Ameliorative Stroke of Selenium Against Toxicological Effects of Mercuric Chloride in Liver of Freshwater Catfish Heteropneustes fossilis (Bloch) Suresh Kothari, Neha Choughule School of Studies in Zoology and Biotechnology, Vikram University, Ujjain 465010, India
Received 7 May 2013; revised 24 January 2014; accepted 24 January 2014 ABSTRACT: Mercury, a prevalent and unrelenting toxin, occurs in a variety of forms in freshwater as well as, in marine life. Mercury is an important inducer of oxidative stress in fish leading to formation of reactive oxygen species. Selenium is an essential micronutrient for animals and has antagonistic effect against mercuric toxicity in fishes. Present study has been made to evaluate toxic effect of HgCl2 (0.15 mg/L) on liver of freshwater catfish Heteropneustes fossilis (Bl.). Protective ability of selenium has been investigated by simultaneous exposure of fish with sodium selenite (0.15 mg/L) along with mercuric chloride. For present study Fishes were divided into three groups of ten fishes each the first group served as control, while the second group fish were exposed to HgCl2. Animals of third group were treated with HgCl2 and Na2SeO3. Results reveal that mercury induced lipid peroxidation and in response to this, antioxidants reduced glutathione (GSH) and Catalase (CAT) were reduced whereas, Glutathione reductase (GR) level was enhanced. These antioxidants scavenge the reactive oxygen radicals. Hg induced histopathological damage and elevation in alkaline phosphatase (ALP) and transaminases and reduction in protein and glucose contents were evidently seen in catfish liver. Intriguingly, results indicate that under stress of mercury, the fish actively generate oxidative stress and antioxidant responses, which can be used as biomarkers of pollution. Simultaneous exposure to Selenium along with Hg suppressed Hg uptake and lipid peroxidation. Histological architecture and all biochemical parameters were maintained C 2014 Wiley Periodicals, Inc. Environ Toxicol 30: near normal in the presence of selenium in liver of the catfish. V 927–936, 2015.
Keywords: mercury toxicity; catfish; histological damage; oxidative damage; antioxidant defense system; indicator enzymes
INTRODUCTION Mercury is an extremely toxic heavy metal. Widely used in chlor-alkali plants, plastics, paper, and pulp industries and effluents from such plants contribute to the entry of mercury into aquatic environments. Mercury pollution causes a serious and complex environment problem resulting in deleteri-
Correspondence to: S. Kothari; e-mail: [email protected] Published online 5 February 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.21967
ous effects on living organisms. Inorganic mercury is the most common form of the metal released in the environment by industries, presenting a stronger acute effect in fish tissues (Sunderland and Chmura, 2000). Liver is a prime established organ in fishes (Braunbeck and Volke, 1993) and plays key roles in uptake, accumulation, biotransformation and excretion of xenobiotics (Couch, 1975; Pentreath, 1976; Gluth et al., 1985; Kohler, 1990). In heavily contaminated localities mercury has been reported to be deposited preferentially in liver (Foster et al., 2000; Havelkova et al., 2008). Various aspects of mercury toxicity in fish species have been reported in the past
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(Kothari et al., 1999, 2003; Scerbo et al., 2005; Kothari, 2008; Hedayati et al., 2010; Thirumavalavan, 2010). Selenium and sodium selenite are reported to have antagonistic effect against acute and chronic toxicity of mercury in fishes (Kim et al., 1977; Siegel et al., 1991). Protective action of selenium against mercury toxicity in animal models is also on record (Chen et al., 2001; El-Demerdazh, 2001; Agrawal and Behari, 2006). Few workers have reported metal induced oxidative damage in animals including fish (Oner, 2000; Ercal et al., 2001; Linden et al., 2008). Having an antioxidative activity selenium has been reported as an effective antagonistic against oxidative stress induced by mercury (Prohaska and Ganther, 1977). Selenium also has higher affinity to bind with mercury forming insoluble mercuryselenides which is assumed to be a mechanism of protection (Moller-Madsen, 1990; Moller-Madsen and Dancher, 1991). The present investigation is aimed to investigate relationship between mercury and selenium in the liver of a fresh water teleost Heteropneustes fossilis (Bl.). Studies on mercury induced oxidative damage in fish organs and protective ability of selenium in fish are very scanty. This study is expected to fill this gap of information and to add the existing knowledge of the subject. This study includes bioaccumulation of Hg, histopathology, lipid peroxidation, antioxidant defense mechanism, alterations in indicator enzymes and metabolites in the liver of the catfish. Simultaneous treatment of selenium along with Hg exposure has been investigated to find out its protective effect against mercury toxicity.
MATERIAL AND METHODS Study Species For present analysis, freshwater catfish Heteropneustes fossilis (Bloch) was chosen, as it is one of endemic species to south eastern region of Asia, especially India. It shows many adaptations in respiratory and cardiovascular systems for obtaining oxygen from both water and air. Both sexes mature when 12 to 13 cm in length. The female occurs two times more in population and grow larger than males. The fish breed once in a year during monsoon period. At this time it exhibits, marked sexual dimorphism. Toxic agent mercuric chloride (CDH (P) Ltd. Mumbai, purity 99.5%) in sublethal concentration was used as a toxicant for this investigation. Sodium selenite (Na2SeO3) (CDH, New Delhi, Purity 99.0%) in very low concentration was tested to find out its protective ability against toxic effects of mercuric chloride (HgCl2). All the reagents used were of analytical grade.
Fish Culture and Experimental Design Living and healthy specimens of H. fossilis procured from local fish market were acclimatized to laboratory conditions
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for 15 days and were treated with 0.01% KMnO4 solution to remove any dermal infection. The average weight and length of fish were 15 6 3 g and 15 6 3 cm, respectively. LC50 values estimated by straight line interpolation method for HgCl2 and Na2SeO3 were 0.75 mg/L and 9 mg/L, respectively. Experimental concentrations of HgCl2 and Na2SeO3 were 0.15 mg/L and 0.15 mg/L, respectively. Fishes were divided into three groups of ten fish each and were kept in glass aquaria, each containing 20 L stored tap water. Physicochemical parameters of the water used for this experimental study were DO 6.4 6 1.3 mg/L, chloride 45.99 6 0.91 mg/L, hardness 82 6 4.2 mg/L, alkalinity 100 6 0.85 mg/L, pH 7.1, turbidity 4 6 0.10 NTV, and temperature 28 6 2 C. First group served as control, while the second group fish were exposed to HgCl2. Animals of third group were treated with HgCl2 1 Na2SeO3. All the three groups of experimental fishes were maintained under similar laboratory conditions. No mortality was recorded during the experimental period of 30 days. The dried and chopped prawns were fed to fishes of all the experimental groups. The daily dose of food for fish was 30 mg/fish/day. Water in all aquaria was changed on every third day and HgCl2 and Na2SeO3 were added afresh each time; after renewal of water. Water in all aquaria was daily aerated by an aquarium pump for 30 min. Fishes of all the experimental groups were dissected on 31st day and liver was separated and processed for different studies.
Mercury Accumulation Mercury was estimated by cold vapor atomic absorptions spectrophotometer in the liver homogenate sample, which was digested with H2SO4 and HNO3 at 80 C followed by oxidation with potassium permanganate and potassium persulfate at 30 C. The absorption of mercury was measured at 253.7 nm.
