Sublethal impacts of heavy metals on antioxidant enzymes and biochemical parameters in rohu (Labeo rohita)

Item Type article

Authors Anjum Zia, M.; Qurat-ul, Ain; Abudullah, S.; Abbas, Kh.; Ahmed, I.

Download date 29/09/2021 19:01:17

Link to Item http://hdl.handle.net/1834/37838 Iranian Journal of Fisheries Sciences 16(2) 668-683 2017

Sublethal impacts of heavy metals on antioxidant enzymes and biochemical parameters in rohu (Labeo rohita)

Anjum Zia M.1*; Qurat-ul-Ain1; Abudullah S.2; Abbas Kh.2; Ahmed I.1

Received: November 2015 Accepted: February 2016

Abstract The present research was designed to study and compare the biochemical parameters in Labeo rohita due to exceptional nutritional value of this fish as a protein source in developing countries. The protein and heavy metal (Zn, Cu, Ni) activities were determined spectrophotometrically. The proximate analysis of L. rohita collected from different localities of Pakistan revealed that the protein (19.97%) and ash contents (1.76%) were highest in hatchery L. rohita while the fat (0.84%), carbohydrate (5.39%) and dry matter contents (24.11%), were maximum in the river L. rohita. In comparison to the hatchery and river L. rohita, the moisture contents (81.42%) were the highest in farmed fish. During enzymatic analysis, maximum activities of the peroxidase, α- amylase and mutarotase were recorded in hatchery fish whereas the catalase and superoxide dismutase activities were found to be the highest in farmed fish. The maximum accumulation of Cu and Ni metals were observed in hatchery fish and least in farmed fish and the highest Zn accumulation was observed in river fish. Analysis of variance on catalase, peroxidase, α-amylase activity and metal accumulation showed statistically significant differences at p<0.05 among sampling sites and fish organs and interaction between sampling sites and fish organs.

Keywords: Aquaculture, Enzymes, Proximate analysis, Antioxidant enzymes, Metals, Labeo rohita.

1-Enzyme Biotechnology Lab., Department of Biochemistry, University of Agriculture, Faisalabad-38040, Pakistan. 2-Fisheries Research Farms, Department of Zoology and Fisheries, University of Agriculture Faisalabad-38040, Pakistan. *Corresponding author's Email: [email protected] 669 Anjum Zia et al., Sublethal impacts of heavy metals on antioxidant enzymes and biochemical …

Introduction etc. to protect them from the oxidative Rapid industrialization is contaminating effect of heavy metals (Basha and Rani, natural freshwaters by the heavy use of 2003). These enzymes provide the first metals mainly iron, zinc, copper, lead, line of defense against oxygen-derived nickel and manganese turning it into a free radicals and protect the fish tissues global problem. The concentrations of from oxidative stress. Antioxidant trace elements are dangerous not only enzymes can be used as biomarkers of to fish growth and reproduction but also exposure to aquatic contamination for human beings. Because of (Ahmad et al., 2000). However, increasing industrial practice, heavy antioxidant enzymes exhibit different metals enter the aquatic environment activities between the cells, tissues and and are transferred to human beings organs of saltwater fish and freshwater through the food chain. Minerals fish depending upon feeding habitat, perform important roles in environmental conditions and other osmoregulation, intermediary ecological conditions. On the other metabolism and in the formation of hand, catalase and superoxide skeleton, healthy scales, teeth and dismutase enzymes may be highly bones (Vutukuru et al., 2007). influenced by a number of conditions Rohu or roho labeo is a species of viz. temperature, season, salt contents, the carp family and is a natural sex, age and feeding habitats. inhabitant of freshwater and is present Labeo rohita is an emerging in the rivers of Asian countries fisheries resource and its highly viable (Pakistan, India, Bangladesh, Burma significance makes it a promising and Nepal). In Pakistan, this species is aquaculture species in Pakistan and mostly available in the province of other Asian countries. The availability Punjab and due to its non-oily nature; it of data regarding antioxidant enzymes is widely consumed as food. Fish is an and nutritional profile with reference to important source of balanced and easily heavy metal stress on this species is digestible protein, carbohydrates, very limited. So, this work was planned polyunsaturated fatty acids, minerals to evaluate various biochemical i.e. copper, iodine, potassium, parameters including proteins, lipids, phosphorus, iron, and vitamin A and D. carbohydrates, heavy metals and Fish proteins have a high biological activity of various enzymes in L. rohita. value and contain all the essential amino acids and are an excellent source Materials and methods of lysine that is limited in vegetable The fish, L. rohita belongs to the family foods (Vladau et al., 2008). Cyprinidae of fresh water fishes, were Fish tissue, generally the kidney and collected from a Fish hatchery at liver, have antioxidant defense Satiana Road, Faisalabad, Fisheries mechanisms consisting of catalase, Research Farms at University of superoxide dismutase and peroxidase Agriculture, Faisalabad and Taunsa Iranian Journal of Fisheries Sciences 16(2) 2017 670

