Seasonal Changes of Oxidative Stress Biomarkers in White Muscle of Longfin Gurnard (Chelidonychthys obscurus) from the Adriatic Sea

Sladjan Pavlović, Slavica Borković-Mitić, Branka Gavrilović, Svetlana G. Despotović, Jelena P. Gavrić and Zorica S. Saičić




University of Belgrade, Institute for Biological Research “Siniša Stanković”, Department of Physiology, 11060 Belgrade, Serbia. e-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it




The aim of this study was to investigate the activity of oxidative stress biomarkers (total superoxide dismutase-Tot SOD, manganese containing superoxide dismutase-Mn SOD, copper zinc containing superoxide dismutase-CuZn SOD, catalase-CAT, glutathione peroxidase-GSH-Px and glutathione reductase-GR), as well as biotransformation phase II enzyme glutathione-S-transferase (GST) in the white muscle of longfin gurnard (Chelidonychthys obscurus) at two localities: Platamuni (PL) and the Estuary of the River Bojana (EB) in the south-eastern Adriatic Sea (Montenegro) in winter and spring seasons. Our study represents the first investigation of this kind in the white muscle of longfin gurnard from the south-eastern Adriatic Sea and shows site specific differences between some investigated enzymes, with seasonal effects being the main influencing factor on investigated enzymes at both localities PL and EB.

Keywords: Longfin gurnard, Chelidonychthys obscurus, oxidative stress biomarkers, seasonal changes, white muscle.




Oxidative stress is an important component of the stress response in marine organisms, which are exposed to wide variety of environmental stressors, such as anthropogenic contamination or seasonal influences (Vinagre et al., 2012). Oxidative stress is the result of the over-production of reactive oxygen species (ROS). ROS, such as superoxide anion radicals, hydrogen peroxide and hydroxyl radicals are natural by-products of aerobic metabolism, but can additionally be produced intracellularly by different xenobiotics. They also can play a beneficial role in cells by contributing to pathways of intracellular signaling and redox regulation (Grim et al., 2013). Their toxicity to the main biological components (proteins, lipids and DNA) is counteracted by the activities of many cell defence mechanisms (Stohs et al., 2000). Cellular antioxidant defence system (AOS) is one of the important biochemical strategies that give protection to cells against deleterious effects of endogenous ROS by keeping their level relatively low (Paital and Chainy, 2010). AOS comprises of both non-enzymatic small antioxidant molecules (reduced glutathione, acsorbic acid, carotenoids) and a cascade of antioxidant defence enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione reductase (GR), as well as phase II biotransformation enzyme glutathione-S-transferase (GST), (Halliwell and Gutteridge, 2007; Van der Oost et al., 2003).

Field studies mainly considered the influence of pollution on antioxidant enzyme activities without examination of the influence of seasonal changes on enzyme activities. An important issue in biomonitoring studies of aquatic organisms is the complexity of biochemical and physiological responses to seasonal variables (Da Rocha et al., 2009). Fishes are thermo and oxygen-conformer organisms and production of ROS levels and antioxidant defence depends of these physical variables (Wilhelm Filho, 2007).

In fish, changes in antioxidant defense enzyme activities can be influenced both by intrinsic factors (age, feeding behavior, food consumption), and also by extrinsic factors, such as toxins present in the water, seasonal and daily changes in dissolved oxygen and water temperature (Bayir et al., 2011). In their natural habitats, fish often have periods of poor food supply as a result of lower environmental temperature, spawning, migration and reproduction (Furné et al., 2009) and changes of these variables are accompanied with seasonal fluctuations. For this reason, investigation of antioxidant enzymes in fish in biomonitoring studies, have also taken into account seasonal effects beside the effects of various pollutants.

Longfin gurnard Chelidonichthys obscurus (Walbaum, 1792) is a perciform fish which occurs in benthic zones and inhabits sand, muddy sand or gravel bottoms. It feeds mainly on fish, crustaceans and mollusks. Longfin gurnard has three isolated rays on the pectoral fin which function as legs on which the fish rests and also help in locating food on the soft bottom (Frimodt, 1995). Moreover, this species maintains a close association with sediments for food and protection and it is therefore more likely to be exposed to sediment-associated pollutants.

