Comparative Studies of Hepatic and Blood Biomarkers in Three Species of Black Sea Elasmobranchs  

Irina I. Rudneva , I.I.  Dorokhova , E.N. Skuratovskaya , N.S. Kuz’minova
Ichthyology Department, Institute of Biology of the Southern Seas National Ukrainian Academy of Sciences, Nakhimov av. 2, Sevastopol, Crimea, 99011, Ukraine
Author    Correspondence author
International Journal of Marine Science, 2014, Vol. 4, No. 44   doi: 10.5376/ijms.2014.04.0044
Received: 25 Feb., 2014    Accepted: 30 Mar., 2014    Published: 08 Aug., 2014
© 2014 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Rudneva et al., 2014, Comparative Studies of Hepatic and Blood Biomarkers in Three Species of Black Sea Elasmobranchs, International Journal of Marine Science, Vol.4, No.44 1-14 (doi: 10.5376/ijms.2014.04.0044)


The investigations of elasmobranchs are very important for the understanding of evolutionary process and mechanisms of fish adaptation. Fishing pressure and global climate changes are affecting elasmobranch population size and structure. Baseline data of these taxa from different locations are useful for the evaluation of their health and for development of the methods of their preservation. In this study Aminotransferases (ALT and AST) activity, oligopeptides, albumin and hemoglobin concentrations were determined in the liver, red blood cells and blood serum of three Black Seaelasmobranch species: Atlantic spiny dogfish Squalus acanthias, buckler skate Raja clavata and stringray Dasyatis pastinaca. Interspecies differences of enzymatic activities depending on fish biology and ecology were shown. Hepatic aminotransferases level and serum oligopeptides concentration were greater in benthic/pelagic active shark, than in benthic sluggish rays. Oligopeptides concentration in red blood cells was the similar in tested elasmobranchs, while serum enzymatic activity and hepatic oligopetides level were higher in R. clavata than in D. pastinaca and S. acanthias inhabiting deeper waters. The results demonstrate that the complex of biochemical characteristics of elasmobranchs together with their ecological specificity can be used to indicate the main strategy of biochemical adaptation of fish evolutionary process. 

Elasmobranchs; Aminotransferases; Antioxidant enzymes; Oligopeptides; Liver; Blood; Black Sea

1 Introduction
There are more 500 species of elasmobranches (sharks, skates and rays) belonging to different ecological groups. These groups are characterized by a specific life cycle, foraging behavior, swimming activity and reproduction (Harms et al., 2002). Elasmobranchs, an ancient taxa with diverse evolutionary history, are interesting organisms for studying comparative physiology and biochemistry. Marine species have high concentrations of plasma urea and trimethylamin oxide (TMO), both of which are essential to maintain plasma osmolality within physiological ranges. A majority of elasmobranchs are predatory carnivorous species at the top of the marine food web. Therefore, they serve as important species to monitor environmental toxicology and contamination of marine ecosystems, not only for the health of marine wildlife, but also human health (Gelsteichter et al., 1998; Sole et al., 2007; 2008; 2009; Haman et al., 2012).

Both sharks and rays have been extensively fished for human consumption (Mathews and Fisher, 2009). The flesh of elasmobranchs often contain significant concentrations of fat-soluble vitamins (D, E and A), essential polyunsaturated fatty acids, and antioxidants (Rudneva, 1997; 1998; 2012), all significant nutritional components of the human diet. Furthermore, the adipose tissue of many elasmobranchs is used in pharmacology like the source of fat-soluble vitamins and the manufacture of feedstuffs for human consumption (Mathews and Fisher, 2009). This fishing pressure on elasmobranchs has had profound impacts on numerous populations of elasmobranchs, especially in the Black Sea (Rudneva et al., 2012). The ability of elasmobranch species to adapt and recover to such pressures, coupled with increased anthropogenic pollution and climate change, remains unknown.

However, the global health and population status of most elasmobranch species is unknown. It is critically important to understand the role of this taxa in marine ecosystems, given their role at the top of the food web. Therefore, the establishment of baseline health parameters and how they differ based on life histories and habitat use is critically important to monitor adaptive processes and population status of wild elasmobranch species, such as S. acanthias, R. clavata and D. pastinaca in the Black Sea.

In recent years the biodiversity in the Black Sea has significantly decreased (Gordina et al., 1999). This decline is associated with anthropogenic pollution, overfishing and the introduction of marine invasive species (Rudneva and Petzold-Bradley, 2001; Rudneva, 2011). Though the diversity of teleost fish species and their population status, particularly those common in economic fisheries, has received great attention. However, the status of elasmobranchs in the Black Sea has received significantly less attention, especially in coastal waters near the interface of riparian zones and anthropogenic development cities. Therefore, the response of key elasmobranch species, such as dogfish S. acanthias, buckler skate R. clavata and stringray D. pastinaca studied here, to the anthropogenic pressures of fishing and pollution remains unknown.