Barrage Indus River. The nine fish were ammonia was collected approximately dissected and the body organs viz. gills, for 2 minutes. Ammonia was titrated kidney and liver were collected in against sulphuric acid (0.1 N) where triplicate for the studies of enzymes and total amount of H2SO4 used was noted heavy metals. and the percentage of nitrogen was calculated. Proximate analysis of fish meat The proximate analyses viz. moisture, Analysis of antioxidant enzymes crude protein, total fat and total ash For enzyme analysis, fish was dissected were assayed as described by and the organs viz. liver, kidney and Association of Official Analytical gills were collected. Tissue samples to Chemists (AOAC, 1990). The moisture be processed for the determination of percentage was calculated as the loss in enzyme activities were homogenized weight of meat samples. Total fat and (1:4, w/v) in phosphate buffer (pH 6.5). ash was calculated from meat samples. Homogenate samples were centrifuged Total fat was calculated by the at 10,000 rpm for 15 min. at 4ºC. A following formula (AOAC, 1990). supernatant fraction was used to check the activity of various enzymes. Percentage of Total fat = The activity of superoxide dismutase Weight of fat × 100 was found by measuring its capacity to Weight of sample inhibit the photoreduction of nitroblue tetrazolium (Worthington, 1988). One Quantitative estimation of protein unit of superoxide dismutase activity is contents the amount which is required for 50% Determination of protein contents was reduction in color and was expressed in carried out by Kjeldahl method units of the enzyme (U/mg) and (AOAC, 1990). In brief, 1 g of dried absorbance was noted on a sample of meat was placed in a spectrophotometer at 560 nm. digestion flask with 5 g of digestion The catalase activity was measured mixture (FeSO4=5 parts, CuSO4=10 according to the method of Chance and parts and K2SO4= 100 parts) and 25-30 Maehly (1995). Catalase enzyme mL H2SO4. The digestion mixture was activity was determined by measuring boiled for 3 hours on digestion its ability to reduce the hydrogen apparatus and the solution was changed peroxide concentration at 240 nm. The into a greenish color. Sample solution reaction mixture (2 mL) contained 1.95 (10 mL) and sodium hydroxide (40%) mL of buffered substrate solution and were placed in the digestion apparatus when 0.05 mL enzyme extract was and distilled with steam. Ammonia was added, the reaction started in the collected in 2% boric acid solution (10 cuvette and absorbance was recorded at mL). When the pink color of 2% boric 240 nm on the spectrophotometer acid changed to golden color, then within 3 minutes. 671 Anjum Zia et al., Sublethal impacts of heavy metals on antioxidant enzymes and biochemical …

The activity of peroxidase was membrane filter (Type HV). The filtrate determined by measuring its capacity to was analyzed for metals viz. Zn, Cu and decrease the hydrogen peroxide Ni following the method of Andaleeb et concentration at 470 nm (Zia et al., al. (2008) by using Atomic Absorption 2011). The spectrophotometer was set Spectrophotometer Analyst 400-Perkin at 470 nm wavelength, after inserting Elmer (USA). the blank solution. Then 0.02 mL of crude extract was added in 1 mL of Statistical analysis buffered substrate solution and the Analysis of variance was performed to absorbance was noted after 3 minutes. find out statistical differences among α-Amylase is measured by its ability to variables. The results were expressed as reduce 3,5-dinitrosalicylic acid (Niaz et mean ± SE. p values<0.05 were al., 2010; Mirza et al., 2013) and regarded as statistically significant. absorbance was noted against a blank solution at 540 nm. Mutarotase is Results another important enzyme that is Proximate analysis of fish meat samples responsible for spontaneous The proximate analysis of fish meat mutarotation of sugars and its activity samples is given in Fig. 1. The in a supernatant of fish samples was maximum protein contents (19.97%) determined according to the method of were analyzed in hatchery L. rohita Mazhar et al. (2012). while, farmed L. rohita exhibited less protein contents (13.76%). The total fat Determination of heavy metals contents were maximum in river In triplicate, fish organs viz. gills, (0.84%) and minimum in farmed fish kidney and liver were removed and (0.64%). digested, separately with concentrated Lipids are the most important nitric acid (HNO3) and HClO4 by 3:1 constituents of fish, which determine ratio. In a 100 ml tube, the sample and the fish meat quality. The fat contents

