The Activity of Antioxidant Defence Enzymes (Glutathione Peroxidase, Glutathione Reductase and Phase II Biotransformation Enzyme Glutathione-S-Transferase) in Some Tissues of Stone Crayfish (Austropotamobius torrentium Shrank) from Krajkovacka River

Slaviša M. Milošević1, Nebojša V. Živić1, Maja V. Milosavljević1 and Tatjana R. Jakšić1

 

1 University of Priština, Kosovska Mitrovica, Faculty of Science and Mathematics, Lole Ribara 29, 38220, Kosovska Mitrovica, Serbia; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it

 

Abstract

The activity of the antioxidant defense enzymes 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 some tissues of stone crayfish (Austropotamobius torrentium) were studied. In our study we investigated the activities of: glutathione peroxidase (GSH-Px), glutathione reductase (GR) and phase II biotransformation enzyme glutathione-S-transferase (GST) in the hepatopancreas, the gills and muscle of Stone crayfish (Austropotamobius torrentium). All enzyme activities were measured spectrophotometrically and presented as specific (U/mg proteins) and total values (U/mg wet mass). The results show strong tissue specificity of investigated enzymes. Specific and total GSH-Ph activity in abdominal muscle was lower than in hepatopancreas and gills. At the same time total GSH-Ph activity in gills was significantly lower in respect to hepatopancreas. The specific and total activity of GR was significantly higher in gills than in muscle and hepatopancreas, as well as in muscle in respect to the hepatopancreas. The specific and total activity of GST was significantly increased in hepatopancreas than in gills and muscle. At the same time, the activity of GST was higher in gills than in muscle. In the presented study our results show that both specific and total glutathione redox cycle enzyme activities GSH-Ph, GR and phase II biotransformation enzyme GST show a strong tissue specificities and present a good environmental markers.

Keywords: Antioxidant defense enzymes, Stone crayfish, Austropotamobius torrentium, Glutathione peroxidase Glutathione reductase, Glutathione-S-transferase, Krajkovacka River.

 

Introduction

Aquatic life is currently being exposed to increasing anthropogenic chemical contamination that can induce many different mechanisms of toxicity, producing various deleterious effects. Increased levels of oxidative stress in an organism can lead to reduced survival. Thus, oxidative damage can be considered a relevant marker of general health status (Beaulieu and Costantini 2014). The responses of stress proteins such as metallothioneins and antioxidant enzymes are the most commonly used markers of intoxication in crustaceans (Lei et al. 2011). All aerobic organisms during their respiratory activity continuously produce reactive oxygen species (ROS) (Perez-Campo et al. 1993; Halliwell & Guterige 1999). Pro-oxidant activity can be used to assess water pollution (Slaninova et al. 2009). Crustaceans are frequently used as bioindicators in various aquatic systems, because they are widely distributed in a number of different habitats including marine, terrestrial and freshwater environments. Therefore, they are suitable candidates for comparative studies. Most studies on antioxidants as biomarkers for the aquatic environment have been carried out on fish (Rodriguez-Ariza et al., 1993; Lemaire & Livingstone 1993) and on marine invertebrates (Livingstone 2001, Regoli & Principato 1995).

The scope of our work was to determine the activity of the following antioxidant defense enzymes: glutathione peroxidase (GSH-Px), glutathione reductase (GR) and the phase II biotransformation enzyme glutathione/S/ transferase (GST ) in the hepatopancreas, the gills and the abdominal muscle of Stone crayfish (Austropotamobius torrentium).

 

Materials and Methods

The specimens were collected by deep nets or by hands from their natural habitat. There were 10 specimens of both sexes similar dimensions and weight. Specimens were sampled in Krajkovacka River, South Serbia (43°17'45.6"N 21°47'45.6"E). Samples were immediately frozen in liquid nitrogen (-196º) and then stored at -80º until further analysis. The isolated tissues were minced and homogenized in 5 volumes (Lioneto 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 & Kunkel, IKA-Werk, Staufen, Germany). (Rossi et al. 1983). The homogenates were sonicated for 30s at 10 kHz on ice and sonicates were then centrifuged at 4°C at 100.000 g for 90 minutes (Takada et al. 1982). The resulting supernatants were used for further biochemical analysis.

The total protein concentration in the supernatant was determined according to the method of Lowry et al. (1951) and presented in mg/g wet mass. The activity of GSH-Px was analyzed by the method of Tamura et al., (1982) and the activity of GR was measured as suggested by Glatzle et al. (1974). GST activity was determined by the method of Habig et al. (1974). All enzyme activities were presented as specific in units/mg proteins and as total in units/g wet mass (Barja de Quiroga et al. 1988). The data are shown as mean ± standard error. The statistical significance of differences was determined by the unpaired t-test considering the significance at the level of p<0.05 as described by Hoel (1966).