In general, various biochemical and health parameters are used for the evaluation of fish health. These often reflect physiological status and the influence of certain biotic (pathogens, parasites, disease state, etc) and abiotic factors (anthropogenic pollution, habitat degradation, etc) (Martinez-Alvarez et al., 2005; van der Oost et al., 2003). Among them blood and hepatic biochemical characteristics are used widely as early warning indicators of changes physiological and health status (Haman et al., 2012). However, in order for these parameters to indicate such physiological change, baseline values from healthy individuals must be established. In this study, we measure dogfish S. acanthias, buckler skate R. clavata and stringray D. pastinaca healthy, wild-caught elasmobranch species. We select several biomarkers such as oligopeptides, antioxidants, aminotransferases, albumin and hemoglobin for the evaluation of fish health and detect them in the red blood cells (RBC), blood serum and liver.

Specifically, oligopeptides are short sequence peptides including from 2 to 20 amino acids. Among them the biological mediators are identified which play an important role in affecting of biological activity (Grune, 2000). To protect against oxidative stress caused many kinds of biotic and abiotic factors, including anthropogenic pollution, aquatic organisms have developed different mechanisms such as the induction of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), peroxidase (PER) and glutathione (GSH) related enzymes (glutathione reductase (GR), glutathione peroxidase (GP) (Livingstone, 2001). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) catalyse the interconversion of amino acids and α-`ketoacids by transfer of amino groups (van der Oost et al, 2003). Both aminotransferases play an important role as a link between carbohydrate and protein metabolism. Along with the study of transaminase activity, several researchers used the method of multivariate approximation. One such approximation is the ratio proposed by de Rytis coefficient (AST / ALT). This index allows us to estimate the functional load on the liver and heart. Changes in the coefficient value associated with pathological processes in the organs and tissues, which cause displacement synthesis, release, and metabolic conversion of aminotransferases. Decrease of this value is indicative of massive hepatocytes, its increase is associated with myocardial infarction (Titov and Bochkova, 1990).

Previously we described variations in blood antioxidants of some Black Sea elasmobranchs and teleosts, thereby reflecting the adaptive strategy of such fish species to environmental changes and oxidative stress (Rudneva, 1997). We also showed the differences of trace elements and nitrozamines accumulation in three tested Black Sea elasmobranch species (Rudneva et al., 2012). The aim of the present study is to detect interspecific differences in blood and hepatic biomarkers in Black Sea elasmobranch species and their relation to the ecological status and exposure to environmental pollution in each of the 3 species. We measured enzyme activities (SOD, CAT, PER, GR, ALT, AST) and concentrations of oligopeptides, albumin and hemoglobin in examined elasmobranch tissues. These data provide baseline information against which comparisons can be made in monitoring the adaptability and response of elasmobranchs in a changing marine ecosystem.

2 Materials and Methods
2.1 Capture and sampling

Biological sampling was performed in the coastal waters of Sevastopol (Figure 1). Three elasmobranch species Atlantic spiny dogfish (Squalus acanthias, n=16), buckler skate (Raja clavata, n=43) and stringray (Dasyatis pastinaca, n=31) were studied in summer period of 2010-2013. Animals were captured by the fishery and immediately transported to the laboratory in the containers with marine water and constant aeration. After blood sampling the animals were decapitated.



Figure 1 Sampling sites of 3 species of elasmobranchs in Sevastopol coastal waters (44°36’N-33°32’E, Sevastopol, Black Sea, Ukraine)

Fish were individually measured and weighed. The total length (from tip of nose to tip of tail), standard length (from tip of nose to precaudal pit.) and total weight were measured according the methods described Pravdin (1966).

2.2 Blood collection, processing, and analysis
Blood (approximately 1~3 mL) was collected from the ventral tail artery using needle syringe or Pasteur pipette. Whole blood was collected and serum was separated within 24 hrs of collection in refrigerator at 4℃.

After blood collection fish were dissected and the liver was quickly removed and stored on ice. The organ was washed in the cold 0.85% NaCl solution several times, then homogenized in a physiological solution (1:5 w/v) using glass homogenizer. The resulting homogenate was centrifuged at 8000 g for 20 min. Biochemical assays were performed immediately after liver preparation.