Histopathology Liver for histopathological study from control and experimental groups was fixed in alcoholic Bouin’s solution for 24 h. Material was then dehydrated and cleared through graded alcohols and xylene, respectively. After infiltration in paraffin wax (58–60 C) for 3 h, paraffin blocks were prepared. Sections of 5 micron thickness were cut and stretched on albumenized slides. The sections processed in routine manner were double stained with haematoxylin and eosin. The permanent slides of liver from different groups were compared with control for histopathological assessment.
Assay for Lipid Peroxidation (LPO) Tissue was immediately removed after exsanguinations and was washed twice in 9% NaCl (saline) solution to remove blood and cellular debris. After decanting saline solution phosphate buffer was added. Tissue was then cut into small pieces and homogenized with glass homogenizer at least 25
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to 30 strokes and supernatant was used for the assay of LPO. Lipid peroxidation level was estimated by the method of Parihar et al. (2004). LPO was determined by the reaction of 2-thiobarbuturic acid with malandialdehyde (MDA), one of the major products formed by peroxidation of lipids. Amount of MDA was measured by taking the absorbance at 535 nm.
Alkaline Phosphatase (ALP)
Evaluation of Antioxidant Defense Mechanism
Transaminases
Catalase (CAT) Tissue was immediately removed after exsanguinations, and then was added 9% NaCl (saline) solution. Tissue was washed twice to remove blood. Then saline was decanted and phosphate buffer was added. Tissue was cut into small pieces and homogenized with glass homogenizer at least 25 to 30 strokes and supernatant was used for the assay of CAT. Catalase activity was estimated by considering the method of Aebi (1984) based on the estimation of amount of hydrogen peroxide (H2O2) decomposed by catalase to H2O and O2. This decomposition of H2O2 can be followed directly by recording the decrease in its absorbance at 240 nm. The difference in extinction (dE240) per unit time is the measure of the catalase activity in the tissue.
Glutathione Reductase Tissue was excised and minced to remove traces of blood if any. After weighing, the tissue was homogenized at 1000 rpm in Teflon-glass homogenizer in 20 volumes of cold 50 mM phosphate buffer (pH 7.4). Homogenate was centrifuged at 3200 3 g for 20 min at 40 C and the supernatant was immediately used for enzymatic assay (Carlberg and Mannervik, 1985).
Reduced Glutathione (GSH) GSH is a part of nonenzymatic defence system. The sulfhydryl group of GSH reacts with DTNB to produce 5-thio-2nitrobenzoic acid (TNB). The amount of TNB produced is directly proportional to the concentration of GSH in the sample. Measurement of the absorbance of TNB at 412 nm provides an accurate estimation of GSH in the sample (Jollow et al., 1974). Liver was immediately removed from sacrificed fishes and washed in ice cold saline water. The adhering blood vessels and other tissue debris were cleaned and tissue was blotted and dried with filter paper. Then a 10% homogenate of liver tissue (w/v) was prepared in 0.25 m sucrose buffer at pH 7.4. This homogenate was used for biochemical studies. For the estimation of indicator enzymes alkaline phosphatase, alanine transaminase, aspartate transaminase, and metabolites protein and glucose, test kits manufactured by Span Diagnostics, India were used.
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The enzyme alkaline Phosphatase was assayed by the method of Kind and King (1957) and optical density was measured at 510 nm. The concentration of ALP was measured from a standard curve which was drawn by taking different concentrations of standard phenol.
The assay for Alanine Transaminase (ALT) and Aspartate Transaminase (AST) were carried out by the method of Reitman and Frankel (1957). The optical density for ALT and AST were measured at 505 nm and 340 nm, respectively.
Metabolites Protein Protein was estimated according to the method of Lowry et al. (1951). The optical density was measured at 550 nm. The concentration of protein was calculated from the standard curve which was drawn by taking different concentrations of Bovine serum Albumin (BSA).
Glucose Glucose was estimated by autozyme STAT reagent or enzymatic, GOD-POD method given by Trinder (1969). Absorbance was read at 505 nm. Optical densities for all parameters were measured by UV VIS spectrophotometer (Model 3A Lambda-Perkin-Elmer).
Statistical Analysis Student’s t-test was used to determine significance of Hg accumulation, LPO, CAT, GR, GSH, ALP, ALT, AST, protein, and glucose as mean 1 SEM and the significance was represented at P < 0.05, P < 0.01, P < 0.001 and nonsignificant levels.
RESULTS LC50 for Mercuric Chloride and Sodium Selenite Lc50 was determined by straight line graphical interpolation method. Lc50 values for HgCl2 and sodium selenite were found to be 0.75 mg/L (96 h) and 9 mg/L (48 h), respectively. Experiment was conducted with a safe concentration of 0.15 mg/L HgCl2 and 0.15 mg/L of sodium selenite for 30 days duration.
Mercury Accumulation Mercury content in liver of control fish was 3.82 mg/kg body wet. In Hg-treated group II the amount of mercury was 30.53 mg/kg wet wt., the increase is statistically significant when compared to group I (P < 0.001 I vs. II). In
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Assay for Lipid Peroxidation
Fig. 1. Mercury contents in the liver of H. fossilis. Data are expressed as mean 1 SEM, n 5 3 for each group, a: P < 0.001, b: P < 0.01, c: P < 0.05, dP 5 NS.
HgCl2 1 Na2SeO3-treated group, metal accumulation was less than Hg alone exposed group (14.44 mg/kg wet wt). Difference in Hg content between II and III group was highly significant (P < 0.001 group II vs. group III) and the difference between group I and III was statistically less significant (P < 0.05 group I vs. III) (Fig. 1).
Histopathology Liver of control fish consists of hexagonal cells arranged in lamini of chords. Each hepatocyte encloses nucleus with distinct nucleoli. Blood sinusoids are seen suspended in hepatic lamini. Mercury caused noticeable histological damage to the liver as revealed by disorganization of hepatic cells showing focal necrosis and cell death. Sinusoidal wall was damaged resulting in infiltration of blood cells. Concomitant treatment of Se along with Hg exposure maintained almost normal histological architecture of hepatocytes (Fig. 2).
Under mercury stress, an increase in LPO was noticed in the liver extract of the catfish. On the other hand, simultaneous treatment with antioxidant (sodium selenite) was found to maintain LPO level near normal. In control group LPO was 2.031nM MDA/mg protein, which was significantly elevated to the 4.855 nm MDA/mg protein, after Hg exposure (P < 0.001 I and II). When fish were simultaneously exposed to mercuric chloride and sodium selenite, noticeable reduction in MDA was found, the value being 2.428 nm MDA/mg protein. The difference in MDA in group II and III was highly significant (P < 0.001). Difference between group I and III was insignificant (P 5 NS) (Fig. 3).
Catalase (CAT) Catalase is an enzyme frequently proposed to be a part of cystolic armory against LPO. CAT activity is based on the estimation of amount of hydrogen peroxide (H2O2) decomposed by catalase to H2O and O2. Under metallic stress decrease in CAT activity was noticed in catfish liver. On the contrary, concomitant administration of sodium selenite along with mercuric chloride retained CAT activity within normal limits. In group II CAT activity decreased significantly as against control (P < 0.001 group I vs. group II). In group III, CAT activity was found to be increased in comparison to group II (P < 0.001 group II vs. III). The difference in CAT activity between groups I and III was statistically insignificant (P 5 NS group I vs. III) (Fig. 4).