10ml concentrated HNO3 were added of fish are variable from species to and heated at 100, 150, 200 and 250˚C species and due to seasonal changes, on a hot plate for 0.5, 0.5, 0.5 and 1.5 physiology, feeding, habitats etc. The hours, respectively. Then HClO4 was carbohydrate contents in hatchery fish added to it. Afterwards we added 2 mL (1.97%) and in river fish (5.39%) were of 1N nitric acid and put it on a hot observed, respectively. plate and heated it until the sample was completely digested and became transparent. The digested samples were then transferred to volumetric flasks (50 mL) and the volume was made up by adding deionized water. The samples were filtered through 0.45 µm Millipore Iranian Journal of Fisheries Sciences 16(2) 2017 672

Figure 1: Proximate composition of Labeo rohita obtained from different habitats.

Ash and moisture contents were found The SOD activity was found lower in highest in hatchery and farmed fish, polluted sites and higher in non- correspondingly. Maximum value of polluted sites that is why farmed fish dry matter (24.11%) and its minimum exhibited higher activity. (18.57%) were noted in farmed fish. Catalase activity: Liver of L. rohita Body composition of L. rohita is from a farmed habitat showed the influenced by a number of factors i.e. highest while the least enzyme activity size, weight and feed intake. was found in gills of hatchery fish (Fig. 3). Analysis of antioxidant enzymes Peroxidase activity: Peroxidase activity Superoxide dismutase activity: in different organs (liver, kidney, gills) Superoxide dismutase (SOD) activity in of L. rohita is given in Fig. 4. Highest different organs (liver, kidney, gills) of activity of peroxidase was observed in L. rohita is given in Fig. 2. Higher kidney (0.701 U mL-1) of hatchery L. activity of superoxide dismutase was rohita and minimum activity was noted observed in kidney (88.4 U mL-1) of in the liver of farmed fish. The overall farmed fish while, the least activity was highest activity of peroxidase was found in liver (74.68 U mL-1) of observed in kidneys of L. rohita of all hatchery fish. The activity of SOD in the three habitats. The activity of organs of L. rohita from different peroxidase in organs of L. rohita from localities showed that an increase in different localities showed an increase activity formed the trend in activity to formed the trend farm>river>hatchery fish. hatchery>river>farmed fish. 673 Anjum Zia et al., Sublethal impacts of heavy metals on antioxidant enzymes and biochemical …

Figure 2: Superoxide dismutase activity in different organs of Labeo rohita.

Statistical analyses reveal that different differences at p>0.05 among fish enzyme (Superoxide dismutase, organs. catalase, peroxidase) activity in L. rohita is highly significant at p<0.05 Determination of heavy metals among sampling sites and fish organs, Zinc accumulation: The leading as well as the interaction between accumulation of zinc was observed in sampling sites and fish organs. the kidney of river fish i.e. 13.11 µg/g α-Amylase activity: Higher α- amylase and smallest amount in liver (3.12 µg/g) activity was found in liver i.e. 7.75 U of farmed fish (Fig. 7). The maximum mL-1 of hatchery fish and lower in meditation of Zn was found in kidney kidney (1.72 U mL-1) of farmed fish and gills of L. rohita. (Fig. 5). Copper accumulation: The maximum Mutarotase activity: Fig. 6 showed the accumulation of copper was recorded in mutarotase activity in liver, kidney and the kidneys (13.17 µg/g) of hatchery gills of L. rohita. Maximum and and minimum of 0.313 µg/g in the liver minimum mutarotase activity was of farmed fish (Fig. 8). In the present found in liver (7.49 U mL-1) of hatchery research work, the maximum L. rohita and in gills (6.04 U mL-1) of concentration of Cu was found in farm fish. However, the interaction kidneys while liver and gills of L. between sampling sites and fish organs rohita also exhibited a significant level. remained statistically at par. In kidney, the highest accumulation of Analysis of variance on mutarotase and metals altered the levels of biochemical α- amylase activity in fish showed parameters for heavy metal significant differences at p<0.01 among detoxification in the body. sampling sites and no significant Iranian Journal of Fisheries Sciences 16(2) 2017 674

Figure 3: Catalase activity in different organs of Labeo rohita.

Figure 4: Peroxidase activity in different organs of Labeo rohita.

675 Anjum Zia et al., Sublethal impacts of heavy metals on antioxidant enzymes and biochemical …

Figure 5: α- Amylase activity in different organs of Labeo rohita.

Figure 6: Mutarotase activity in different organs of Labeo rohita.