Specifically, the sediments of red blood cells (RBC) were washed three times with cold 0.85% NaCl solution and then lysed by addition of 5 vol of distilled water and stored for 24 hrs at 4℃ as we described previously (Rudneva, 1997). The enzyme activity was then determined in the RBC lysates immediately after preparation. Spectrophotometer Specol-211 (Carle Zeiss, Germany) was used for all biochemical determinations.

2.3 Biochemical assays
Oligopeptide concentrations (OP): The concentration of oligopeptides (OP) was detected separately from RBCs, serum and liver extracts of the 3 species. Specifically, 0.25 mL Trichloracetic acid (TCA) was added to 0.5 mL of the sample and centrifuged at 9000 g for 30 min. Next, 0.3 mL of the supernatant was mixed in 3.7 mL 3% NaOH and 0.2 mL Benedict reagent. The mixture was incubated for 15 min at room temperature and then optical density (OD) was measured at 330 nm (Karyakina and Belova, 2004). We express the results in arbitrary units (OD at 330 nm per mg protein ×10-3).

Antioxidant activity (SOD, CAT, PER, GR): Antioxidant activities in the liver extracts and red blood cells from the 3 species in this study were determined according to methods described previously (Rudneva, 1997), with a few minor modifications. Specifically, Superoxide dismutase (SOD) was assayed on the basis of inhibition of the reduction of nitroblue tetrasolium (NBT) with NADH mediated by phenazine methosulfate (PMS) under basic conditions (Nishikimi et al., 1972). All measurements were performed in 0.017 M sodium pyrophosphate buffer pH 8.3 at 25℃. The reaction mixtures contained 5 µM NBT, 78 µM NADH, 3.1 µM PMS, and a 0.1 mL sample; the final volume was 1.5 mL. The reactivity was measured using OD at 560 nm.

Catalase (CAT) was measured using a previously described method (Asatiani, 1969) that involves a hydroperoxide reduction.

Peroxidase (PER) activity was detected by spectrophotometry at 600 nm using benzidine reagent (Litvin, 1981). Specifically, the reaction mixture contained 1 mL acetate buffer pH 5,4, 0.4 mL 0.09% benzidine, 0.2 mL 0.03% H2O2, and 0.2 mL sample.

Glutathione reductase (GR) activity was assayed spectrophotometrically using a method modified after Goldberg and Sparner (Goldberg and Sparner, 1987). The reaction mixture contained 0.1 mL mM NADPH, 0.5 mL 7.5 mM oxidized glutathione, 0.2 mL mM EDTA, and 2 mL 0.05 M phosphate buffer pH 8.0. After incubation for 10 min (at what tempt), the OD of the mixture was determined at 340 nm.

Aminotransferases activity: Aminotransferases (ALT and AST) activity was also determined spectrophotometrically with 2,4-dinitrophenylhydrazine using the standard kit (Filicit-Diagnosis, Ukraine). Specifically, 0.2 mL substrate-buffer solution was added to 0.04 mL of liver extract or serum and incubated at room temperature for 1 h. The reaction was stopped by 0.2 mL 1,4-dinitrophenylhydrazine and the solution was incubated for 20 min. Then 2 mL 0.4 N NaOH was added to the mixture and the OD of the sample was measured at 500~530 nm. The ratio AST/ALT (de Rytis coefficient) was calculated also (Catalogue, 2005).

Total soluble protein, albumin and hemoglobin concentrations, Total soluble protein was quantified spectrophotometrically according the method described by Lowry et. al. (1951). Albumin (Alb) concentration was assayed spectrophotometrically on the basis by Bromokresol green reaction, used the standard commercial kit (Felicit-Diagnosis, Ukraine). Hemoglobin (Hb) concentration was measured by spectrophotometric method on the basis of cyanide reaction (Young, 1997). The enzyme activities were calculated per mg protein in liver extracts, RBC and serum.

2.4 Statistical analysis
Biochemical measurements were detected in duplicate for each sample. The number of tested individuals ranged from 5 to 12 animals, depending on species (Table 1). Simple, descriptive statistics were performed using an ANOVA to determine means (+/- SE) (Halafyan, 2008). P value of 0.05 was used for determination of statistical significance in all cases. The graphs were made using the computer program EXELL. Statistical correlations between studied biochemical parameters in tested animals and tissues were calculated by the least-squares method using the computer program CURVEFIT (Version 2.10-L).



Table 1 Number of analyses of examined biochemical characteristics of elasmobranch species caught in Sevastopol coastal waters (Black Sea, Ukraine)

3 Results
3.1 Biometric characteristics

Biometric characteristics of examined individuals are present in Table 2. They are varied in examined animals depending on fish species (Black Sea, Ukraine).