Glutathione Reductase (GR) In the presence of Hg fish tissue exhibited more oxidative stress, which was estimated by the level of glutathione reductase enzyme. This enzyme is responsible to provide
Fig. 2. T.S. liver of control fish showing normal hepatic cells (3400) (Group I). T.S. liver of mercury treated fish showing degenerating zones, cell death and damaged sinusoids (3400) (Group II). T.S. liver of Hg1 Se treated fish showing normal hepatocytes and blood sinusoids. Few scattered damaged hepatocytes are also seen (3400) (Group III). BC: blood cells, BS: blood sinusoid, DLS: damaged lining of sinusoid, HC: hepatic cell, HN: hepatic nuclei, L: liver, NZ: necrotic zone.
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Fig. 3. Lipid peroxidation activity in the liver of different experimental groups of catfish. Data are expressed as mean 1 SEM, n 5 3 for each group a: P < 0.001, b: P < 0.01, c: P < 0.05, dP 5 NS.
Fig. 5. Glutathione reductase activity in the liver of different experimental groups of catfish. Data are expressed as mean 1 SEM, n 5 3 for each group a: P < 0.001, b: P < 0.01, c: P < 0.05, dP 5 NS.
more GSH by reduction of GSSG (stored) in the body for reducing the oxidative stress efficiently. In the presence of selenium, which is a GSH synthesizer, reduction in GR activity was recorded (P < 0.001 II vs. III). Which was significantly increased due to Hg caused oxidative stress (P < 0.001 group I vs. II). Difference in GR activity between group I and III was insignificant (P 5 NS group I vs. III) (Fig. 5).
group III). The difference in GSH value between I and III group was statistically insignificant (P 5 NS group I vs. III) (Table I).
Reduced Glutathione (GSH) Reduced glutathione has been described as the most important nonenzymatic cellular antioxidant. reduction in liver GSH was noticed due to mercury intoxication as compared to control group (P < 0.001 group I vs. group II). On the other hand, antioxidative activity of sodium selenite caused an increase in the reduced gultathione content and maintained GSH level near normal (P < 0.001 group II vs.
Indicator Enzymes Mercury intoxication induced significant increase in the level of ALP, ALT, and AST enzymes (Table II), which are indicators of structural damage in organ system of animals.
Alkaline Phosphatase (ALP) Hg caused significant elevation in ALP activity as against the control group (P < 0.001 group I vs. II). In group III, ALP activity was significantly lower than group II (P < 0.001) group II vs. III). Although, ALP value was slightly higher in HgCl2 1 Na2SeO3 treated group, but the difference was statistically insignificant (P 5 NS group I vs. III) (Table II).
Alanine Transaminase (ALT) ALT activity which was significantly enhanced due to Hg (P < 0.001 group I vs. II) was maintained near normal in Hg 1 Se treated group (P 5 Ns group I vs. III) and was TABLE I. Reduced glutathione level Experimental Groups
GSH Level (mmol/gm)
Control (I) Exposed to 0.15 mg/L HgCl2 (II) Exposed to 0.15 mg/L, HgCl2 6 0.15 mg/L, Na2Seo3 (III) Fig. 4. Catalase activity in the Liver of different experimental groups of H. fossilis. Data are expressed as mean 1 SEM, n 5 3 for each group a: P < 0.001, b: P < 0.01, c: P < 0.05, dP 5 NS.
115.90 6 2.707 70.44 6 0.870a 113.95 6 2.571a,b
Expression levels of reduced glutathione (GSH) in different experimental groups in H. fossilis liver. All values are mean 6 SE (three replicates). a P < 0.00 5 I vs. II, II vs. III, b P 5 NS (not significant) I vs. III.
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TABLE II. Indicator enzymes level Experimental Groups
ALT Level (Mg Pyruvate/gm pro)
ALP Level (Mg Phenol/gm pro)
AST Level(lU/L)
64.66 6 1.76 146 6 2.40a 69.66 6 3.53a,b
28.33 6 0.33 112 6 3.50a 51.33 6 1.20a,b
42.33 6 2.02 115.33 6 1.76a 53.00 6 2.31a,b
Control (I) Exposed to 0.15 mg/L HgCl2 (II) Exposed to 0.15 mg/L, HgCl2 6 0.15 mg/L, Na2Seo3 (III)
Expression levels of indicator enzymes (ATL, ALP, and AST) observed in catfish liver. All values are mean 6 SE (three replicates). a P < 0.00 5 I vs. II, II vs. III. b P 5 NS (not significant) I vs. III.
lesser than Hg alone treated group (P < 0.001 group II vs. III) (Table II).
Aspartate Transaminase (AST) AST activity was significantly elevated in liver after exposure to Hg (P < 0.001group I vs. II). In HgCl2 1 Na2SeO3 treated fish AST level was maintained at a significantly lower level incomparison to group II (P < 0.001 group II vs. III). Although, normal enzyme value was not attained in presence of Se, which was still higher than control group, but the difference was not significant (P 5 NS group I vs. III) (Table II).
Metabolites Effect of mercuric chloride on protein and glucose contents and effectiveness of sodium selenite was estimated in the liver of the catfish.
Protein Content Significant decline in protein content was observed in mercury intoxicated liver due to cellular damage (P < 0.001 group I vs. II). In the presence of selenium protein content was maintained at a higher level, as against mercury intoxicated group (P < 0.001 group II vs. III). The difference in protein content between control and selenium treated group was insignificant (P 5 NS group I vs. III) (Table III).
DISCUSSION Findings of this investigation reveal that toxic effects of mercury (0.15 mg/L) induce noticeable structural damage in fish liver due to significant increase in lipid peroxidation. Changes in ALP, ALT, and AST levels are indicative of stress condition of fish. Mercury induced significant alterations were also noticed in antioxidant enzyme activities, whereas protein and glucose contents of liver were significantly declined. Mercury substantiates and significantly hampers histological architecture and biochemical parameters through reactive oxygen species mediated oxidative damage. Simultaneously treatment of HgCl2 exposed fishes with Na2Seo3 was able to maintain all these parameters near normal value. Results indicate that selenium plays protective role against mercury induced histological, biochemical and oxidative damage in catfish liver. Mercury caused histopathological changes in fish liver have been reported by several investigators. Kendall (1977) reported inflammation at the hepatic capsular surface, hydropic degeneration, cytoplasmic vacuoluation, and hepatic necrosis due to Hg intoxication. Hg induced other structural damages included degeneration of hepatic cells, elongation of blood vessels and swelling of hepatic nuclei
TABLE III. Protein and glucose level
Glucose Content Glucose content in liver of different experimental groups was variable. It significantly decreased in group II, as against control (P < 0.001 group I vs. II). The value of glucose in HgCl2 1 Na2SeO3 treated group was significantly higher than Hg alone treated group (P < 0.001 group II vs. III). The difference in glucose content between group I and II was non significant (Table III). Mercury adversely affects histology and other biochemical parameter in the liver of catfish, whereas selenium provided protection against all these toxic changes of mercury.
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Experimental Groups Control (I) Exposed to 0.15 mg/L HgCl2 (II) Exposed to 0.15 mg/L, HgCl2 6 0.15 mg/L, Na2Seo3 (III)
Protein (mg/gm Wet Weight)
Glucose (mg/dL)
47.66 6 2.02 21.00 6 1.15a
48.33 6 2.02 23.00 6 1.55a
47.33 6 3.21a,b
37.00 6 1.73a,b
Table showing glucose and protein contents in different experimental groups (I, II, and III) in liver of H. Fossilis. All values are mean 6 SE (three replicates). a P < 0.00 5 I vs. II, II vs. III. b P 5 NS (not significant) I vs. III.
