Biochemical Markers of Oxidative Stress in Brain of Zebrafish Danio rerio Exposed to Different Heavy Metals Lead and Cobalt

Aquatic organisms have been considered to concentrate metals several times greater than environmental levels. Fishes have been used for many decades to evaluate the pollution status of water and thus considered as excellent biological indicator of heavy metals in aquatic environments. Heavy metals are natural tress components of the aquatic environment, but their levels have increased due to domestic, industrial, mining and agricultural activities. These heavy metals when accumulated in the fish tissues, they damage and weaken the mechanisms concerned leading to physiological, pathological and biochemical changes. The lead is non essential element while cobalt is an essential element for living organisms but its presence in fresh water in higher concentration are toxic to organism’s brain, liver, ovary, kidney and gills of the fish. The present study was aimed to investigate the changes due to two heavy metals (lead & cobalt) on the activity of the antioxidant enzyme, Catalase (CAT), Reduced glutathione (GSH), and Lipid peroxidation (LPO) in the brain of Danio rerio during 5, 10, 15 and 20 days of exposure period. For this study adult fishes were exposed to four different concentrations viz., 20, 30, 40 and 50 mg/l of cobalt and 5, 9, 13, and 17 mg/l of lead. Key-wordsZebrafish, Lead, Cobalt, Catalase, LPO, Glutathione, Heavy metals INTRODUCTION Heavy metals are produced from a variety of natural and anthropogenic sources [1] . In aquatic environments, heavy metal pollution results from direct atmospheric deposition, geologic weathering or through the discharge of agricultural, municipal, residential or industrial waste products, also via wastewater treatment plants [2-4] . The contamination of heavy metals and metalloids in water and sediment, when occurring in higher concentrations, is a serious threat because of their toxicity, long persistence, and bioaccumulation and bio magnification in the food chain [5] . Generally, metals can be categorized as biologically essential and non-essential. The nonessential metals for example, aluminum (Al), cadmium (Cd), mercury (Hg), tin (Sn) and lead (Pb) have no proven biological function also called xenobiotics or foreign elements and their toxicity rises with increasing concentrations [6] . Essential metals for example, copper (Cu), zinc (Zn), chromium (Cr), nickel (Ni), cobalt (Co), Molybdenum (Mo) and iron (Fe) on the other hand, have Access this article online Quick Response Code Website: www.ijlssr.com DOI: 10.21276/ijlssr.2017.3.6.10 known important biological roles [7] , and toxicity occurs either at metabolic deficiencies or at high concentrations [8] . The deficiency of an essential metal can therefore cause an adverse health effect, whereas its high concentration can also result in negative impacts which are equivalent to or worse than those caused by non-essential metals [9] . The river system may be extremely contaminated with heavy metals released from domestic, industrial, mining and agricultural effluents. Heavy metal contamination may have disturbing effects on the ecological balance of the recipient environment and a diversity of aquatic organisms [10-11] . Among animal species, fishes are inhabitants that cannot escape from the detrimental effects of these pollutants [12] . The impact of metals, as well as other pollutants, on aquatic biota can be evaluated by toxicity test, which are used to detect and evaluate the potential toxicological effects of chemicals on aquatic organisms. However, little research has been done on the impact of contaminations on tropical ecosystems [13] . Fish are widely used to evaluate the health of aquatic ecosystems because pollutants build up in the food chain and are responsible for adverse effects and death in the aquatic systems. Fish can obtain their trace elements, either directly from the water through the gills or indirectly from food through the alimentary tract [14] . Heavy metals have been recognized as strong biological poisons because of their persistent nature, tendency to RESEARCH ARTICLE Int. J. Life. Sci. Scienti. Res., 3(6):1484-1494 November 2017 Copyright © 2015-2017| IJLSSR by Society for Scientific Research is under a CC BY-NC 4.0 International License Page 1485 accumulate in organisms and undergo food chain amplification [15] , they also damage the aquatic fauna. The contamination of freshwaters with a wide range of pollutants has become a matter of great concern over the last few decades. Lead is a persistent metal which is commonly used in various industrial processes. It is toxic to living systems and may stay in the environment for a prolonged period of time, due to its persistency; it exists as a free metal in various compounds. Lead is a widespread environmental and occupational xenobiotic and is hazardous to humans and various ecosystems [16] . Its exposure to humans is mainly by ingestion through the mouth and inhalation