Table 2 Biometrical characteristics of elasmobranch fish caught in Sevastopol coastal waters (Black Sea, Ukraine)

3.2 Hepatic biomarkers
Oligopeptide concentrations in the liver of tested elasmobranch species were higher in buckler skate than in dogfish (P<0.01) and stringray (P<0.05) (Figure 2). No significant differences were shown in the hepatic oligopeptides in shark and stringray.



Figure 2 Oligopeptides concentration (mean ± SE, ×10-3 mg protein-1) in the liver of elasmobranch species caught in Sevastopol Bay (Black Sea, Ukraine)

Antioxidant enzyme activities in the liver of elasmobranch species are presented in Figure 3. SOD activity was significantly higher (P<0.001) in the liver of both skates as compared with the shark. Hepatic enzymatic activity was greater in buckler skate than in stringray (P<0.05). The highest CAT activity was observed in the liver of buckler skate, it was significantly greater (P<0.01) than in both examined elasmobranchs.



Figure 3 Antioxidant enzyme activities (mean ± SE., mg protein-1 min-1) in the liver of elasmobranch fish species caught in Sevastopol Bay (Black Sea, Ukraine)

PER and GR activities were significant higher (P<0.05 and P<0.001 respectively) in both skates as compared with shark, and no significant differences were shown between skates.

High correlations were shown between the activity of SOD and CAT (r=0.91), SOD and PER (r=0.94) and between PER and GR (r=0.87). The relationships between SOD and GR and CAT and PER were lower (r=0.66 and r= 0.72 respectively).

Aminotransferase activities and de Rytis coefficient are shown in Figure 4. Both ALT and AST levels were significantly higher in the dogfish than in both species of ray (P<0.05). However, the de Rytis coefficient was higher in both dogfish and buckler skate as compared with stringray. As expected, high correlation was shown between hepatic ALT and AST activity (r=0.95).



Figure 4 Aminotransferase activities and de Rytis coefficient (mean ± SE, mg protein-1 h-1) in the liver of elasmobranch species caught in Sevastopol Bay (Black Sea, Ukraine)

There was no significant difference in albumin concentration in the liver of the examined elasmobranchs (Figure 5). Though not significant, we did note a decreased concentration of albumin in the liver of stringray as compared with both dogfish and buckler skate.



Figure 5 Albumin concentration (mean ± SE) in the liver of elasmobranch species caught in Sevastopol Bay (Black Sea, Ukraine)

3.3 Blood and serum biomarkers
Oligopeptide concentrations in RBCs of examined elasmobranchs varied insignificantly while in serum the level was greater in dogfish (P<0.001) than in either skate species (Figure 6).



Figure 6 Oligopeptides concentration (mean ± SE, ×10-3 mg protein-1) in the serum and red blood cells of elasmobranch fish species caught in Sevastopol Bay (Black Sea, Ukraine)

Antioxidant enzyme activities in RBCs of tested elasmobranch species are presented in Figure 7. SOD activity was significantly higher (P<0.001) in both skates as compared with the dogfish. SOD activity was similar in buckler skate and in stringray. CAT activity was very low in dogfish and in several individuals we did not detect CAT activity in RBCs. CAT activity in RBCs was insignificantly greater in buckler skate than in stringray. PER and GR activities were higher in the dogfish as compared with both skates (P<0.001). No significant differences were documented between the two species of skate. Overall, RBC antioxidant enzymatic activities were higher in skates than in shark at the case of SOD and CAT, PER and GR activity was higher in dogfish. The differences between the two species of skate were less than between shark and skates, with the exception of CAT activity.



Figure 7 Antioxidant enzyme activities (mean ± SE., mg protein-1 min-1) in the red blood cells of elasmobranch fish species caught in Sevastopol Bay (Black Sea, Ukraine)

High negative correlations were observed between SOD and PER (r=-0.98), SOD and GR (r=-0.98) and a positive relationship was shown between PER and GR activities (r=0.91).

Serum ALT and AST activities were significantly higher (P<0.01) in buckler skate as compared with stringray and dogfish. Enzyme activities were the similar in stringray and shark (Figure 8). Values of de Rytis coefficient varied insignificantly in tested elasmobranch species. High correlation was shown between serum ALT and AST levels (r=0.94).



Figure 8 Aminotransferase activities and de Rytis coefficient (mean ± SE, mg protein-1 h-1) in the serum of elasmobranch fish species caught in Sevastopol Bay (Black Sea, Ukraine)

Serum albumin concentrations in dogfish and stringray was lower than in buckler skate (P<0.01) (Figure 9). Hemoglobin concentrations were similar in all tested elasmobranch species.



