1Department of Zoology, Sri Krishnadevaraya University, Anantapur - 515003, Andhra Pradesh, India, 2Department of Life sciences, Universidad de las Fuerzas Armadas-ESPE, Sangolqui, Quito, Ecuador, South America, 3Department of Environmental Engineering, Universidad Estatal Amazonica, Puyo 160150, Ecuador, South America.
Email: manjubhargav2010@gmail.com
Received: 01 Jan 2015 Revised and Accepted: 25 Jan 2015
ABSTRACT
Objective: The present study was carried out to investigate the influences of sublethal toxicity of potassium cyanide (KCN) exposure to Oreochromis niloticus with special reference to blood biochemistry.
Methods: Three groups of fish (25 in each group) were treated with different concentrations of 0 (control), 0.1 and 0.2 mg/L KCN for 2 weeks. The Blood samples were drawn after 2 weeks of exposure and serum biochemical analysis including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), gamma-glutamyl transferase (GGT), glucose, triglyceride, cholesterol, creatinine, total protein, and albumin activities were measured.
Results: KCN exposure at both concentrations caused significant (P<0.05) elevation of AST and LDH activities and creatinine concentration as compared to the control. Indeed, ALT, ALP and glucose levels in the fish treated with 0.2 mg/L KCN were significantly higher than those levels from controls. On the other hand, the biochemical parameters including GGT, triglyceride, cholesterol, total protein, and albumin did not change significantly following cyanide exposure.
Conclusion: The current study has clearly indicated the alterations in some of the measured serum biochemical parameters which reflect tissue damages, especially in liver and kidney, due to chronic cyanide intoxication in Oreochromis niloticus and may be used for better understanding the pathophysiology of this toxicity and as an aid in the diagnosis of cyanide poisoning in fishes.
Keywords: Nile tilapia, Oreochromis niloticus, Cyanide toxicity, Serum biochemical profile.
INTRODUCTION
Aquatic vertebrates live in an intimate contact with the environment, making them more susceptible to pollutants. Among aquatic species, fish are the major target for the action of toxicants. Fishes are largely being used for the assessment of the quality of an aquatic environment and as such can serve as bioindicators of environmental pollution [1]. The primary site of action of cyanide is presumed to be the central nervous system (CNS) [2]. Cyanide inhibits the mitochondrial enzyme cytochrome oxidase in the respiratory electron transport chain of the mitochondria, impairing both oxidative metabolism and the associated process of oxidative phosphorylation [3]. Additionally, a number of other enzymatic processes are also inhibited which exacerbate the cyanide toxicity [1].
Cyanide is one of the most toxic chemical substances on Earth. The extreme toxicity of cyanide arises from its readiness to react with other elements and hence interfere with normal biological processes [4]. The sensitivity of aquatic organisms to cyanide is highly species specific, and also influenced by water pH, temperature and oxygen content, as well as the life stage and conditions of the organism. Fish and aquatic invertebrates are particularly sensitive to cyanide exposure [5]. Cyanide is a potent and rapid-acting asphyxiant; it induces tissue anoxia through inactivation of cytochrome oxidase, causing cytotoxic hypoxia in the presence of normal hemoglobin oxygenation. The effect of the hypoxia causes depression of the central nervous system that can result in respiratory arrest and leading to death [6].
The use of cyanide compounds in mining industries, coupled with limitations in the current analysis and monitoring of these compounds, raises serious concerns regarding public safety and environmental protection at mine sites using cyanide processing. Cyanide and its salts are used extensively in electroplating, extraction of ores (Silver and gold), metal processing, photographic processes, production of synthetic rubber, chemical synthesis, manufacturing of plastic, pesticides, dehairing of hides, laboratory processes, manufacturing of dyes and pigments [7-9] and are also found in water and food consumed by animals and humans. Chronic exposure to low levels of cyanide is suspected to be responsible for various neuropathic and thyrotoxic conditions in humans. However, cyanide also tends to react readily with many other chemical elements, and is known to form, at a minimum, hundreds of different compounds [10]. Many of these breakdowns compounds, while generally less toxic than the original cyanide, are known to be toxic to aquatic organisms. In addition, they may persist in the environment for long periods of time, and there are evidences that some forms of these compounds could be accumulated in plants [11] and fish tissues [12]. The Nile tilapia, Oreochromis niloticus (Linnaeus, 1758) is a widely cultured species because it grows and reproduces in a wide range of environmental conditions and tolerates stress induced by handling [13]. The fish is currently ranked second only to carps in global production [14]. Previously, tilapia was consumed mainly in Africa and Asia but nowadays it has been touted as the “new white fish” replacing the depleted ocean stocks of cod and heck, leading to a worldwide demand for the fish [15]. The toxicity of cyanide to fish can be influenced by a variety of factors including concentration, environmental temperature, dissolved oxygen content, pre-exposure and age [16]. Although a number of studies have investigated the effects of sublethal cyanide exposure on the serum biochemical parameters in various animal species [4, 17-21], very little is known about fish species. Thus, the objective of the present study was to investigate the effects of sublethal exposure to potassium cyanide (KCN) on serum biochemical changes in Oreochromis niloticus.