from fumes and dust in the atmosphere [17] . Exposure to lead is mainly from anthropogenic sources due to its widespread usage. The form in which lead exists determines how toxic it is in the environment. Several studies link inorganic lead like lead acetate compounds to increased incidence of diseases in various organisms. Lead toxicity has been linked to incidence of neurological disorders, hypertension, cognitive impairments etc [18] . Chen et al. [19] reported that exposure of low doses of developmental lead to the embryo of zebra fish resulted in embryonic toxicity, behavioral alteration, and adult learning/memory deficit in zebrafish. It’s accumulation in sediment is of significance for aquatic organisms. It is not a transition metal and cannot readily undergo valence changes, it can induce oxidative damage through direct effects on the cell membrane, interactions between lead and haemoglobin, which increase the auto-oxidation of hemoglobin, auto-oxidized δ-aminolevulinic acid, interactions with glutathione reductase, or through the formation of complexes with selenium, which decrease glutathione peroxidase activity [20] . Lead deposits in various fish organs like liver, brain, kidneys, spleen, digestive tract and gills [21] . Cobalt is an essential nutrient for man and is an integral part of vitamin B12. It performs important biochemical function but its higher concentration in aquatic ecosystems becomes toxic to fish as it interferes with the enzyme systems [22] . It is reported to be a potential carcinogenic compound and has been included recently in group 2A carcinogens i.e., probably carcinogenic to humans. Cobalt can be absorbed from the surrounding water through the gills as well as from the diet. The uptake of waterborne cobalt increased with a rise in temperature and decrease in waterborne calcium. Also, heavy metals are known to induce oxidative stress and carcinogenesis by mediating free radicals e.g. reactive oxygen species [23] . They deplete glutathione, resulting enhanced production of Reactive Oxygen Species (ROS) such as catalase. ROS are considered as critical mediators for the metal-triggered tissue injuries and apoptosis. To prevent oxidation induced damage, there must be effective anti-oxidation system enzyme including free radical scavenging enzymes, such as Superoxide Dismutase (SOD) and Catalase (CAT) changes in the activity of enzymes and other biomarkers are the possible tool for aquatic toxicological research [24] . Zebrafish can be used for bio-indicator of environmental contamination. MATERIALS AND METHODS The present work was conducted in the Zebrafish laboratory, Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur, India in the duration of April 2017. Zebrafish, recommended by International Organization for Standardization (IOS, 1976) [25] and the Organization for Economic Co-operation and Development (OECD) [26] were collected and acclimatized for a month, stocked and reared under laboratory conditions. The aquariums were aerated continuously through stone diffusers connected to a mechanical air compressor and the water temperature was maintained at 25 ±2 o C. The fishes were fed twice daily alternately with raw and chopped goat liver and shrimp powder. Tubifex worm, Tetrabit and spirulina granules purchased from pets shop were also supplemented. For the present study, mature adult zebrafish approximately 3.5 cm in length and 1 g in weight were procured from stock aquarium and exposed to four different concentrations viz., 20, 30, 40 and 50 mg/l of cobalt and 05.00, 09.00, 13.00 and 17.00 mg/l of lead calculated from our previous toxicity test. The concentrations of heavy metals were decided for exposures were below the range of 80% 96-h LC50 as calculated earlier Singh and Ansari [27] . Low concentrations were selected since fish can survive the stress of the toxicant. Twenty fishes were exposed to each concentration. The water in the aquarium was replaced daily with fresh treatment of metals. Each experiment was accompanied by their respective control. After exposure periods of 5, 10, 15 and 20 days, required number of treated fish were removed from the experimental and control groups. Their brain were removed and processed. Biochemical AssayThe activity of CAT (EC 1.11.1.6) was estimated according to procedures by Sinha [28] . This method is based on the fact that in acetic acid dichromate is reduced to chromic acetate when heated in the presence of H2O2 with the formation of perchromic acid as an unstable intermediate. The chromic acetate is measured colorimetrically at 620 nm. The catalase preparation is allowed to split H2O2 at different time intervals by the addition of a dichromic acetic acid mixture and the remaining H2O2 is determined colorimetrically. The results were expressed as μM H2O2 utilized/min/mg protein. Glutathione (GSH) content in the brain was estimated according to the method of Paglia et al. [29] . Tissue (brain) was lyses with 2.0 ml of 1g/l EDTA (ethylene diamine tetraacetic acid) solution and 1.5 ml