Figure 9 Serum albumin and hemoglobin concentration (mean ± SE) in the blood of elasmobranch species caught in Sevastopol Bay (Black Sea, Ukraine)

4 Discussion
The aim of the present study was to investigate differences between specific health variables, known to show physiological responses to environmental conditions, in different elasmobranch species in the Black Sea. Given the increased anthropogenic development, pollution, introduction of invasive species, and resulting loss of biodiversity in the Black Sea, understanding the physiological responses and adaptations to these changes in elasmobranchs is of utmost importance. Our findings demonstrated significant differences between serum, RBC, and hepatic biochemical parameters in three species of Black Sea elasmobranchs. These differences may be the result of species differences in relation to their ecology, life history, and biology. This discussion will focus on two main points: 1. to discuss possible explanations for the interspecies differences; and 2. to detect potential strategies of biochemical adaptations in the tested species to their living conditions and anthropogenic impacts.

Atlantic spiny dogfish Squalus acanthias is gregarious benthic-pelagic fish which abundance is at the depth of 180-200 m. However, it migrates in the upper waters for feeding. This shark is carnivorous species, and it consumes primary fish (horse mackerel, pickerel, sand smelt, red mullet, and etc.). The dogfish shark is a viviparous species, its maturation begins at the age of 13-14 years and at the body length of 1 m. Incubation period of the embryos continues 2 years, each female produces 10-12 embryos and 18 eggs. Usually the length of the adults is estimated as 150~208 cm and the weight is 14 kg. Fish life span continues approximately 25 years, number of males in the catches are higher than females. Liver mass of the adult sharks is estimated as 17.9~29.6% of the total body weight (Svetovidov, 1964).

Buckler skate Raja clavata is a benthic species; it is abundant at the depth of 100 m at the bottom sediments or at the soil. Females spawn at spring time at the shelf. Individual female spawns from ten to one hundred eggs on the bottom. The period of the embryogenesis continues during 4.5~5.5 months. The length of the male is 70 cm and the length of the female is 125 cm. It consumes primary benthic fish species (scomber, pickarel) and invertebrates, such as crustacean and mussels.

Stringray Dasyatis pastinaca is abundant at the depth of 200 m at the bottom sediments or at the soil. The size of adults is estimated as 60-70 cm and the mass is 6-10 kg. It is viviparous; the development of the embryo continues 4 months. Each female produces 4-12 embryos and 12-32 developmental eggs. The food includes small fish species, crustacean and mussels. Thus, the general prey categories, and trophic level, are very similar between these species (with S. acanthias is known to be an active predator of fast-swimming prey).

The liver plays an important role in metabolism (synthesis and degradation of various biomolecules, biotransformation of xenobiotics), and its biochemical characteristics reflect directly the adaptation of the organism to the living conditions. Blood biochemistry is also important in evaluation of fish physiological and ecological status and biochemical adaptations to environment. Besides that, blood biochemical parameters directly reflect health status of the animals, which is very important at the case of wild populations, including elasmobranchs (Haman et al., 2012). Therefore, the observed significant differences between skates and the dogfish indicate the specificity of adaptation mechanisms of the fish to environmental conditions.

The liver is characterized by an increased metabolic rate and includes a majority of physiological and biochemical pathways, including those that produce oligopeptides. Specifically, oligopeptides are formed via protein degradation and biotransformation of xenobiotics, thus generating multiple low molecular weight molecules. An increase of oligopeptide levels in tissues is associated with an increase in protein degradation coupled with a decrease in protein synthesis and inhibition of proteasomal function (Vysotskaya and Nemova, 2008). Currently, the use of protein peptide fragments is a rapidly growing field of research. Studies suggest that various biologically active peptides can result in favorable or negative outcomes on organism, and the role of biologically active short sequence peptides as potential agents through the modulation of age-, sex- and pathologies -dependent biochemical pathways is very important (Grune, 2000; Karyakina and Belova, 2004). Thus, oligopeptides concentration in animal tissues serve be biomarkers of physiological status.

In our study oligopeptides concentration in the liver of examined elasmobranchs was significantly higher in buckler skate compared with dogfish and stringray. In the serum the greatest value was indicated in dogfish, while no significant differences shown in red blood cells of examined fish species. We suggest the observed difference could be explained by the specificity of metabolic rate of the species. High level of oligopeptides in shark serum could be associated with its active swimming and feeding behavior, transport and excretory function of blood serum, while ray and skate are more sluggish and their metabolic rate is slower.