MATERIALS AND METHODS
Collection of experimental fish
The Nile tilapia (Oreochromis niloticus; total n=75), weighing 60±10 g, were obtained from the Fisheries Department, Anantapur, Andhra Pradesh, India. Fish was acclimatized for 7 days before the commencement of the experiment and were fed daily with commercial fish feed at 2.5% total body weight at a fixed time. Physicochemical conditions of the water during the experimental period were dissolved oxygen, 5.8–6.3 ppm; temperature, 25±2°C; pH, 7.1±0.5. Photoperiod was a 12:12 light–dark cycle. The water in aquariums was renewed every 24 h.
Experimental design
After acclimation the Oreochromis niloticus fish were divided randomly into three groups of 25 each and held in three glass aquaria, each containing 250 L fresh water. Experimental groups were exposed either to 0 (control), 0.1 and 0.2 ppm potassium cyanide (Sigma-Aldrich Pty. Ltd. 12 Anella Avenue, Castle hill nsw 2154, Australia) for 14 days. At the end of each exposure, 15 fish starved for 24 h were collected randomly from each aquarium and anesthetized in diluted tricaine methanesulfonate. Blood samples (approximately 0.8 mL) were collected from the caudal vena. Blood serum separation was done by centrifugation at 750 g for 20 min and serum samples were stored at −80 °C further analysis.
Biochemical assays
Serum biochemical analysis including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), gamma-glutamyl transferase (GGT), glucose, triglyceride, cholesterol, creatinine, total protein, and albumin were done using commercial colorimetric kits (Cayman chemicals-USA).
Statistical analysis
All calculations were performed using SPSS/PC software. All results were analyzed using one way analysis of variance (ANOVA), followed by Duncan's multiple comparisons test. The level of significance was set at P<0.05.
RESULTS
The effects of KCN exposure on the values (mean ± SEM) of the measured biochemical parameters in serum of Oreochromis niloticus are presented in table 1. As shown in table 1, KCN exposure at both concentrations caused significant (P<0.05) elevation of AST and LDH activities and creatinine concentration as compared to controls. Indeed, ALT, ALP and glucose levels in the Oreochromis niloticus treated with 0.2 ppm KCN were significantly higher than those levels from control. On the other hand, serum levels of other measured biochemical variable including GGT, triglyceride, cholesterol, total protein, and albumin did not change significantly following KCN exposure as compared to control (table 1).
DISCUSSION
Fish is a heterogeneous group of aquatic animals. Since the fish are ectothermic aquatic vertebrates, having economic values as natural resources of proteins and other fish products, their physiologic, pathologic and toxicologic studies are emerging fields at the present days. Such fields pertaining to fish physiology, pathology and toxicology are further worth attention due to the significance of fish in human nutrition, as people of almost every nation agree to include fish in their normal diet as a rich source of quality protein [22]. It is informed that important elements responsible for normal physiology of animals and their intact structure of various organs for physiological life sustaining processes are hematopieotic tissue, hematological parameters, blood serum biochemistry, lipid profile and circulating blood [23].
The toxic substances cause fluctuations in hematological parameters, either by enhancing their number or concentration by promoting their biosynthetic activities or their fall in number or concentration by suppressing their biosynthetic sites [24]. In clinical and veterinary medical sciences, the hematological parameters are considered as good health indicators of animals as well as for environment [25].
Cyanide is one of the most toxic chemical to fish, as fish are one thousand times more sensitive to cyanide than human [26]. This active sensitivity of fish to cyanide therefore makes fish an excellent biomarker for the presence of cyanide in water [27]. Pollution of the aquatic ecosystem stresses the animals and disturbs their metabolism by altering the enzyme activity, damage and dysfunction the tissues and hindering growth all that associated with biochemical changes [28]. Analysis of biochemical parameters could help to identify the target organs of toxicity as well as the general health status of animals. It may also provide an early warning signal in stressed organism [29, 30].