Iranian Journal of Fisheries Sciences 16(2) 2017 676

Figure 7: Concentration of Zn in different organs of Labeo rohita.

Figure 8: Concentration of Cu in different organs of Labeo rohita.

Nickel accumulation: The highest normal range expected for Ni. The dose accumulation of nickel was observed in of a toxic metal that one obtains from kidney of hatchery fish and the detailed fish however, not only depends on the results are shown in Fig. 9. The concentration of specific metals in fish, maximum concentration of Ni was but also on the quantity of fish found in liver and kidney of L. rohita consumed. Although levels of heavy whereas gills also showed a significant metals are not high, care must be taken level. considering some people regularly consume large quantities of fish. Daily consumption safety Our study demonstrated that the concentrations of heavy metal were in 677 Anjum Zia et al., Sublethal impacts of heavy metals on antioxidant enzymes and biochemical …

Table 1: Standard levels and maximum range of concentrations of heavy metals (μg/g wet weight) in fish described in literature. Metals Organization/Country Ni Cu Zn FAO (1983) - 30 30 FAO/WHO limits (1989) - 30 40 Turkish guidelines (Dural et al., 2007) - 20 50 Range of metal in present study 2.25-3.43 0.313-13.17 3.12-13.11 All tissue concentrations are in μg/g wet weight.

Figure 9: Concentration of Ni in different organs of Labeo rohita .