Marine elasmobranchs are osmoconforming hypoionic regulators, with extracellular fluids containing lower ionic levels than the surrounding marine water. Yet, the plasma osmolality is hyperosmotic to the external medium due to the retention of urea. (Ip et al., 2009). Taking this into account, we propose that oligopeptides in elasmobranch serum may be involved in the regulation of osmolality, together with urea and trimethylamine oxide (TMO) (Filho and Boveris, 1993; Harms et al., 2002; Metcall and Gemmell, 2005; Lopez-Cruz et al., 2010).

No correlations were observed between oligopeptides level in the serum, red blood cells and in the liver, indicating they fluctuate independently in elasmobranchs. However, data pertaining to oligopeptide concentrations in fish tissues are very limited, and further investigations are needed to understand the relationship of interspecies differences to the ecology and life history of the species in question.

Antioxidant defense of the organism include several enzymes: SOD protects against oxidative damage by catalyzing the reaction of dismutation of the superoxide anion to H2O2, which degrades by CAT to H2O. PER includes peroxide-degrading enzymes both specific and nonspecific, which reduce both hydrogen peroxide and hydroperoxides. GR maintains a ratio of GSH/GSSG under oxidative stress; it is a major regulatory enzyme that regenerates GSH from GSSG (Livingstone, 2001; Lesser, 2006). Alterations in the antioxidant enzyme activities of aquatic animals in response to environmental changes are used to indicate the potential for more severe hazards.

In our study antioxidant enzyme activities in the liver of the ray and skate were higher than in dogfish. Given the sluggish nature of skates and rays, these results contradict several other studies demonstrating that antioxidant levels in fish species depended on metabolic oxygen consumption or swimming activity (Filho et al., 1993; Sole et al., 2009; Lopez-Cruz et al., 2010). Furthermore, antioxidant enzyme activities differ between tissue type and function (Filho and Boveris, 1993; Filho et al., 1993; Rudneva, 2012; Velez-Alavez et al., 2013).

Our findings show a different trend of antioxidant levels in elasmobranch liver and red blood cells. In dogfish blood SOD and CAT activities were significantly lower than in skates, while PER and GR level demonstrated the opposite trend. No correlations between antioxidant enzyme activities in liver and red blood cells of tested elasmobranchs were observed with the exception of strong positive correlation between hepatic and blood CAT activity (r=0.93). We therefore conclude that antioxidant enzyme levels vary independently in the liver and in red blood cells of tested elasmobranchs. It could be associated with different functions of blood and liver.

Like the increased concentrations of oligopeptides in the dogfish, the increased concentration of PER and GR in shark blood compared with skate/rays may be associated with the increased swimming activity of the dogfish. However, in our previous study no differences in glutathione-S-transferase (GST, the phase II biotransformation enzyme) activity were observed between examined Black Sea ray and skate. Because GST involves in antioxidant defense of the organism also, and taking into account the peculiarities of metabolic pathways of elasmobranchs we proposed the presence of specific GST activity in their tissues (Rudneva et al., 2010a, b).

A possible explanation for the high hepatic antioxidant activities in both the ray and skate, as compared with the dogfish, may be the hypoxic conditions of benthic environment of the Black Sea. The ray and skate species included in this study are classified as benthic dwelling species. In general, benthic biotops are characterized by oxygen deficient conditions when compared with the top water layers. Several researchers have noted that in hypoxic conditions antioxidant defense is significantly enhanced in fish (Hermes-Lima et al., 2001; Welker et al., 2013). This is an evolutionary adaptation, that may play an important role when species venture into habitats with low concentrations of oxygen, such as when foraging or dwelling on the bottom. This adaptation allows fish to cope with oxidative stress arising from tissue reoxygenation (Lushchak and Bagnyukova, 2006).

More research regarding the antioxidant system of elasmobranchs, especially in deep habitats characterized by hypoxic and/or anoxic conditions are needed to understand the development of antioxidant defenses in aquatic animals. This is extremely important for the Black Sea ecosystem as it is one of the largest anoxic seas in the world. Furthermore, its deep waters contain high concentrations of hydrogen sulphide (Fashchuk and Sapozhnikov, 1999) or, perhaps, other unknown toxins that triggers antioxidant defenses. Benthic fish, including the elasmobranchs in this study migrate or dwell entirely in anoxic/hypoxic zones in the sea, and high level of antioxidants in their tissues may be a physiological adaptation to prevent oxidative stress (Welker et al., 2013). Despite these correlations, further investigations of the biochemical strategies for dealing with oxidative stress in fish inhabiting deep waters, and those containing hydrogen sulphides are needed.

