Role of Copper Sulphate on Oxidative and Metabolic Enzymes of Freshwater Fish; Channa Punctatus

Present day heavy metal pollution has become global concern. The early detection of heavy metal ions, especially bioavalable metal ion, in the environment is very important to safeguard to human health. Copper is an essential trace metal in small quantity for several fish metabolic functions. Essentiality of copper arises from its specific incorporation into a variety of enzymes such as peroxidase, xanthine oxidase, invertase, glucose oxidase and protease papain and bromelain [1,2] which play important roles in physiological processes (e.g. enzymes involved in cellular respiration, free radical defense, neurotransmitter function, connective tissue biosyntheses and other functions), as well as, proteins. Copper is used today a chemotherapeutic agent in aquaculture however, the increased level of copper in aquatic environments coming from sewage, industries (electroplating, mining and metallurgy) and agricultural wastes [3,4]. Metals can either increase or decrease hepatic enzyme activities and can lead to histo-pathological hepatic changes, depending on the metal concentration, fish species, and length of exposure period [5]. Transaminase enzymes play vital role in carbohydrate-protein metabolism in fish tissues [6]. Changes in enzymes activity and other biomarkers have been studied as possible tools for aquatic toxicological research [7].


Introduction
Present day heavy metal pollution has become global concern. The early detection of heavy metal ions, especially bioavalable metal ion, in the environment is very important to safeguard to human health. Copper is an essential trace metal in small quantity for several fish metabolic functions. Essentiality of copper arises from its specific incorporation into a variety of enzymes such as peroxidase, xanthine oxidase, invertase, glucose oxidase and protease papain and bromelain [1,2] which play important roles in physiological processes (e.g. enzymes involved in cellular respiration, free radical defense, neurotransmitter function, connective tissue biosyntheses and other functions), as well as, proteins. Copper is used today a chemotherapeutic agent in aquaculture however, the increased level of copper in aquatic environments coming from sewage, industries (electroplating, mining and metallurgy) and agricultural wastes [3,4]. Metals can either increase or decrease hepatic enzyme activities and can lead to histo-pathological hepatic changes, depending on the metal concentration, fish species, and length of exposure period [5]. Transaminase enzymes play vital role in carbohydrate-protein metabolism in fish tissues [6]. Changes in enzymes activity and other biomarkers have been studied as possible tools for aquatic toxicological research [7].
Increasing population, industrialization and agricultural production has resulted in increasing the number of freshwater systems which are being impaired by the contaminants present in wastewater releases [8,9]. Micronutrient interact with toxic metals at several points in body, absorption and excretion of toxic metals, transport of metals in body, binding to target proteins, metabolism and sequestration of toxic metals, and finally in secondary mechanisms of toxicity such as oxidative stress. Copper speciation is directly affected by water pH, and the free cupric ion concentration is higher in water with low pH, while a copper hydroxide complex prevails in water with high pH [10,11].
The aim of present study was to evaluate effect on metabolic indicators by sub-acute concentration of copper sulphate to a freshwater fish, Channa punctatus. The effects on organ were assessed by ALAT, ASAT, CAT and protein content.

Experimental groups
The metabolic indicator effect of copper was studied by static bioassay using tap water (University water supply) as dilution medium was estimated by the method of APHA [12]. The changes in physicochemical characteristics, such as temperature, pH, TDS (total dissolved solids), DO (dissolved oxygen), hardness, alkalinity, chloride and iron of experimental water were recorded throughout the experimental period. The small size freshwater fish, Channa punctatus, weighing 15±2 g and measuring11±2 cm, were collected with the help of local fisherman from water bodies located in the sub-region of Lucknow. The fish was properly washed in tap water and treated with 0.02% KMNO 4 and 0.004% formalin solution to remove external infection of fungi and algae. Prior to the experimentation the normal uninfected healthy fishes were selected for experiment. The fish were acclimatized to laboratory conditions 15 days before taken for experimentation. The animals were fed fish (TOKYO) made in Japan on each day in the evening. The LC 50 was estimated employing Trimmed Spearman Karber Method [13] as 3.60 mg l -1 . The sub-lethal dose (0.36 mg l -1 ) was related for exposure to fish for 15, 30 and 45 days.

