic heavy metals.Also, M a c F a r l a n e et al. (2006) found that heavy metals induce increase of GSH content in the crab Parasesarma erythodactyla, while naphthalene induces decrease of GSH content in the hepatopancreas, ovary, and gills of the crab Scylla serrata (V i � a y a v e l and B a l a s u b r a m a n i a n , 2006).It has been established that GSH content and the GSH/GSSG ratio are related to the survival rate of mussels exposed to fenitrothion (P e n a -� l o p i s et al., 2002).Although pollution influence on GSH content is regulated by a feedback mechanism and the liver, the GSSG/GSH ratio can be used as a potential biomarker in fish (Va n d e r O o s t et al., 1996).
The glutathione redox index represents a marker of the GSH/GSSG ratio, i.e., it defines the glutathione redox status of the cell.We found the highest GSH RI in the hepatopancreas and the lowest in muscle.In gills, the highest GSH RI was found in O. limosus, while in muscle it was recorded in A. astacus.B e n z i et al. (1988) showed, that GSH content in the rat brain decreases and GSSG content increases with age, which means that the total GSH RI profile is age-linked and decreases with time.
In conclusion, the present study represents the first report treating the contents of tGSH, GSH, and GSSG and values of GSH RI in the investigated tissues (hepatopancreas, gills, and muscle) of two native (A.astacus and A. torrentium) and one introduced (O.limosus) freshwater crayfish species collected from three different Serbian rivers (the Southern Morava, the Kra�kovačka, and Danube Rivers).The obtained results show tissue differences in the investigated species and indicate that the studied parameters can be considered a suitable biomarkers of the cellular glutathione redox status in freshwater crayfish species from different localities.

ГЛУТАТИОН KAО ПОГОДАН
Tissues were minced and homogenized in 5 volumes (� i o n e t t o et al., 2003) of 25 mmol/� sucrose containing 10 mmol/� Tris-HCl, pH 7.5, at 4°C with an Ultra-Turrax homogenizer (Janke and Kunkel, IKA-Werk, Staufen, Germany) (R o s s i et al., 1983).The homogenates were sonicated for 30 s at 10 kHz on ice (Ta k a d a et al., 1982) and then used for determination of the contents of total GSH, reduced GSH, and oxidized glutathione.