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(Kumar and Kothari, 1990). Sinusoid damage, infiltration, and focal necrosis (Geed and Kothari, 1994) and necrosis of liver parenchyma and dilation of biliary passages (Kothari, 2008) are also on record. Histological damage noticed in catfish liver during this investigation is in accordance with these earlier reports. Co-administration of Se was able to maintain normal structure of hepatocytes in the catfish liver. At cellular level the plasmic membrane may be considered as a complex system of potential binding sites for Hg (Boudou et al., 1983). Higher concentration of Hg in fish liver has been reported by Benson et al. (2007) and Fatma and Nahed (2008). During this study a fair amount of Hg was noticed in cat fish liver, which was significantly reduced (P < 0.001 II vs. III group) when fish were simultaneously given Se exposure. Mercury induced significant increase in the level of lipid peroxidation as noticed during this study is also reported by few workers in fish tissues (Rana et al., 1995; Stohs and Bagchi, 1995; Monteiro et al., 2010). Catalase is an antioxidant enzyme produced naturally in the body and any reduction in this enzyme results in lipid peroxidation. Reduction in CAT activity under stress of heavy metals is a known phenomenon (Thirumavalavan, 2010; Rajkumar and Miton, 2011). Reduction in CAT activity as observed during this study is in accordance with the earlier reports. Increase in glutathione reductase (GR) activity under stress of heavy metals and other pollutants has been reported in fish liver and other fish tissues (Karakoc et al., 1997; Stephensen et al., 2000) as an adaptation to oxidative stress. Increase in the GR activity in catfish liver under Hg stress suggests that GR belongs to the defense system protecting the organism against oxidative stress, as also suggested by Tekman et al. (2008). Reduced Glutathione (GSH) is one of the most abundant and essential thiol for scavenging reactive oxygen species (Dekant et al., 1994; Dringen, 2000). Hg induced inhibition of GSH, as seen during this study in catfish has also been reported by other workers (Elia et al., 2000; Zhao et al., 2009; Mieiro et al., 2010; Patniaik et al., 2010; Bashandy et al., 2011). Metal induced decrease in GSH has been attributed to the direct binding of metal to GSH through its SH group or due to enhanced oxidation of this thiol (Elia et al., 2003). Findings of this study reveal that, while Hg induces the lipid peroxidation causing alterations in the CAT, GR, and GSH activities, concomitant administration of selenium maintains all these parameters near normal in fish liver. Little information is available on the preventive action of Se against Hg induced lipid peroxidation (Talas et al., 2008). This study supports the view that Se functions as an antioxidant against toxic action of Hg. Enzymes are biochemical molecules that control metabolic processes of organisms. Slight variation in enzyme activities would affect the organisms (Roy, 2002). Thus, by
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estimating the enzyme activities in an organism, we can easily identify disturbance in its metabolism. Changes in ALP, ALT, and AST activities in fish have been frequently used as stress biomarkers to assess histological, physiological and functional alterations of organ system and tissue damage (Bernet et al., 2001). Under stress of toxicants including heavy metals, elevated level of ALP (Oluah, 1999; Bernet et al., 2001; Jiraungkoorskul et al., 2003; Kothari, 2008), ALT (Yang et al., 2003; Oner et al., 2007) and AST (Sharma, 1999; Begum, 2004) are well documented. These enzymes may be released into blood plasma and serum level may increase due to cellular damage (Oluah, 1999; Bernet et al., 2001; Yang et al., 2003; Begum, 2004; Kothari, 2008). The involvement of ALP in active transport (Denielli, 1972), protein synthesis (Pilo et al., 1972) and glycogen metabolism (Gupta and Rao, 1974) is on record. Increased transaminase activity may be associated with rapid breakdown of carbohydrate and proteins to compensate the increased energy crisis due to Hg intoxication (Sharma, 1999; Kothari, 2008). Increased activity of indicator enzymes due to Hg poisoning was maintained near normal values in HgCl2 1 Na2SeO3 treated fish liver. Depletion of total protein content as noticed in this study may also be attributed to the destruction or necrosis of cellular structures and consequent impairment in protein synthesis machinery (David et al., 2004) and also due to either arrested metabolism in the liver or to use it to build up new cells or enzymes to reduce the stress (Sakr and Al Lail, 2005). Decrease in the glucose content in Hg exposed cat fish is in accordance with that reported by Martin and Arivoli et al. (2008). This decrease may be due to glucose utilization to meet excess energy demand imposed by severe anaerobic stress of mercury intoxication (Margarat et al., 1999). Depletion in glucose content may also be attributed to the reduction in normal food uptake due to Hg poisoning as noticed in catfish during this study, which is in agreement with earlier reports (Sharma, 1996; Martin and Arivoli, 2008). In HgCl2 1 Na2SeO3 treated group metal accumulation in liver was significantly low as compared to Hg alone treated group. Consequently in the presence of Se histological architecture, oxidative stress and activities of antioxidant enzymes, indicator enzymes and metabolites were maintained near normal values. It is well recognized that mercury and sulfur bind together to form complex. This binding property is the basis of chelating therapy used as a treatment in cases of acute Hg poisoning. Se has higher affinity to bind with Hg and form insoluble mercury-selenides (Moller-Madsen, 1990; MollerMadsen and Dancher, 1991), thus reducing the free concentration of the toxic ion, as suggested by Gasiewicz and Smith (1976). Selenoproteins (Selp) also have their anti-oxidative effect. Thus, by selenium treatment, fish may be able to constitute the selp, which helps in reducing the rate of formation of lipid radicals. which is another mechanism of protective
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action of selenium. Effectiveness of Se against increased LPO and also improved antioxidative enzyme activities induced by heavy metal toxicity have also been reported by earlier workers (Jin et al., 1999; Chen et al., 2006). Thus, the findings of this study reveal that application of selenium prevents Hg uptake on one hand and on the other; helps to maintain normal histoarchitecture and biochemical machine along with lipid peroxidation, glutathione content, and antioxidative enzyme activities in the liver of fresh water teleost H. fossilis. On the basis of this study it is concluded that, while Hg induces oxidative stress in liver as evidenced by an increase in lipid peroxidation, the antioxidants GSH and CAT were inhibited, whereas GR level was increased. Selenium not only maintained these antioxidants near normal but structural damage, indicator enzymes and metabolites activities were also comparable with those of control. Consequently normal histoarchitecture of catfish liver was maintained in the presence of Se. All the parameters taken together; suggest that Selenium effectively protects against various histophysiological disturbances caused in liver due to HgCl2.
REFERENCES Aebi H. 1984. Catalase in vitro. Methods Enzymol 105:121–126. Agarwal R, Behari JR. 2007. Role of selenium in mercury intoxication in mice. Ind Health 45:388–395. Bashandy SA, Alhazza IM, El-desoky GE, Al-othman ZA. 2011. Hepato protective and hypolipidemic effects of spirulina platensis in rats administered mercuric chloride. Afr J Pharm Pharmacol 5:175–182. Begum G. 2004. Carboferan insecticide induced biochemical alteration in liver and muscle tissue of the fish Clarias batrachus (linn) and recorery response. Aquat Toxicol 66:83–92. Benson NU, Essien JP, Williams AB, Bassey DE. 2007. Mercury accumulation in fishes from tropical aquatic ecosystems in the niger delta of nigeria. Curr Sci 92:781–785. Bernet D, Schmidt H, Wahli T, Burhardt-Holm P. 2001. Effluent from a sewage treatment works causes changes in serum chemistry of brown trout (Salmo trutta L.). Ecotoxicol Environ Saf 48:140–147. Boudou A, Ribeyre F. 1983. Contamination of a aquatieciocenoses by mercury compounds: An experimental ecotoxicological approach. In: Nriagu JO, editor. Aquatic Toxicology. New York: Wiley. Chapter 3. pp 117–136. Carlberg I, Mannervik B. 1985. Glutathione reductase. Methods Enzymol 113:484–90. Chen C, Yu H, Zhao J, Li B, Qu L, Liu S, Zhang P, Chai Z. 2006. The roles of serum selenium and selenoproteins on mercury toxicity in environmental and occupational exposure. New evidence on variations of human body burden of methylmercury from fish consumption. Environ Health Perspect 114:297–301. Chen YW, Belzile N, Gurn JM. 2001. Antagonistic effect of selenium on mercury assimilation by fish population near sudbury metal smelters. J Limnol Oceanogr 46:1814–1818.