Another explanation for the increased antioxidant enzyme activities in the liver of skates as compared with dogfish may be explained by the high concentration of low molecular weight scavengers (vitamins E, A, K, carotenoids, and glutathione) in shark tissues combined with the high concentrations of lipid peroxidation substances (Filho and Boveris, 1993; Filho et al., 1993). These low molecular weight scavengers may have evolved to serve as an antioxidant defense in sharks (Martinez-Alvarez et al., 2005). Therefore, the shark, unlike the skate, may not require the same elevated levels of antioxidants in the liver since the low molecular weight molecules serve the same evolutionary role.

An increase of aminotransferase enzyme activity in the blood serum, plasma and other extracellular fluids was shown in the tissues of the organisms impacted unfavorable conditions, organ dysfunction or internal lesions in tissues. The damaged cells release their contents (including aminotransferases) towards the blood stream, and the level of these enzymes enhances in serum (Martinez-Porchas et al., 2011). AST is used as clinic diagnostic tool, and it is associated with cell necrosis of the liver and skeletal or cardiac muscle, starvation and lacking of vitamin E. Plasma ALT is an acute hepatic damage good marker (Coppo et al., 2001~2002).

Our findings also demonstrate interspecies differences of aminotransferases (ALT and AST) activity in fish liver: in dogfish the enzyme (both ALT and AST) activity was the highest, in stringray and in buckler skate it was lower. We propose that these interspecies differences depend on fish ecological status; rays and skates are generally benthic or epibenthic slow swimming forms, and dogfish is an active pelagic animal. An increase of aminotransferase activity in the dogfish may be explained by the high metabolic rate and swimming activity of this species, while ray and skate have lower metabolic rates.

The de Rytis coefficient was higher in the skate and ray compared with shark which could be associated with lack of oxygen in bottom biotops. This agrees with the observations of several researchers who showed that enzyme activity ranges in fish widely, and depends on species biological specificity and swimming activity (Goto et al., 2003; Treberg et al., 2003), age (Coppo et al., 2001-2002), sex, maturation stage (Mehdi et al., 2011), period of reproduction (Svoboda et al., 2001) and animals adaptation to different salinities (Ip et al., 2009). Thus, the de Rytis coefficient (AST/ALT) differences in sharks vs rays/skates reflect the specificity of habitats of examined fish, particularly the oxygen concentration in waters because the lack of oxygen in the environment and in tissues led their damage and enzyme activity changes.

Aminotransferases activity in blood serum of buckler skate was the highest as compared with the values of dogfish and stringray. However, several authors have shown that the level of plasma aminotransferases in rays was lower than in sharks. For instance, AST activity in plasma of the ray Dasyatis americana was approximately 3-fold lower than in shark Sphyrna tiburo (Cain et al., 2004; Harms et al., 2002). We propose that the differences of aminotransferases activity in blood serum may be linked with the specificity of fish habitat, foraging ecology, and life history. For instance, aminotransferase activity in the muscle of elasmobranchs decreases significantly with the depth at which they exist. For example, muscular enzymatic activity in black dogfish Centroscyllium fabricii caught at a depth of 500~1000 m was significantly lower than in S. acanthias caught at the depth of 180 m (Treberg et al., 2003). Our findings support this; the concentration of aminotransferases is lower in the serum of dogfish and stringray (abundant at the depth of 180-200 m) than in buckler skate (abundant at the depth of 100 m).

Hemoglobin concentration was the similar in red blood cells of examined elasmobranchs. Serum albumin-like proteins level ranged from 2.4 g/L in D. pastinaca to 6.2 g/L in R. clavata and 2.95 g/L in S. acanthias. In the liver it varied lower. Several researchers also showed variations of serum albumin-like proteins in elasmobranch, which were fluctuated between 3-5 g/L (Harms et al., 2002).

Finally, interspecies variations of tested hepatic and blood biochemical parameters of Black Sea elasmobranchs may be the result of different sensitivities to pollution. Previously we described the significant anthropogenic impact in Sevastopol Bay (Black Sea, Ukraine) and the resulting negative consequences for fish health (Rudneva, 2011; Rudneva and Petzold-Bradley, 2001). In our recent publication (Rudneva et al., 2012) we have also shown that the concentration of several trace elements in buckler skate and stringray was higher than in dogfish (Table 3). Specifically, the concentrations of Cu, As and Hg were significantly higher in the skate’s and ray’s tissues than in shark. Furthermore, the concentration of nitrosamines in tissues of buckler skate and stringray was also greater than in dogfish (1.8+0,1, 1.9+0.01 and 1.6+0.05 ng/kg wet weight respectively) (Rudneva et al., 2012) because skates and rays live on the bottom, they are exposed to toxicants, including heavy metals, which tend to concentrate in the bottom sediments.