 

Results and Discussion

The specific GSH-Ph activity was significantly higher in the hepatopancreas than in abdominal muscle (p<0.05) of the Austropotamobius torrentium. At the same time, specific GSH-Ph activity in gills was significantly higher in respect to muscle (p<0.05). The significant difference was not found in the specific GSH-Ph activityes between hepatopancreas and gills (Fig. 1). The total GSH-Ph activity in the hepatopancreas was significantly higher than that found in gills (p<0.05) and abdominal muscle (p<0.05). The significant difference was not recorded in the total GSH-Ph activities between gills and abdominal muscle (Fig. 2).

 

Fig01
Figure 1: The specific activity of the glutathione peroxidase (GSH-Px) in the hepatopacreas, gills and abdominal muscle of Stone crayfish  (Austropotamobius torrentium).

 

Fig02
Figure 2: The total activity of the glutathione peroxidase (GSH-Px) in the hepatopacreas, gills and abdominal muscle of Stone crayfish  (Austropotamobius torrentium).

 

In the gills specific GR activity was significantly higher than in other two investigated tissues, hepatopancreas (p<0.05) and abdominal muscle (p<0.05), as well as in muscle in respect to the gills (p<0.05). (Fig. 3). The same trend was obtained for the total GR activity in the gills, which was significantly increased when compared to two other investigated tissues hepatopancreas (p<0.05) and abdominal muscle (p<0.05). At the same time, total GR activity in muscle was significantly higher in respect to hepatopancreas (p<0.05). (Fig. 4).

 

Fig03
Figure 3: The specific activity of the glutathione reductase (GR) in the hepatopacreas, gills and abdominal muscle of Stone crayfish  (Austropotamobius torrentium).

 

Fig04
Figure 4: The total activity of the glutathione reductase (GR) in the hepatopacreas, gills and abdominal muscle of Stone crayfish  (Austropotamobius torrentium).

 

The specific and total activity of phase II biotransformation enzyme was significantly higher in the hepatopancreas of Stone crayfish in comparison to the gills (p<0.05) and muscle (p<0.05), as well as in gills in respect to the abdominal muscle (p<0.05). (Fig. 5). At the same time, specific and total GST activity in the gills was significantly higfer in respect to muscle (p<0.05). (Fig. 6).

 

Fig05
Figure 5: The specific activity of the glutathione-S-transferase (GST) in the hepatopacreas, gills and abdominal muscle of Stone crayfish  (Austropotamobius torrentium).

 

Fig06
Figure 6: The total activity of the the glutathione-S-transferase (GST in the hepatopacreas, gills and abdominal muscle of Stone crayfish  (Austropotamobius torrentium).

 

All aerobic organisms during their respiratory activity continuously produce reactive oxygen species (ROS) (Halliwell & Gutteridge 1999; Perez-Campo et al. 1993). ROS induce many cellular disturbances, such as oxidation of protein sulphydryl groups and peroxidation of fatty acids, depolymerization of polysacharides and nucleic acids. (Stohs et al. 2000). The enzymes of the antioxidant defense system can interact for mutual modulation of activity (de Assis et al. 2013). The antioxidant defense system includes enzymes such as glutathione peroxidase, catalase, superoxide dismutase, glutathione reductase and glutathione-S-transferase (Menezes et al. 2011). Glutathione reductase plays an essential role in cell defense against reactive oxygen metabolites. GR maintains the reduced status of glutathione, which is necessary for GSH-Ph activity; hence GR regulates homeostatic oxido-reductive balance in the cell (Djordjevic et al. 2010). Many studies have shown positive correlations between levels of antioxidant defenses and the presence of xenobiotics (Orbea et al. 2002). According to the Environmental Risk Assessment (ERA), the components of antioxidant defense 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). Reduced glutathione (GSH) is a physiologically useful scavenger if the reaction with superoxide is prevented (Munday & Winterbourn 1989). GST, one of phase II biotransformation enzyme systems, has been used as a biomarker of organic industrial effluents (Sheehan et al. 1995). Although many compounds possess antioxidant, defense is provided by functional overlapping of antioxidant enzymes and correlation between their activities. This means, if cellular antioxidant defense 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.

 

Conclusion

In our research we've come to the conclusion that both specific and total enzyme activities of GSH-Px and GR and activity of phase II biotransformation enzyme GST show a strong tissue specificities of the Stone crayfish (Austropotamobius torrentium), and represent good environmental influences biomarkers to this organism.

Specific and total GSH-Ph activity in abdominal muscle was lower than in hepatopancreas and gills. At the same time total GSH-Ph activity in gills was significantly lower in respect to hepatopancreas. The specific and total activity of GR was significantly higher in gills than in muscle and hepatopancreas, as well as in muscle in respect to the hepatopancreas. The specific and total activity of GST was significantly increased in hepatopancreas than in gills and muscle. At the same time, the activity of GST was higher in gills than in muscle.

 

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