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Couch JA. 1975. Histopathological effects of pesticides and related chemicals on the liver of fishes. In: Ribelin WE, Magaki G, editors. Pathology of Fishes. Madison: University of Wisconsin Press. pp 559–584. David M, Mushigeri SB, Shivkumar R, Philip GH. 2004. Response of Cyprinus carpio (Linn) to sublethal concentration of cypermethrin: Alterations in protein metabolism profiles, Chemosphere 56:347–352. Dekant W, Vamvakas S, Anders MW. 1994. Formation and fate of nephrotoxic and cytotoxic glutathione-s-conjugates: Cysteine conjugate b-lyase pathway. In Anders WA, Dekant W, editors. Advances in Pharmacology, Vol. 27. San Diego, CA: Academic Press. pp 115–162. Denielli JF. 1972. Structural factors in cell permeability and secretion. Symp Soc Exp Biol 6:1–15. Dringen R. 2000. Metabolism and Functions of glutathione in brain. Prog Neurobiol 62:649–71. El-Demerdash FM. 2001. Effect of Selenium and mercury on the enzymatic activities and lipid peroxidation in brain, liver and blood of rats. J Environ Sci Health B 36:489–499. Elia AC, Dorr AJM, Mantilacci L, Taticchi MI, Galarini R. 2000. Effects of mercury on glutathione and glutathione-dependent enzymes in catfish (Ictalurus Melas R.) In Markert B, Friese K, editors. Trace Element—Their Distribution and Effects in the Environment: Trace Metals in the Environment. Amsderdam: Elsevier Science. pp 411–421. Elia AC, Galarini R, Taticchi MI, Dorr AJM, Mantilacci L. 2003. Antioxidant responses and bioaccumulation in Ictalurus melas under mercury exposure. Ecotoxicol Environ Saf 55:162–167. Ercal N, Gurer OH, Aykin-Burns N. 2001. Toxic metals and oxidative stress part I: Mechanisms involved in metal induced oxidative damage. Curr Top Med Chem 1:529–539. Fatma ASM, Nahed SG. 2008. Environmental pollution-induced biochemical changes in tissues of Tilapia Zillii, Solea vulgaris and Mugil capito from Lake Qarun, Egypt. Global Veterinaria 2:327–336. Foster EP, Drake DL, Didomenico G. 2000. Seasonal changes and tissue distribution of mercury in largemouth bass (Micropterus salmoides) from Dorena Reservoir, Oregon. Arch Environ Contam Toxicol 72:733–740. Gasiewicz TA, Smith JC. 1976. Interaction of cadmium and selenium in rat plasma in vivo and in vitro. J Biochem Biophys Acta 428:113–122. Gluth G, Freitag D, Hank W, Korte F. 1985. Accumulation of pollutant in fish. Comp Biochem Physiol 81:273–277. Gupta V, Rao G. 1974. Histological studies on the chloride plexes of the goat embryos II. Histological distribution of acid and alkaline phosphatase. Acta Histochem 49:60–63. Havelkova M, Dusek L, Nemethova D, Poleszczuk G, Svobodova Z. 2008. Comparison of mercury distribution between liver and musc—A biomonitoring of fish from lightly and heavily contaminated localities. Sensors (Basel) 8:4095–4109. Hedayati A, Safahieh A, Savari A, Marammazi JG. 2010. Assessment of aminotransferase enzymes in yellowfin sea Bream (Acanthopagrus latus) under experimental condition as
SELENIUM PROTECTION AGAINST MERCURY TOXICITY
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biomarkers of mercury pollution. World J Fish Marine Sci 2: 186–192.
mullet (Liza aurata) as an effect of mercury exposure. Ecotoxicology 19:1034–1045.
Jin L, Guo P, Xu X. 1999. Effect of selenium on mercury methylation in facultative lake sediments. J Toxical Environ Chem 69: 255–261.
Moller-Madsen B, Danscher G. 1991. Localization of mercury in CNS of the rat IV. The effect of selenium on orally administered organic and inorganic mercury. Toxicol Appl Pharmacol 108:457–473.
Jiraungkoorskul W, Upatham ES, Kruatrachue M, Shaphong S, Vichasri-Grams S, Pokethityook P. 2003. Biochemical and histopahtoloical effects of glyphosate herbicide on. Nile tilapia (Oreochromis niloticus). Environ Toxicol 18:260–267. Jollow DJ, Mitchell JR, Zampaglione N, Gillette JR. 1974. Bromobenzene-induced liver necrosis. Protective role of glutathione and evidence for 3,4-bromobenzene oxide as the hepatotoxic metabolite. Pharmacology 11:151–169. Karakoc FT, Hewer A, Phillips DH, Gaines AF, Yuregir G. 1997. Biomarkers of marine pollution observed in species of mullet living in two eastern mediteranean harbours. Biomarkers 2:303–309. Kendall MW. 1977. Acute effects of methylemercury toxicity on Channel Catfish (Ictalurus punctatus) liver. Bull Environ Contam Toxicol 18:143–151. Kim JH, Birks E, Heisinger JF. 1977. Protective action of selenium against mercury in northern creek chubs. J Bull Environ Contain Toxicol 17;132–136. Kohler A. 1990. Identification of contaminant induced cellular and sub cellular lesions in the liver of flounder (Platychthys fleus L.) caught at differently polluted estuaries. Aquat Toxicol 16:271–294. Kothari S, Bhalerao S, Jat D. 2003. Protection by herbal compound against toxic effects of mercuric chloride in fish intestine. J Ecophysiol Occup Health 3:51–58. Kothari S, Bhalerao S, Sharma SK. 1999. Effect of an herbal compound on mercury induced biochemical changes in the liver of Catfish. Indian J Environ and Toxicol 9:30–32. Kothari S. 2008. Protective action of essential phospholipids accumulation and toxicity of mercuric chloride in fish liver. In: Mishra PK, Maudar KK, editors. Status of Medical Biotechnology Research in the State of Madhya Pradesh: Past Accomplishments, Current Trends and Challenges Ahead. Bhopal, India: MEDBIO 216–230. Kumar MR, Kothari SC. 1990. Histological and histochemical studies in the liver of Heteropneustes fossilis (Bloch) treated with mercuric chloride. Environmental Series. 3:127–132. Linden A, Gulden M, Martin HJ, Maser E, Sibert H. 2008. Peroxide induced cell death and lipid peroxidation in glioma cells Toxicol In Vitro 22:1371–75. Lowry OW, Rosenbrough NJ, Farr AK, Randall RJ. 1951. Protein measurement with the folin phenol reagent. J Biochem 193: 265–275. Margarat A, Jagadeesan G. 1999. Abtodotal recovery ion carbohydrate metabolism of mercury in toxicated mice musculus(L). Uttarpradesh J Zool 19:196–198. Martin PDP and Arivoli S. 2008. Biochemical study of freshwater fish Catla-Catla with reference to mercury chloride. Iran J Environ Health Sci Eng 5:109–116. Mieiro CL, Ahmad I, Pereira ME, Duarte AC, Pacheco M. 2010. Antioxidant system breakdown in brain of feral guldan grey