Design of sub-lethal toxicity study
The fishes were divided into 4 equal groups consisting of 10 each and each group was transferred separately to glass aquaria of 100 L volume. While the Group I fishes were maintained as control without any treatment, the Group II, III and IV fishes were exposed to sub-

Abstract
The effect of copper sulphate on liver, gills and kidney of fish Channa punctatus was observed as metabolic indicators. The fish were exposed to sub-lethal (0.36 mg l -1 ) concentrations for 15, 30 and 45 days. The observations revealed that in the presence of copper sulphate the metabolic enzymes aspirate amino transferases (ASAT) and alanine amino transferases (ALAT) can enhance their activity, while catalase activity significantly reduced in response to elevated level of superoxide production in exposed fish as compared to healthy subjects. The major effect of copper sulphate toxicity on exposed fish shows as reduced amount of protein content as compared to unexposed fish. However, ASAT level have a significant negative correlation between catalase activity (r = 0.833, t = 6.3872, p 0.05) and protein content (r = 0.8916, t = 8.3540, p 0.05) in the exposed organs. These findings suggest that the significant increase of transaminase activity while reduced amount of catalase and protein content might be the consequences of tissue damage in Channa Punctatus.
lethal concentration (0.36 mg l -1 ) of copper sulphate for 15, 30 and 45 days. The waste products were removed from aquaria water by using good quality of aquaria water filter. The solution of CuSO 4 .5H 2 O (MERCK India) was freshly prepared distilled water before mixing in aquaria water. At the end of the exposure period i.e. 15, 30 and 45 days, the control and treated fish were killed and tissues collected for biochemical studies.

Biochemical analysis
The fish tissues liver, gills and kidney taken out were homogenized for the estimation of aspirate amino transferases (ASAT) and alanine amino transferases (ALAT) method of Reitman and Frankel [14] using a kit procured from Span Diagnostics (Surat, Gujrat), catalase by the method of Euller and Jopshon [15] and protein by the method of Lowry et al [16].

Statistical analysis
The data observed in the experiment were statistically analyzed for the calculation of standard error of mean (SEM). One way ANOVA and Duncan Multiple range test for individual group wise comparison was administrated for testing the hypothesis [17]. The data shown are the average of three replicates ± SEM and statistical significance was tested at p<0.05 level.

Toxicity of copper sulphate on enzyme activity and protein content of exposed fish
The present study has revealed alteration in aspirate amino transferases (ASAT), alanine amino transferases (ALAT), catalase activity and protein content of treated fish (Figure 1-4). ASAT and ALAT activity progressively significant (p<0.05) increased with increase copper exposure period 15, 30 and 45 days as compared to control, respectively. ASAT activity was highly found on 45 days of kidney in comparison to treated group of liver and gills organs in fish (Figure 1), while ALAT activity was highly shows in liver and kidney on 45 days of exposed to copper in comparison to gills organs of Channa punctatus ( Figure 2).
The result clearly indicated catalase activity in liver, gills and kidney were found reduced on 15, 30 and 45 days of exposed fish. Catalase activity was significantly (p<0.05) reduced in gills and kidney on 45 days in comparison to treated group of liver in fish, however 15 and 30 days of copper exposure period catalase activity was nearly found in treated group of liver and kidney in fish, respectively (Figure 3). Protein contents in liver, gills and kidney were found significantly (p<0.05) reduced on 15, 30 and 45 days of copper exposure period in comparison to control, respectively (Figure 4).
Significant negative correlation of asat with both catalase and protein content of copper sulphate exposed organs ASAT  was inhibited in gills, liver and kidney of the freshwater fish Wallago attu (Bl. and Schn.). Asztolos et al. [38] observed increased lactate dehydrogenase (LDH), glutamic oxialoactetic trasmination (GOT) and glutamate dehydrogenase (GIDH) enzyme activity in Cyprinus carpio. Abdel Hameid [39] was observed assayed of enzyme alanine and aspartate amino transferases activity increase and protein contents was significantly reduced in fish Oreochromis aureus.
It is a fact that copper containg materials, capable of entering the body via the food chain, are harmful to the ecosystem. A way of analyzing these illnesses is to highlight the effects of these harmful chemicals on the enzymes. The changes in aspirate amino transferases (ASAT) and alanine amino transferases (ALAT), catalase and protein levels in gill, liver and kidney and following different exposure periods of sub-lethal copper concentration suggested that Channa punctatus showed adaptive elevation in the activity of enzymes in these tissues. These enzymes have a very important role in the metabolical process since they are biological catalysts. The enzymes could be successfully used as potential biomarker of fish health. Their deficiency or surplus indicates damage of body organs in fish. Intake of such type of food materials is very harmful to human beings.
catalase and protein content of all the exposed organs of fish. The ASAT level of fish liver has significant negative correlation with catalase (r = -0.7483, t = 3.67, p< 0.05) and protein content (r = -0.906, t = 6.765, p< 0.05) (Figure 5a-5c). Although a negative correlation observed between level of ALAT with catalase and protein content but it was not significant. Both gills and kidney of exposed fish shows highly significant negative correlation for observed value of ASAT with catalase and protein content (Figure 6a-6c).

