Theme: 1. Environmental problems. Subtheme: Climatic change /pollution.
Antioxidative enzyme in Mugil cephalus as a Bioindicators for aquatic Metal pollution
Sneha More*, Dr. Smita Durve
Department of Zoology",
SIES College of Arts, Science and Commerce, Sion (W).
Metals act as an inducer of generating Reactive oxygen species (ROS) which cause oxidative stress in marine fish. Most of the Antioxidant enzymes are sensitive and respond differently towards accumulating metals in different organs of fish. These studies aim at assessing the effect of accumulated metals on the activities of antioxidant enzymes like superoxide dismutase (SOD) and Catalase (CAT) in the different organs like muscle and liver of fish from the natural aquatic environment. Assessment of accumulated heavy metals from tissues was carried out using Inductively Coupled Plasma –Atomic Emission Spectroscopy (ICP-AES) and Inductively Coupled Plasma Mass Spectroscopy ICP-MS. The antioxidant activity of metalloenzyme like the one SOD was measured based on its inhibition of reduction of Nitro blue tetrazolium using a spectrophotometric assay. Whereas CAT activity was measured by a decrease in the absorption of hydrogen peroxide with time before and after decomposition by it at 240nm. The result of this study considers SOD and CAT as a sensitive bio indicator of oxidative stress caused by accumulated metals.
Metals, ROS, Oxidative stress, ICP-AES, ICP-MS, SOD, CAT
Aquatic metal pollution is mostly caused due to untreated sewage; agricultural runoff along with pesticides and herbicides, effluent discharges from metal, processing and manufacturing industries is dumped into the aquatic ecosystem (Sanchez, Palluel, & Meunier, 2005). (Batista, et al., 2014). Heavy metal like Fe, Cu, Zn & Mn from marine water has the tendency to bio accumulated in the marine organism and can cause oxidative stress by generating highly reactive oxygen species (ROS), such as superoxide radical, hydroxyl radical and hydrogen peroxide which often cause damage to cell structure or cell death which can be revealed by studies using light microscopy and scanning electron microscopy. Accumulation of metal in different organ at different concentration depends on the route of exposure to metal and may show different activity of metallo enzymes i.e CAT and SOD (Mahurpawar, 2015) (Pandey, et al., 2008) (Farombi, Adelowo, & Ajimoko, 2007) (Vieira, Gravato, Soares, Morgado, & Guilhermino, 2009). To combat with this oxidative stress and metal poising, antioxidant enzymes like superoxide dismutase (SOD) and Catalase (CAT) provide antioxidant defence system (ADO) against reactive oxidative species (ROS) within organism and plays an important role to cope with free radical in several ways (Atli & Canli, 2010) (Jackim, Hamlin, & Sonis, 1970) (Hansen, Romma, Garmo, Olsvik, & Andersen, 2006). SOD and antioxidant enzyme catalyse the reduction of superoxide radical into hydrogen peroxide, where CAT eliminates hydrogen peroxide a non-radical oxygen species that can cause tissue damage and can inactivate enzymes. CAT mostly get stimulated by most of the metals like Cd, Zn, Cu & Cr and there is a sharp decrease in the activity of CAT by Ag accumulation (Atli, Alptekin, Tukel, & canli, 2006). Liver and kidney are the main organ endowed with antioxidant defence system consisting of the antioxidant enzymes which protect these organs with oxidative stress and prevent this organs from damage (Jorgensen , 2010) (Kanak, Dogan, Eroglu, & Atli, 2014).
SOD are of three types depend on their active metals sites : copper and zinc (Cu/Zn-SOD) has ligands for copper and zinc, which are six histidine and one aspartate side-chain; one histidine is bound between the two metals, Manganese (Mn-SOD) and Iron (Fe-SOD) mostly in eukaryotes (Petkar, Pillai, Kulkarni, Bondre, & Roa, 2013) whereas CAT contain four iron- containing heme group that allow the enzyme to react with the hydrogen peroxide therefore this enzyme are also considered as metalloenzymes (Tainer, Getzoff, Richardson, & Richardson, 1983).
This research sheds light on the activity of antioxidant enzymes CAT and SOD in Mugil cephalus, which are influenced by the accumulated heavy metals due the metal pollution in aquatic environments. The accumulated metals from different organs of Mugil cephalus here estimated by Inductively Coupled Plasma-Atomic Emission spectrometry (ICP-AES) and Inductively Coupled Plasma- Mass spectrometry (ICP-MS) and enzyme activity of antioxidant and metallo enzymes like CAT and SOD were measured. Enzyme activities are considered as sensitive biochemical indicators of oxidative stress due to metal accumulation, hence they can consider as biomarkers for oxidative stress and metal pollution in aquatic environments.