Table 1: Serum biochemical parameters in Oreochromis niloticus exposed to different concentrations of potassium cyanide (KCN) for two weeks (n=15 in each group)
| Parameter | Control | KCN 0.1 mg/L | KCN 0.2 mg/L |
| AST (U/L) | 109.75±6.89 a | 161.39±12.63 b | 172.21±12.03b |
| ALT (U/L) | 39.03±4.11 a | 51.29±5.17a,b | 58.25±4.39b |
| ALP (U/L) | 18.25±0.69 a | 22.18±1.17 a | 27.47±1.21 b |
| GGT (U/L) | 2.13±0.08 | 2.27±0.16 | 2.01±0.15 |
| LDH (U/L) | 395.68±12.15 a | 472.19±8.19 b | 460.35±9.89 b |
| Glucose (mg/dL) | 50.89±2.25 a | 51.83±2.41a | 59.65±3.17 b |
| Triglyceride (mg/dL) | 153.75±11.04 | 131.15±12.09 | 151.19±16.01 |
| Cholesterol (mg/dL) | 181.36±17.45 | 162.34±30.41 | 168.01±12.05 |
| Total protein (g/dL) | 3.25±0.17 | 3.17±0.21 | 4.09±0.35 |
| Albumin (g/dL) | 1.65±0.25 | 1.81±0.18 | 1.30±22.01 |
| Creatinine (mg/dL) | 0.49±0.07 a | 0.74±0.08 b | 0.85±0.06 b |
a,b mean±SEM in each row with no common superscript differ significantly (P<0.05)
Transaminases are important enzymes known to play a key role in mobilizing L-amino acids for gluconeogenesis and function as links between carbohydrate and protein metabolism under altered physiological, pathological conditions [30, 31]. Increase in the levels of these enzymes in liver, muscle and gills of fishes can be considered as a response to the stress induced by cyanide to generate ketoacid-like ketoglutarate and oxaloacetate for contributing to gluconeogenesis and/or energy production necessary to meet the excess energy demand under the toxic manifestation [32]. In the present study, AST and ALT levels were found to be increased following cyanide exposure at both concentrations, although the increase was not significant for ALT activity at 0.1 mg/L KCN. In agreement with these results, [33]showed significant increases of AST and ALT enzyme activities in rats drinking water contaminated with cyanide. Similar findings were also observed by Naveed et al., [34] in C. punctatus. Agrahari et al., [35] observed an increase in the ALT and AST of the catfish exposed to pesticide and opined that this increase in the activity of these enzymes is an indicator of cellular damage.
Indeed, ALT activities in pigs treated with 6.0 mg/kg of KCN were significantly increased [21]. On the other hand, there was no statistically significant difference in the serum activities of AST in rabbits [17] and both AST and ALT activities in goats [20] and rats [36] following cyanide administration. These differences could be due to variation in the dose, duration of exposure, and the species. The increased activities of AST and ALT in the present study would indicate the deleterious hepatic and renal damages of cyanide exposure in Oreochromis niloticus that is reminiscent of previously reported pathological findings in liver and kidney of several animal species and humans exposed to cyanide [4, 36, 37].
Tissue distribution of alkaline phosphatase is virtually ubiquitous especially within cell membranes and would easily leak out of the cell in cyanide-induced tissue damage [17]. Elevation of serum ALP has been also reported due to liver damage from poisoning by some chemicals in humans [38]. In the present study serum ALP activity was increased significantly following exposure to 0.2 ppm KCN as compared to control. In agreement with this finding, increased serum alkaline phosphatase has been reported in the rabbits fed mash plus cyanide compared to controls fed mash alone [4, 17]. Significant increases in serum ALP and ALT activities in addition to histopathological changes in liver, lung and kidney tissues has been also reported following chronic cyanide intoxication in rabbits [19].
LDH, the terminal enzyme in vertebrate anaerobic glycolysis, is one of the enzymes that have been employed for diagnosing liver, muscle, and gill damages caused by pollutants in fish [39]. Increased activity of LDH is a characteristic feature of a shift from aerobic to anaerobic metabolism leading to an elevated rate of pyruvate conversion into lactate, resulting in lactic acidosis [40]. In the present study, significant rise in LDH activity was observed in cyanide-exposed Oreochromis niloticus compared to control group. Increases in serum and tissue levels of LDH activities are characteristic features of lactic acidosis resulting from the inhibitory effect of cyanide on aerobic metabolism [19] and increased LDH activity in liver of Cyprinus carpio was reported by Kamalaveni et al., [41]. Similarly, increased LDH activity in freshwater fish by arsenic treatment was reported by Shobha Rani et al., [42].
Cyanide is also known to alter glucose metabolism [43]. There are some literatures available that report diabetes as a toxic effect produced by ingesting cassava, a cyanogenic plant, in various species [44-47]. Based on the present results, cyanide exposure at the dose of 0.2 mg/L caused the significant increase in glucose concentration in Oreochromis niloticus that is reminiscent to the results reported previously in swine and rats [48, 36]. On the other hand, no alterations in plasma glucose were observed following chronic cyanide exposure in goats, rats and rabbits [17, 18, 20].
Based on the result of the present study, increased creatinine concentration was observed following cyanide exposure that might be associated with kidney damage due to cyanide exposure. In line with this finding significant increases in serum creatinine concentrations have been reported following KCN administration in rats [33] and pigs [21]. Okolie and Osagie, have reported that degenerative changes in the kidney sections of the cyanide-fed rabbits may be responsible for the significant increases in serum urea and creatinine [4].
CONCLUSION
The observed alterations in some measured serum biochemical parameters would reflect tissue damages, especially in liver and kidney, due to chronic cyanide intoxication in Oreochromis niloticus and may be used for better understanding the pathophysiology of this toxicity and as an aid in the diagnosis of cyanide poisoning in fish species.
CONFLICT OF INTEREST
The authors declare that we have no conflict of interest.
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