Table 3 Concentration of trace elements in skeletal muscle (mg/kg, mean ± SE) of Black Sea elasmobranchs (Rudneva et al., 2012)

Elasmobranchs are at the top of the food web and as such, they are at a greater risk of ingesting pollutants, including trace elements that have been biomagnified up the food web (Da Rocha et al., 2009; Sole et al., 2009). Increased levels of trace metals and nitrozamines in ray and skate may the result of increased exposure due to ingestion of toxicant-containing sediments and food (Goksoyr et al., 1996; Porte et al., 2000; Da Rocha et al., 2009; Sole et al., 2009). Dietary differences among ray, skate and shark seemed to be the most important causes for differences in their trace elements and nitrosamine levels in the body, which in turn modify biochemical parameters such as those investigated in this study.

Many investigators reported the highest antioxidant enzyme activities in fish liver, because it plays the major part in the detoxification processes of xenobiotics and endogenously generated metabolites that can not be metabolized by the other organs (Goksoyr et al., 1996; Sole et al., 2009). In this study, we show that hepatic antioxidant enzymes are in fact suitable biomarkers for monitoring physiological adaptations to habitat type, forage, and contaminants exposure. The interspecies variations of hepatic antioxidants in elasmobranchs in this study may reflect the specific adaptations to the oxidative stress and protective mechanisms against oxidative damage caused environmental pollution. Through multiple studies, we have shown that the ray and skate are more polluted than dogfish (Sole et al., 2009; Rudneva et al., 2012). In this study we document the antioxidant defense response of benthic elasmobranchs was higher than in dogfish.

The differences in hepatic antioxidant defense of Black Sea elasmobranch species correlated with the differences of oligopeptides concentration and aminotransferase level. High relationships were indicated between oligopeptides level and SOD (r=0.92), CAT (r=0.99) and PER (r=0.75). Significant links were observed also between SOD and ALT (r=0.58) and SOD and AST (r=0.69), high correlations were shown between CAT and ALT (r=0.98), PER and ALT (r=0.82), PER and AST (r=0.89) and negative relationships were noted between GR and both aminotransferases (r=-0.99). Toxicants, including trace elements may bind carboxyl, amino, sulfhydryl, phosphate and other groups of proteins, and modify enzyme activities, membrane structure and its permeability. The changes of aminotransferase activities cause the disturbances of Kreb’s cycle (Van der Oost et al., 2003). Thus, decrease of enzymatic activity in the liver of skates may result from increased exposure to them due to the ingestion of toxicant-containing sediments and food, because accumulation of toxicants in fish tissues, particularly in the liver, may damage hepatocytes, enzyme structure and inhibit their activity.

5 Conclusions
The interspecies variations of hepatic antioxidant enzyme activities between sharks and rays may be connected with high pollution and deficient oxygen concentrations in the benthic environment. Specifically, the increased hepatic antioxidant enzyme activity in the two benthic species in this study may be the result of increased protection to decreasing oxygen and hypoxic conditions. The hepatic aminotransferase activity was higher in dogfish than in ray and skate. The differences between ray, skate and shark are very important from an evolutionary and ecotoxicological view point, because ray and skate are benthic forms and could be used as biomonitors in the Black Sea ecosystem. In terms of hypoxic/anoxic conditions in benthic biotops of Black Sea three tested elasmobranchs may be considered suitable for understanding the adaptation of the marine organisms to the oxygen deficit in deep sea, and especially to the high concentrations of hydrogen sulphyde in the case of Black Sea.

A number of environmental factors, both biotic and abiotic, influence fish metabolic rate and biochemical characteristics. Pollutants may induce changes in metabolism and oxidative stress in aquatic organisms and modulate their biochemical characteristics. Thus, the analysis of enzyme activities in fish tissues is important to understand the adaptive ability of elasmobranchs to survive in increasing amounts of marine pollution. In conclusion, the biochemical and physiological variables in this study may be suitable for monitoring mechanisms of adaptation in the marine environment, not just for the species in the Black Sea, but also for elasmobranchs across the world’s oceans.

We would like to express our gratitude to Dr. Katherine H. Haman, University of Georgia for all her efforts for improving this manuscript, great help, scientific comments, edition it and support. Thank for anonymous referees. We would like to thank to the fishermen of the Institute of the Biology of the Southern Seas (Sevastopol, Ukraine) for providing the examined fish species.

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