Genarally, in the present study, it was Discussion observed that in farmed fish the Proximate analysis of fish meat samples accumulation of heavy metals was not The maximum protein contents of fish to a great extent and was considered were due to the good food consumption suitable for consumers as compared to and its conversion into the protein in wild and hatchery fish. The meditation the fish meat as previously reported of heavy metals in kidney was highest (Mahboob et al., 2004). It was observed than its corresponding values in other that there was a significant increase in tissues. The metal accumulation was protein contents with an increase in varied in different tissues within the body weight of fish. same fish affecting the activity of Carbohydrates are also important but antioxidant enzymes. Although levels they are present in very less amounts in of heavy metals are not high, care must fish. The percentage of moisture is a be taken considering some people good indicator to find lipids and protein regularly consume large quantities of contents in fish. The lower the fish. percentage of moisture, the greater the lipids and protein contents (Dempson et Iranian Journal of Fisheries Sciences 16(2) 2017 678 al., 2004). Vladau et al. (2008) reported and wild fish. Presence of heavy metals that fish has protein contents in the showed inhibitory effect on catalase range of 13 and 25%, which accounts activity. McFrarland et al. (1999) for 80 to 90% of the energy content of reported that catalase activity was the fish. The surveillance indicated that found lower in the contaminated black variations in proximate compositions of river fish as compared to the least L. rohita were due to different habitats. contaminated environment. Peroxidase activity: In fish, generally Analysis of antioxidant enzymes the kidney and liver have antioxidant Superoxide dismutase activity: Gills defense systems to defend them from showed higher activity than liver and oxidative stress of heavy metals (Basha kidney tissues. Gills have direct contact and Rani, 2003). Peroxidase activity with the different environmental factors was varying among the cells, tissues and they are the primary route for the and organs, depending upon ecological entry of toxicants, heavy metals and conditions, feeding habitat and other pesticides. According to Jordanoska et environmental factors (Winston and Di al. (2008) the superoxide dismutase Giulio, 1991). activity differs from one species to The liver is a major site of another, from one tissue to another and detoxification and the first target of from freshwater to marine environment ingested oxidants and a very important as well. Depending on the degree of tissue in the study of the role of water pollution, fish have the ability to peroxidase in protection from lipid retain large amounts of pollutants in peroxidation. Velma and Tchnounwou their tissues, either directly from the (2010) made an attempt to evaluate water by breathing or through their diet. organ specific toxic effects of Catalase activity: Catalase activities hexavalent chromium in kidney and may be decisively influenced by a liver of freshwater gold fish during number of environmental factors such acute exposure. Their results indicated as temperature, salinity, season and that activity of peroxidase in both feeding habitats, age, sex. Shen et al. kidney and liver showed a significant (2007) observed the catalase activity in increase and altered as compared to the Brocarded carp liver under pollutant control. Bray and Bettger (1990) exposure and the results showed that observed the activity of peroxidase in there is a decrease in catalase activity liver of G. brasiliensis and results due to the fluctuation of superoxide showed that the activity of enzymes is radicals. Their findings agreed with the affected by the presence of zinc and present research work, whereas the copper potent activators. highest catalase activity was found in α-Amylase activity: Change in digestive liver of farmed fish and then due to the enzyme activity is affected by accumulation of heavy metals the biochemical composition of feed and highly significant decrease in hatchery feeding behavior of fish. The digestive 679 Anjum Zia et al., Sublethal impacts of heavy metals on antioxidant enzymes and biochemical … process in fish is not well recognized as and lipid contents in muscles and gills in mammals, although the data obtained in the three fish species. McFrarland et in fish so far have shown that the al. (1999) found that Zn is strictly digestive enzymes studied were connected to catalytic, structural and qualitatively similar to those observed regulatory functions. It is a cofactor of in other vertebrates. superoxide dismutase and at the Agrawal et al. (1975) determined physiological level it has been required the activities of digestive enzymes as a factor in protein synthesis for (amylase, sucrase, protease and lipase) growth and development. Madhusudan in carnivorous fish Wallago attu, et al. (2003) determined the omnivorous fish Clarias batrachus bioaccumulation of zinc and cadmium and herbivorous fish L. rohita. in freshwater fishes. The activity of amylase is higher in the Garg et al. (2009) studied the effects L. rohita than in the W. attu and C. of exposure of zinc for 45 days on rohu batrachus. The adaptations of the fish. Zinc and copper accumulates digestive system of different fish mainly in metabolic organs liver and species exhibit correlation with their kidney which store metals for diet. This might be due to differences in detoxification by producing their food components in both habitats. metallothionein. Zn in certain Mutarotase activity: Bailey et al. concentrations is desirable for the (1969) studied the mutarotase content growth of freshwater animals but its in kidneys of freshwater fish and over accumulation is dangerous to saltwater fish. The results showed that exposed organisms as well as to those the mutarotase content of kidneys in who consume them directly or seven species of freshwater fish was indirectly through the food chain significantly higher than in nine species (Sultana and Rao, 1998). of saltwater fish. There is no more Copper accumulation: Cu and Zn are literature available on the study of essential for fish metabolism and gills mutarotase in fish. The present research act as the first line of defense and a showed that mutarotase activity prime site for metal accumulation decreased in all organs of farmed fish whereas liver has the role in metal as compared to that in hatchery and metabolism and detoxification river fish. (Jayakumar and Paul, 2006). Radhakrishnaiah (1988) reported that Determination of heavy metals copper concentration increased Zinc accumulation: Zinc is an essential significantly in the gills, liver, brain and for body growth, maturation and muscle of rohu. Deb and Santra (1997) development, tissue repair and investigated the bioaccumulation of resistance to disease. copper in the liver, intestine, ovary, It was concluded that exposure of muscles and brain in various fish. heavy metals reduces the carbohydrate Copper accumulate in all fishes when Iranian Journal of Fisheries Sciences 16(2) 2017 680 they are reliant on a sewage-fed the majority of which were concerned ecosystem, in contrast to their either with different fish species freshwater control fish population. collected from the same water body or Nickel accumulation: Nickel affects with the same fish species collected succinate dehydrogenase activity in from different localities. It is well liver, kidney, gills and muscles of L. known that, copper, nickel and zinc are rohita thereby inhibiting the energy essential elements, required by a wide yielding metabolic pathways. variety of enzymes and other cell Moorthikumar and Muthulingam, components and having vital functions (2011) observed the decreasing trend of in all living organisms, but very high succinate dehydrogenase activity in intakes can cause adverse health gills, liver, kidney, brain and muscle of problems (Demirezen and Uruc, 2006). fish, due to the effect of nickel. It is also recommended to conduct Begum et al. (2009) reported the continuous monitoring of commercial momentous level of nickel fish in Pakistan markets to ensure that concentration in liver, kidney, gills and the concentrations of metals remain muscles of freshwater L. rohita and within the prescribed worldwide limits. recorded the maximum concentration of heavy metals in kidney and liver. Acknowledgment Abdullah et al. (2011) studied the metal This research work was carried out by bioaccumulation patterns in major carps Qurat-ul-Ain for her MPhil thesis in during acute toxicity. Before Ni Enzyme Biotechnology Lab., revelation, the L. rohita showed 3.58 Department of Biochemistry and at µg/g concentration of nickel. Tariq et Fisheries Research Farms, UAF under al. (1994) worked on commercially the supervision of Muhammad Anjum important fish species, along with Zia. relevant water and sediment samples, from non-polluted and polluted sites of References the river Ravi and analyzed Cd, Cr, Cu, Abdullah, S., Javed, M., Yaqub, S. Ni, Pb, Zn and Hg by using atomic and Ahmad, N., 2011. Metal absorption spectrophotometer. 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