Material and Methods:
Estimation of Metal by ICP-AES and ICP-MS in fish tissues.
Commercially significant fish Mugil cephalus were selected for the experiment and were collected from Sassoon Dock station by various fishing methods by local fishermen. Six fish sample from each six fish catch were collected. The specimens were stored in an ice box and transported to the laboratory. Total length (cm) and weight (g) were measure before dissection table.1. Organs from the fish were dissected out and acid digestion of organs like Liver and the muscle were carried out using conc. HNO3 and conc. HClO4 and sample after digestion was filtered using membrane syringe filter of 25MM (TARSONS SYRINGE FILTER 25 MM (PSF) LOT No. A020216, product no.521090). TDS was adjusted with digital TDS meter (HM Digital Aquapro water tester TDS Meter). Heavy metals analysis for Zn, Cu, Mn was carried out from digested sample using ICP-AES (Model-ARCOS (simultaneous ICP Spectrometer)).
Table: 1. Total weight, length and width of Mugil cephalus from Sassoon Dock
Total length (cm)
Diurnal coastal, estuaries, rivers and mud bottoms
Production of homogenates:
Mugil cephalus from Sassoon dock were collected, liver and muscle was dissected. 5% homogenate were prepared in 0.25 M sucrose in phosphate buffer of pH 7.6 and store in deep freezer and further used for enzyme assay.
Catalase Activity (Luck, 1974) .
Catalase enzyme assay was carried out using 50 mM potassium phosphate buffer pH 7.6, 0.036% (w/w) Hydrogen peroxide solution as substrate where used with absorbance of 0.550 to 0.520 at λ max 240nm. The reaction started by adding enzyme extract to mixture. The decomposition of hydrogen peroxide by catalase result in decrease in absorption with time and time were recorded for the decrease in absorbance. Catalase activity were calculated in U/ml i.e one unit will decompose 1 µ mole of H2O2 per minute at pH 7 at 25 °C, while the H2O2 concentration falls from 10.3 mM to 9.2 mM. The rate of disappearance of H2O2 was followed by observing the rate of decrease in the absorbance at 240 nm.
Superoxide Dismutase .
Super oxide radical scavenging modified assay (Madamanchi, Donahue, Cramer, Alscher, & Pedersen, 1994)
In modified assay for SOD using 0.1M Phosphate buffer (pH 7.8), 65mM methionine, 750 uM nitroblue tetrazolium, 0.2 mM riboflavin, 0.001 mM EDTA and enzyme extract. Reaction were initiated after the addition of enzyme and riboflavin and were incubated under tube light source for 15 mins. Second sets of tubes was kept in dark as control. And absorbance was measured at 560 nm. One unit of enzyme activity is defined as the amount of SOD capable of inhibiting 50% of nitrite formation under assay conditions.
The Cu and Zn content is highest in the Liver i.e 1.26 & 3.91µg/g at the same time Activity of CAT was also found to high at 160 U/ml in liver and 17.25 U/ml in Muscle of Mugil cephalus for respective Conc. of Cu & Zn i.e 0.08 and 0.49 µg/g. The lowest accumulation was found in the muscle of Cu & Zn i.e 0.03 & 0.12 µg/g respectively, which also corresponds to lower activity of CAT in respective Conc. i.e 6.9 & 6.9 U/ml respectively. Table 2 and Fig 1 & 2
Table 2. CAT enzyme activity U/ml and content of Metal µg/g in the Liver and Muscle in Mugil cephalus form Sassoon Dock
Liver CAT U/ml
Muscle CAT U/ml
Fig 1: CAT activity U/ml in the liver of Mugil cephalus under the influence of Cu & Zn µg/g from Sassoon Dock
Fig.2 : Catalase activity in Muscles of Mugil cephalus under the influence of Cu &Zn µg/g from Sassoon Dock
Super oxide dismutase Activity:
The Cu, Zn and Mn content are highest in the Liver i.e. 1.21, 3.91 and 0.55 µg/g at the same time Activity of SOD was also found to be high at 5.35 U/ml in liver and 5.06 U/ml in Muscle of Mugil cephalus for respective Conc. of Cu, Zn & Mn i.e. 0.05, 0.2 & 0.15 µg/g. The lowest accumulation of Cu, Zn & Mn was found in the muscle i.e. 0.03, 0.12 & 0.09 µg/g, respectively, but it did not correspond to lower activity of SOD in respective coins. i.e. 3.30 U/ml for Cu metal whereas SOD activity was lowest with respective to lower concentration of Zn and Mn in the muscle of Mugil cephalus.Table No.3 Fig 3 & 4.