Moller-Madsen B. 1990. Localization of mercury in CNS of the rat intraperitoneal injection of methylmercuric chloride (CH3HgCl2) and mercuric chloride (HgCl2). Toxicol Appl Pharmacol 103:303–323. Monteiro DA, Rantin FT, Kalinin AL. 2010. Inorganic mercury exposure: Toxicological effects, oxidative stress biomarkes and bioaccumulation in the tropical freshwater fish matrinxa, Brycon amazonicus (Spix and agassiz, 1829). Ecotoxicology 19: 105–123. Oluah NS. 1999. Plasma asparate aminotransferase activity in the Catfish Clarias albopuntatus exposed to sublethal Zinc and mercury. Bull Environ Contam Toxicol 63:343–349. Oner M, Atli G, Canli M. 2007. Changes in serum Biochemical parameters of fresh water fish Oreochromis Niloticus following prolonged Metal (Ag Cd Cr Cu Zn) Exposures. Environ Toxicol Chem 27:360–366. Parihar MS, Choudhary M, Shetty R, Hemnanai T. 2004. Susceptibility of hippocampus and cerebral cortex to oxidative damage in streptozotocin treated mice prevention by extracts of withania somnifera and aloe vera. J Clin Neurosci 11:397–402. Patnaik BB, Roy A, Agarwal S, Bhattacharya S. 2010. Induction of oxidative stress by non-lethal dose of mercury in rat liver: Possible relationships between apoptosis and necrosis. J Environ Bio 31:413–416. Pentreath RJ. 1976. The accumulation of mercury from food by the plaice Pleuronectes platessa L. J Exp Marine Biol 25:51–65. Pilo B, Ansari MV, Shah RV. 1972. Studies of wound healing and repair in pigeon liver III. Histochemical studies on acid and alkaline phosphatase activities during the process. J Anim Morphol Physiol 9:205–212. Prohaska JR, Ganther HE. 1977. Interactions between Selenium and methylmercury in rat brain. Chem, Biol Interact 16:155–167. Rajkumar JSI, John Milton MC. 2011. Studied biochemical markers of oxidative stress in Mughil cephalus exposed to cadmium, copper, lead and zinc. Int J Pharma Biosci 2:41–49. Rana SVS, Rekha S, Seema V. 1995. Mercury induced lipid peroxidaiton in kidney, brain and gills of a fresh water fish, Channa punctuatus, Japanese. J Ichthyol 42:255–259. Reitman S, Frankel SA. 1957. Colorimetric method for determination of serum glutamic oxaloacetic and glumatic pyruvic transminase. Am J Clin Pathol 28:56–63. Roy SS. 2002. Some toxicological aspects of chlorpyrifos to the inter tidal fish Boleopthlmus dissumeieri. PhD Thesis. India: University of Mumbai. pp 52–71. Sakr SA, Al Lail JSM. 2005. Fenvalerate induced histopathological and histochemical changes in the liver of the catfish Clarias Gariepinus. J Appl Sci Res 1:263–267.
Environmental Toxicology DOI 10.1002/tox
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Scerbo R, Ristori T, Stefanini B, De Ranieri S, Barghiani C. 2005. Mercury assessment and evaluation of its impact on fish in the Ceciana River basin. J Environ Pollut 134:179–186. Sharma B. 1999. Effect of carbaryl on some biochemical constituents in the blood and liver of C batrachus a freshwater teleost. J Toxicol Sci 24:157–164. Sharma R. 1996. Toxicological effects of HgCl2 on the enzymes of kidney and ovary of Channa punctatus. J Nacton 8:53–56. Siegel BZ, Siegel SM, Correa T, Dagan C, Galvez G, Leeloy L, Padua A, Yaeger E. 1991. The protection of invertebrates, fish and vascular plants against inorganic mercury poisoning by sulfur and Selenium derivatives. Arch Environ Contam Toxicol 20:241–246. Stephensen E, Svavarsson J, Sturve J, Ericson G, Adolfsson-Erici M, Forlin L. 2000. Biochemical indicators of pollution exposure in shorthorn sculpin (Myxocephalus scorpius), caught in four harbours on the southwest coast of iceland. Aquat Toxicol 48:431–442. Stohs SJ, Bagchi D. 1995. Oxidative mechanisms in the toxicity of metals ions. Free Radic Biol Med 2:321–336. Sunderlaland EM, Chmura GL. 2000. An inventory of historical mercury emission of Matritime Canada: Implication for present and future contamination. Sci Tot Environ 256:39–57.
Environmental Toxicology DOI 10.1002/tox
Talas ZS, Orun I, ozdemir I, Erdogan K, Aldan A, Yilmaz I. 2008. Studied Antioxidative role of selenium against the toxic effect of heavy metals (Cdp2, Crp3) on liver of rainbow trout (Oncorhynchus Mykiss Walbaum 1972). J Fish Physiol Biochem 34: 217–222. Tekman B, Ozdemir H, Senturk M, Ciftci M. 2008. Purification and characterization of glutathione reductase from rainbow trout (Oncorhynchus mykiss) liver and inhibition effects of metal ions on enzyme activity. Comp Biochem Physiol C 148: 117–121. Thirumavalavan R. 2010. Effect of Mercury on lipid peroxidation and antioxidants in fresh water fish labeo rohita. J Int J Curr Res 8:108–112. Trinder P. 1969. Determination of glucose in serum, plasma and CSF, GOD/POD method. Ann Clin Biochem 6:24. Yang JH, Chen H. 2003. Serum metabolic enzyme activities and hepatocyte ultrastructure of common carp after Gallium exposure. Zool Stud 42:455–461. Zhao JQ, Wen YF, Bhadauria M, Nirala SK, Sharma A, Shrivastava S, Shukla S, Agrawal OP, Mathur R. 2009. Protective effects of propolis on inorganic mercury induced oxidative stress in mice. Indian J Exp Biol 47:264–269.
Received 7 May 2013; revised 24 January 2014; accepted 24 January 2014 ABSTRACT: Mercury, a prevalent and unrelenting toxin, occurs in a variety of forms in freshwater as well as, in marine life. Mercury is an important inducer of oxidative stress in fish leading to formation of reactive oxygen species. Selenium is an essential micronutrient for animals and has antagonistic effect against mercuric toxicity in fishes. Present study has been made to evaluate toxic effect of HgCl2 (0.15 mg/L) on liver of freshwater catfish Heteropneustes fossilis (Bl.). Protective ability of selenium has been investigated by simultaneous exposure of fish with sodium selenite (0.15 mg/L) along with mercuric chloride. For present study Fishes were divided into three groups of ten fishes each the first group served as control, while the second group fish were exposed to HgCl2. Animals of third group were treated with HgCl2 and Na2SeO3. Results reveal that mercury induced lipid peroxidation and in response to this, antioxidants reduced glutathione (GSH) and Catalase (CAT) were reduced whereas, Glutathione reductase (GR) level was enhanced. These antioxidants scavenge the reactive oxygen radicals. Hg induced histopathological damage and elevation in alkaline phosphatase (ALP) and transaminases and reduction in protein and glucose contents were evidently seen in catfish liver. Intriguingly, results indicate that under stress of mercury, the fish actively generate oxidative stress and antioxidant responses, which can be used as biomarkers of pollution. Simultaneous exposure to Selenium along with Hg suppressed Hg uptake and lipid peroxidation. Histological architecture and all biochemical parameters were maintained C 2014 Wiley Periodicals, Inc. Environ Toxicol 30: near normal in the presence of selenium in liver of the catfish. V 927–936, 2015.