Discussion
Fish prefers optimal environmental conditions for their growth and reproduction. Any change in environmental conditions causes stress on fish health. To maintain productivity of the ecosystem, a healthy aquatic environment and production of sufficient fish food organisms in water body is necessary. The liver, gills and kidney enzymes were unexpected since organs damage is indicated by an inhibition of these enzymes took place or a change in the metabolism of the fish. The metals have adverse effect on enzyme activity of fresh water fishes [7,18,19]. The fish developed a protective defense against the deleterious effect of essential and nonessential metals and other xenobiotics that produced degenerative changes like oxidative stress in the fish body [7,20,21]. The result of the present study shows reduced level of protein in liver, gills and kidney similar to the observation recorded by Mastan [22], Kumar and Gopal [23] on depletion level of protein in different organs of fish C. punctatus under the stress of copper and distillery effluents. Dinodia et al. [24] also carried out effect of cadmium toxicity on fresh water species Labeo rohita, Cirrhinus mrigala and Cyprinus carpio as evidenced by reduction in the body tissue and residual protein in all the fish species after 45 days of exposure, which may be due to dysfunction of several physiological and biochemical processes in the body.
Oxygen in its molecular state O 2 , is essential for many metabolic processes that are vital to aerobic life. Aerobic organisms cannot exist without oxygen, which nevertheless is inherently dangerous to their lives. Like all aerobic organisms, fish are also susceptible to the effects of reactive oxygen and have inherent and effective of different biotic and abiotic factors (age, phylogenetic position, feeding behavior, environmental factors, oxygen, temperature, presence of xenobiotics) on antioxidant defenses in fish [25]. The catalase activity decreased all organs with increases copper exposure period at 15, 30 and 45 days of exposure. Jee and Kang [26] also observed effect of phenanthrene exposure on Paralichthys olivaceus after 4 week incubation decreased catalase activity in the liver, gill and kidney tissues, however some study showed that there were no alteration catalase activity [27]. High concentrations of copper reduced catalase activity in the liver, gill and muscle, and 100 ppm ZnSO 4 in the gill and muscle [28,29].
The activity of catalase decreased in liver of fish (Cyprinous carpio) in the Karakaya Dam Lake observed by Yilmaz et al. [30]. Ali et al. [31] observed similar result decreased catalase (CAT), activities liver and gills by fly ash leachate (FAL) exposued on Channa punctata. Buet et al. [32] Juvenile rainbow trout (Oncorhynchus mykiss) were carried out antioxidant enzymes like catalase were decreased in liver throughout the 10 days of uranium exposure. Copper Sulfate stimulation is potential biomarkers of exposure to an oxidative stress [33]. Trace metals, and high temperature, high salinity and light duration significantly decreased catalase activity of Mytilus galloprovincialis [34,35]. Santos et al. [36] the enzymatic and nonenzymatic antioxidants catalase, glutathione peroxidase, glutathione S-transferase and nonenzymatic antioxidant molecule such as glutathione in Anguilla anguilla L. gill, kidney and liver in decreased response to bleached kraft pulp mill effluent (BKPME). Pandey et al. [37] also observed catalase activity