Table 3. SOD enzyme activity U/ml and content of Metal µg/g in the Liver and Muscle of Mugil cephalus from Sassoon Dock
Liver SOD U/ml
Liver Cu µg/g
Liver Zn µg/g
Liver Mn µg/g
Muscle SOD U/ml
Muscle Cu µg/g
Muscle Zn µg/g
Muscle Mn µg/g
Fig. 3: SOD activity in Muscles of Mugil cephalus under the influence of Cu, Zn & Mn µg/g from Sassoon Dock
Fig .4: SOD activity in Muscles of Mugil cephalus under the influence of Cu, Zn & Mn µg/g from Sassoon Dock
This work is on SOD and CAT in the population of Mugil cephalus with different concentration of heavy metal accumulation in the liver and the muscle from Sassoon Dock. The highest concentration of metal was found in the liver i.e of Zn 3.91 µg/g, Cu i.e 1.26 µg/g followed by Mn 0.12 µg/g. Where as the lowest concentration of metal was found in the muscle i.e Cu 0.03 µg/g followed by Mn i.e. 0.09 µg/g and Zn i.e. 0.12 µg/g.Table no.2 & 3. Liver show highest accumulation of most of heavy metals in it (Malik, Biswas, Qureshi, Borana, & Virha, 2010).
Bioaccumulation of this metal in the organs of Mugil cephalus have thought to produce oxidative stress in the organs of fish and to combat with this oxidative stress, antioxidative enzymes like SOD and CAT provide a defense mechanism against reactive oxidative species (ROS) within the tissues of fish and plays an important role to cope with free radicals in several ways. Exposure to tissues to heavy metals increases SOD and CAT activity to cope up with stress condition. Research on it has also suggested that increased activity of SOD and CAT can be a warning sign of oxidative stress and tissue damage hence SOD and CAT activity may be considered as a sensitive bio indicator for the antioxidant defence system due to oxidative stress caused due to metal pollutants (Atli, Alptekin, Tukel, & canli, 2006).
In this study response of CAT activity and SOD activity in different tissues, i.e. the liver and the muscle with respect to bio accumulated metals like. Cu, Mn and Zn were studied. Bioaccumulation of metals in the tissue of Mugil chephalus is the main concerned, which indicated metal pollution in the aquatic environment. In addition, to monitor metal toxicity CAT and SOD activity were considered as a sensitive biomarker for aquatic metal pollution. (Farombi, Adelowo, & Ajimoko, 2007)
The results of this study show significant elevation in the activity of CAT and SOD in the liver i.e. 160 U/ml and 5.35 U/ml, respectively, with corresponding high accumulation of metals like Cu, Zn and Mn i.e. 1.26, 3.91 and 0.55 ug/g. Due to accumulation of metals in the tissue had led to oxidative stress, which has increased in activity of CAT and SOD in liver of Mugil chephalus. Whereas the activity of CAT and SOD in the muscle i.e 17.25 U/ml and 5.06 U/ml increased with respective to increase in the concentration of accumulated metals in the muscles i.e. Cu 0.08 ug/g, Zn 0.49 ug/g. The lowest accumulation was found in the muscle of Cu & Zn i.e 0.03 & 0.12 µg/g respectively, which also corresponds to lower activity of CAT in respective Conc. i.e 6.9 & 6.9 U/ml. Table 2 and Fig 1 & 2. The lowest accumulation of Cu, Zn & Mn was found in the muscle i.e. 0.03, 0.12 & 0.09 µg/g, respectively, but lowest Cu conc. i.e. 0.03 µg/g did not correspond to lower activity of SOD i.e. 3.30 U/ml, whereas SOD activity was lowest with respective to lower conc. of Zn and Mn in the muscle of Mugil cephalus.Table No.3 Fig 3 & 4. Therefore, it can be concluded that the lowest concentration of Cu is enough to increase the activity of SOD by increasing oxidative stress in the tissue of Mugil cephalus from Sassoon Dock.
Atli, G., & Canli, M. (2010). Response of antioxidant system of freshwater fish Orechromis niloticus to acute and chronic metal (Cd, Cu, Cr, Zn, Fe) exposures. Exotoxicology and Environmetla Safety, 1884-1889.
Atli, G., Alptekin, O., Tukel, S., & canli, M. (2006). Response of catalase activity to Ag, Cd, Cr, Cu and Zn in five tissues of freshwater fish Oreochromis niloticus. Comparative Biochemistry and Physiology, Part C , 218-224.
Batista, M., Rodrigues, E., Feijo-Oliveira, M., Ribeiro, A., Suda, C., & Vani, G. (2014). Tissu levels of the anitoxidant enzymes superoxide dismutase and catalase in fish Astyanax bimaculatus from the Una River Basin. Amblente & Agua - An Interdisciplinary Journal of Applied Science, 621-631.