Keywords: mercury toxicity; catfish; histological damage; oxidative damage; antioxidant defense system; indicator enzymes
INTRODUCTION Mercury is an extremely toxic heavy metal. Widely used in chlor-alkali plants, plastics, paper, and pulp industries and effluents from such plants contribute to the entry of mercury into aquatic environments. Mercury pollution causes a serious and complex environment problem resulting in deleteri-
Correspondence to: S. Kothari; e-mail: [email protected] Published online 5 February 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.21967
ous effects on living organisms. Inorganic mercury is the most common form of the metal released in the environment by industries, presenting a stronger acute effect in fish tissues (Sunderland and Chmura, 2000). Liver is a prime established organ in fishes (Braunbeck and Volke, 1993) and plays key roles in uptake, accumulation, biotransformation and excretion of xenobiotics (Couch, 1975; Pentreath, 1976; Gluth et al., 1985; Kohler, 1990). In heavily contaminated localities mercury has been reported to be deposited preferentially in liver (Foster et al., 2000; Havelkova et al., 2008). Various aspects of mercury toxicity in fish species have been reported in the past
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(Kothari et al., 1999, 2003; Scerbo et al., 2005; Kothari, 2008; Hedayati et al., 2010; Thirumavalavan, 2010). Selenium and sodium selenite are reported to have antagonistic effect against acute and chronic toxicity of mercury in fishes (Kim et al., 1977; Siegel et al., 1991). Protective action of selenium against mercury toxicity in animal models is also on record (Chen et al., 2001; El-Demerdazh, 2001; Agrawal and Behari, 2006). Few workers have reported metal induced oxidative damage in animals including fish (Oner, 2000; Ercal et al., 2001; Linden et al., 2008). Having an antioxidative activity selenium has been reported as an effective antagonistic against oxidative stress induced by mercury (Prohaska and Ganther, 1977). Selenium also has higher affinity to bind with mercury forming insoluble mercuryselenides which is assumed to be a mechanism of protection (Moller-Madsen, 1990; Moller-Madsen and Dancher, 1991). The present investigation is aimed to investigate relationship between mercury and selenium in the liver of a fresh water teleost Heteropneustes fossilis (Bl.). Studies on mercury induced oxidative damage in fish organs and protective ability of selenium in fish are very scanty. This study is expected to fill this gap of information and to add the existing knowledge of the subject. This study includes bioaccumulation of Hg, histopathology, lipid peroxidation, antioxidant defense mechanism, alterations in indicator enzymes and metabolites in the liver of the catfish. Simultaneous treatment of selenium along with Hg exposure has been investigated to find out its protective effect against mercury toxicity.
MATERIAL AND METHODS Study Species For present analysis, freshwater catfish Heteropneustes fossilis (Bloch) was chosen, as it is one of endemic species to south eastern region of Asia, especially India. It shows many adaptations in respiratory and cardiovascular systems for obtaining oxygen from both water and air. Both sexes mature when 12 to 13 cm in length. The female occurs two times more in population and grow larger than males. The fish breed once in a year during monsoon period. At this time it exhibits, marked sexual dimorphism. Toxic agent mercuric chloride (CDH (P) Ltd. Mumbai, purity 99.5%) in sublethal concentration was used as a toxicant for this investigation. Sodium selenite (Na2SeO3) (CDH, New Delhi, Purity 99.0%) in very low concentration was tested to find out its protective ability against toxic effects of mercuric chloride (HgCl2). All the reagents used were of analytical grade.
Fish Culture and Experimental Design Living and healthy specimens of H. fossilis procured from local fish market were acclimatized to laboratory conditions
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for 15 days and were treated with 0.01% KMnO4 solution to remove any dermal infection. The average weight and length of fish were 15 6 3 g and 15 6 3 cm, respectively. LC50 values estimated by straight line interpolation method for HgCl2 and Na2SeO3 were 0.75 mg/L and 9 mg/L, respectively. Experimental concentrations of HgCl2 and Na2SeO3 were 0.15 mg/L and 0.15 mg/L, respectively. Fishes were divided into three groups of ten fish each and were kept in glass aquaria, each containing 20 L stored tap water. Physicochemical parameters of the water used for this experimental study were DO 6.4 6 1.3 mg/L, chloride 45.99 6 0.91 mg/L, hardness 82 6 4.2 mg/L, alkalinity 100 6 0.85 mg/L, pH 7.1, turbidity 4 6 0.10 NTV, and temperature 28 6 2 C. First group served as control, while the second group fish were exposed to HgCl2. Animals of third group were treated with HgCl2 1 Na2SeO3. All the three groups of experimental fishes were maintained under similar laboratory conditions. No mortality was recorded during the experimental period of 30 days. The dried and chopped prawns were fed to fishes of all the experimental groups. The daily dose of food for fish was 30 mg/fish/day. Water in all aquaria was changed on every third day and HgCl2 and Na2SeO3 were added afresh each time; after renewal of water. Water in all aquaria was daily aerated by an aquarium pump for 30 min. Fishes of all the experimental groups were dissected on 31st day and liver was separated and processed for different studies.
Mercury Accumulation Mercury was estimated by cold vapor atomic absorptions spectrophotometer in the liver homogenate sample, which was digested with H2SO4 and HNO3 at 80 C followed by oxidation with potassium permanganate and potassium persulfate at 30 C. The absorption of mercury was measured at 253.7 nm.
Histopathology Liver for histopathological study from control and experimental groups was fixed in alcoholic Bouin’s solution for 24 h. Material was then dehydrated and cleared through graded alcohols and xylene, respectively. After infiltration in paraffin wax (58–60 C) for 3 h, paraffin blocks were prepared. Sections of 5 micron thickness were cut and stretched on albumenized slides. The sections processed in routine manner were double stained with haematoxylin and eosin. The permanent slides of liver from different groups were compared with control for histopathological assessment.
Assay for Lipid Peroxidation (LPO) Tissue was immediately removed after exsanguinations and was washed twice in 9% NaCl (saline) solution to remove blood and cellular debris. After decanting saline solution phosphate buffer was added. Tissue was then cut into small pieces and homogenized with glass homogenizer at least 25
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to 30 strokes and supernatant was used for the assay of LPO. Lipid peroxidation level was estimated by the method of Parihar et al. (2004). LPO was determined by the reaction of 2-thiobarbuturic acid with malandialdehyde (MDA), one of the major products formed by peroxidation of lipids. Amount of MDA was measured by taking the absorbance at 535 nm.
Alkaline Phosphatase (ALP)
Evaluation of Antioxidant Defense Mechanism
Transaminases
Catalase (CAT) Tissue was immediately removed after exsanguinations, and then was added 9% NaCl (saline) solution. Tissue was washed twice to remove blood. Then saline was decanted and phosphate buffer was added. Tissue was cut into small pieces and homogenized with glass homogenizer at least 25 to 30 strokes and supernatant was used for the assay of CAT. Catalase activity was estimated by considering the method of Aebi (1984) based on the estimation of amount of hydrogen peroxide (H2O2) decomposed by catalase to H2O and O2. This decomposition of H2O2 can be followed directly by recording the decrease in its absorbance at 240 nm. The difference in extinction (dE240) per unit time is the measure of the catalase activity in the tissue.