The aim of the present work was to compare the activity of oxidative stress biomarkers in the white muscle of longfin gurnard (Chelidonichthys obscurus) between two localities, Platamuni and the Estuary of the River Bojana (south-eastern Adriatic Sea) in two seasons: winter and spring. We examined the activities of total superoxide dismutase (Tot SOD), manganese containing superoxide dismutase (Mn SOD, EC, copper zinc containing superoxide dismutase (CuZn SOD), catalase (CAT, EC, glutathione peroxidase (GSH-Px, EC, glutathione reductase (GR, EC, as well as the activity of biotransformation phase II enzyme glutathione-S-transferase (GST, EC



Materials and Methods

The specimens of longfin gurnards (Chelidonychthys obscurus) were caught by trawling in winter (February) and late spring (May) at two localities: Platamuni (PL) and the Estuary of the River Bojana (EB), (Fig. 1). These localities receive different amounts of industrial and agricultural discharges and were selected in order to compare the activity of oxidative stress biomarkers between sites, as well as between periods of lower metabolic activity (winter) and higher metabolic activity (spring). At Platamuni, 20 specimens (10 in the winter and 10 in the spring) and at the Estuary of the River Bojana, 20 specimens (10 in the winter and 10 in the spring) of longfin gurnard were collected. Specimens of fish were collected and immediately transferred to seawater tanks where they were identified. Fish were killed on board by severing the spinal cord and dissected within 3 minutes on ice. The white muscle was rapidly dissected from each sample, washed in ice-cold 0.65% NaCl and frozen in liquid nitrogen (-196°C) before storage at -80°C. White muscle was ground and homogenized in 5 volumes of 25 mmol/L sucrose containing 10 mmol/L Tris-HCl, pH 7.5 (Lionetto et al., 2003) using Janke & Kunkel (Staufen, Germany) IKA-Werk Ultra-Turrax homogenizer at 4°C (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 100000 g for 90 minutes. The resulting supernatants were used for 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 antioxidant defence enzymes was measured simultaneously in triplicate for each sample using a Shimadzu UV-160 spectrophotometer and a temperature controlled cuvette holder. The total activity of SOD was assayed using the epinephrine method (Misra and Fridovich, 1972) and expressed as specific activity (U/mg of protein). For the determination of Mn SOD activity the assay was performed after the preincubation with 8 mmol/L KCN. CuZn SOD activity was calculated as a difference between total SOD and Mn SOD activities. CAT activity was evaluated by the rate of hydrogen peroxide (H2O2) decomposition and expressed as μmol H2O2/min/mg protein (Claiborne, 1984). The activity of GSH-Px was determined following the oxidation of nicotine amide adenine dinucleotide phosphate (NADPH) as a substrate with t-butyl hydroperoxide (Tamura et al., 1982) and expressed in nmol NADPH/min/mg protein. The activity of GR was assayed as described by Glatzle et al. (1974) and expressed as nmol NADPH/min/mg protein. The activity of biotransformation phase II enzyme GST towards 1-chloro-2,4-dinitrobenzene (CDNB) was measured using the method of Habig et al. (1974) and expressed as nmol GSH/min/mg protein. All chemicals were the products of Sigma (St. Louis, MO, USA).

The data are expressed as mean ± S.E. (standard error). The non-parametric Mann-Whitney U-test was used to seek significant differences between means. A minimum significance level of p<0.05 was accepted. In addition, Principal Component Analysis (PCA) was employed to detect variables that significantly contributed to differences in the activities of investigated enzymes between the examined sites and seasons. Analytical protocols described by Darlington et al. (1973) and Dinneen and Blakesley (1973) were followed.



Figure 1: The geographical position of two investigated localities: Platamuni (PL) and the Estuary of the River Bojana (EB) in the south-eastern Adriatic Sea (Montenegro).