Farombi, E., Adelowo, O., & Ajimoko, Y. (2007). Biomarkers of Oxidative Stress and Heavy Metal Levels as Indicators of Environmental Pollution in African Cat Fish (Clarias gariepinus) from Nigeria Ogun River. Internation Jorunal of Environmental Research and Public Health, 158-165.
Hansen, B., Romma, S., Garmo, Olsvik, P., & Andersen, R. (2006). Antioxidative stress proteins and their gene expression in brown trout (salmo trutta) from three rivers with different heavy metals levels. Comparative Biochemistry and Physiology, 263-274.
Jackim, E., Hamlin, J., & Sonis, S. (1970). Effects of Metals poisoning on Five Liver Enzymes in the Killifish (Fundulus heteroclitus). J. Fish. Res, 383-390.
Jorgensen , S. (2010). A derivative of encyclopedia of ecology . Ecotoxicology, 390.
Kanak, E., Dogan, Z., Eroglu, A., & Atli, G. (2014). Effects of fish size on the response of antioxidant systems of Orechromis niloticus following metal exposures. Fish Physiol Biochem.
Luck, H. (1974). Methods in Enzyme Analysis 2. In Bergmeyer. Academic press New York.
Madamanchi, N., Donahue, J., Cramer, C., Alscher, R., & Pedersen, K. (1994). Differential response of Cu, Zn superoxide dismutase in two pea cultivars during a short-term exposure to sulfur dioxide. Plant Molecular Biology, 95-103.
Mahurpawar, M. (2015). Effects of Heavy Metals on Human Health. International journal of Research Granthaalayah, 1-7.
Malik, N., Biswas, A., Qureshi, T., Borana, K., & Virha, R. (2010). Bioaccumulaltion of heavy metals in fish tissues of a freshwater lake of Bhopal. Environ Monit Assess, 267-276.
Pandey, S., Parvez, S., Ansari, R., Ali, M., Kaur, M., Hayat, F., . . . Raisuddin, S. (2008). Effects of exposure to multiple trace metals on biochemical, histological and ultrastrual features of gills of a freshwater fih, channa punctata Bloch. Chemico-Biological Interactions, 183-192.
Petkar, M., Pillai, D., Kulkarni, A., Bondre, S., & Roa, D. (2013). Purification and Characterization of Superoxide Dismutase Isolated From Sewage Isolated E.coli. Microbial & Biochemical Technology, 102-106.
Sanchez, W., Palluel, O., & Meunier, L. (2005). Copper-induced oxidative stress in three-spined stickleback:relationship with hepatic metal levels. Enviornmental Toxiocology and Pharmacology, 177-183.
Tainer, J., Getzoff, E., Richardson, J., & Richardson, D. (1983). Structure and mechanism of Copper, Zinc superoxide dismutase. Nature, 284-290.
Vieira, L., Gravato, C., Soares, A., Morgado, F., & Guilhermino, L. (2009). Acute effects of copper and mercury on the estuarine fish pomatoschistus microps: Linking biomarkers to behaviour. Chemosphere, 1416-1427.
SOD activity in Muscle of Mugil cephalus under the influence of Cu, Zn & Mn
Muscle U/ml 5.0588235294117645 3.3 2.5 1.7 1.5 4.9411764705882346 Cu µg/g 0.05 0.03 0.06 0.04 0.04 0.08 Zn µg/g 0.2 0.21 0.32 0.12 0.25 0.3 Mn µg/g 0.15 0.1 0.13 0.09 0.12 0.49
SOD activity U/ml
Metal Conc. µg/g
CAT activity in Liver of Mugil cephalus under the influence of Cu & Zn
Liver CAT U/ml 35.567010309278352 151.64835164835165 69 138 160.46511627906978 45.999999999999993 Cu µg/g 0.5 1.21 0.9 1 1.26 0.8 Zn µg/g 1.5 3.52 2.1 2.6 3.91 1.8
CAT activity U/ml
Metal Con. µg/g
CAT activity in Muscle of Mugil cephalus under the influence of Cu & Zn
Muscle CAT U/ml 6.9 6.9 8.625 6.9 6.9 17.25 Cu µg/g 0.05 0.03 0.06 0.04 0.04 0.08 Zn µg/g 0.2 0.21 0.32 0.12 0.25 0.49
CAT activity U/ml
Metal Conc. µg/g
SOD activtiy in Liver of Mugil cephalus under the influence of Cu, Zn &Mn
Liver SOD U/ml 3 5.35 4.5 2 4.9000000000000004 5.24 Cu µg/g 0.5 1.21 0.9 1 1.1599999999999999 0.8 Zn µg/g 1.5 3.91 2.1 2.6 3.52 1.8 Mn µg/g 0.12 0.55000000000000004 0.3 0.32 0.42 0.45
SOD activity U/ml
Metal Conc. µg/g