Glutathione Reductase Tissue was excised and minced to remove traces of blood if any. After weighing, the tissue was homogenized at 1000 rpm in Teflon-glass homogenizer in 20 volumes of cold 50 mM phosphate buffer (pH 7.4). Homogenate was centrifuged at 3200 3 g for 20 min at 40 C and the supernatant was immediately used for enzymatic assay (Carlberg and Mannervik, 1985).
Reduced Glutathione (GSH) GSH is a part of nonenzymatic defence system. The sulfhydryl group of GSH reacts with DTNB to produce 5-thio-2nitrobenzoic acid (TNB). The amount of TNB produced is directly proportional to the concentration of GSH in the sample. Measurement of the absorbance of TNB at 412 nm provides an accurate estimation of GSH in the sample (Jollow et al., 1974). Liver was immediately removed from sacrificed fishes and washed in ice cold saline water. The adhering blood vessels and other tissue debris were cleaned and tissue was blotted and dried with filter paper. Then a 10% homogenate of liver tissue (w/v) was prepared in 0.25 m sucrose buffer at pH 7.4. This homogenate was used for biochemical studies. For the estimation of indicator enzymes alkaline phosphatase, alanine transaminase, aspartate transaminase, and metabolites protein and glucose, test kits manufactured by Span Diagnostics, India were used.
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The enzyme alkaline Phosphatase was assayed by the method of Kind and King (1957) and optical density was measured at 510 nm. The concentration of ALP was measured from a standard curve which was drawn by taking different concentrations of standard phenol.
The assay for Alanine Transaminase (ALT) and Aspartate Transaminase (AST) were carried out by the method of Reitman and Frankel (1957). The optical density for ALT and AST were measured at 505 nm and 340 nm, respectively.
Metabolites Protein Protein was estimated according to the method of Lowry et al. (1951). The optical density was measured at 550 nm. The concentration of protein was calculated from the standard curve which was drawn by taking different concentrations of Bovine serum Albumin (BSA).
Glucose Glucose was estimated by autozyme STAT reagent or enzymatic, GOD-POD method given by Trinder (1969). Absorbance was read at 505 nm. Optical densities for all parameters were measured by UV VIS spectrophotometer (Model 3A Lambda-Perkin-Elmer).
Statistical Analysis Student’s t-test was used to determine significance of Hg accumulation, LPO, CAT, GR, GSH, ALP, ALT, AST, protein, and glucose as mean 1 SEM and the significance was represented at P < 0.05, P < 0.01, P < 0.001 and nonsignificant levels.
RESULTS LC50 for Mercuric Chloride and Sodium Selenite Lc50 was determined by straight line graphical interpolation method. Lc50 values for HgCl2 and sodium selenite were found to be 0.75 mg/L (96 h) and 9 mg/L (48 h), respectively. Experiment was conducted with a safe concentration of 0.15 mg/L HgCl2 and 0.15 mg/L of sodium selenite for 30 days duration.
Mercury Accumulation Mercury content in liver of control fish was 3.82 mg/kg body wet. In Hg-treated group II the amount of mercury was 30.53 mg/kg wet wt., the increase is statistically significant when compared to group I (P < 0.001 I vs. II). In
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Assay for Lipid Peroxidation
Fig. 1. Mercury contents in the liver of H. fossilis. Data are expressed as mean 1 SEM, n 5 3 for each group, a: P < 0.001, b: P < 0.01, c: P < 0.05, dP 5 NS.
HgCl2 1 Na2SeO3-treated group, metal accumulation was less than Hg alone exposed group (14.44 mg/kg wet wt). Difference in Hg content between II and III group was highly significant (P < 0.001 group II vs. group III) and the difference between group I and III was statistically less significant (P < 0.05 group I vs. III) (Fig. 1).
Histopathology Liver of control fish consists of hexagonal cells arranged in lamini of chords. Each hepatocyte encloses nucleus with distinct nucleoli. Blood sinusoids are seen suspended in hepatic lamini. Mercury caused noticeable histological damage to the liver as revealed by disorganization of hepatic cells showing focal necrosis and cell death. Sinusoidal wall was damaged resulting in infiltration of blood cells. Concomitant treatment of Se along with Hg exposure maintained almost normal histological architecture of hepatocytes (Fig. 2).
Under mercury stress, an increase in LPO was noticed in the liver extract of the catfish. On the other hand, simultaneous treatment with antioxidant (sodium selenite) was found to maintain LPO level near normal. In control group LPO was 2.031nM MDA/mg protein, which was significantly elevated to the 4.855 nm MDA/mg protein, after Hg exposure (P < 0.001 I and II). When fish were simultaneously exposed to mercuric chloride and sodium selenite, noticeable reduction in MDA was found, the value being 2.428 nm MDA/mg protein. The difference in MDA in group II and III was highly significant (P < 0.001). Difference between group I and III was insignificant (P 5 NS) (Fig. 3).
Catalase (CAT) Catalase is an enzyme frequently proposed to be a part of cystolic armory against LPO. CAT activity is based on the estimation of amount of hydrogen peroxide (H2O2) decomposed by catalase to H2O and O2. Under metallic stress decrease in CAT activity was noticed in catfish liver. On the contrary, concomitant administration of sodium selenite along with mercuric chloride retained CAT activity within normal limits. In group II CAT activity decreased significantly as against control (P < 0.001 group I vs. group II). In group III, CAT activity was found to be increased in comparison to group II (P < 0.001 group II vs. III). The difference in CAT activity between groups I and III was statistically insignificant (P 5 NS group I vs. III) (Fig. 4).
Glutathione Reductase (GR) In the presence of Hg fish tissue exhibited more oxidative stress, which was estimated by the level of glutathione reductase enzyme. This enzyme is responsible to provide
Fig. 2. T.S. liver of control fish showing normal hepatic cells (3400) (Group I). T.S. liver of mercury treated fish showing degenerating zones, cell death and damaged sinusoids (3400) (Group II). T.S. liver of Hg1 Se treated fish showing normal hepatocytes and blood sinusoids. Few scattered damaged hepatocytes are also seen (3400) (Group III). BC: blood cells, BS: blood sinusoid, DLS: damaged lining of sinusoid, HC: hepatic cell, HN: hepatic nuclei, L: liver, NZ: necrotic zone.
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Fig. 3. Lipid peroxidation activity in the liver of different experimental groups of catfish. Data are expressed as mean 1 SEM, n 5 3 for each group a: P < 0.001, b: P < 0.01, c: P < 0.05, dP 5 NS.
Fig. 5. Glutathione reductase activity in the liver of different experimental groups of catfish. Data are expressed as mean 1 SEM, n 5 3 for each group a: P < 0.001, b: P < 0.01, c: P < 0.05, dP 5 NS.
more GSH by reduction of GSSG (stored) in the body for reducing the oxidative stress efficiently. In the presence of selenium, which is a GSH synthesizer, reduction in GR activity was recorded (P < 0.001 II vs. III). Which was significantly increased due to Hg caused oxidative stress (P < 0.001 group I vs. II). Difference in GR activity between group I and III was insignificant (P 5 NS group I vs. III) (Fig. 5).
group III). The difference in GSH value between I and III group was statistically insignificant (P 5 NS group I vs. III) (Table I).
Reduced Glutathione (GSH) Reduced glutathione has been described as the most important nonenzymatic cellular antioxidant. reduction in liver GSH was noticed due to mercury intoxication as compared to control group (P < 0.001 group I vs. group II). On the other hand, antioxidative activity of sodium selenite caused an increase in the reduced gultathione content and maintained GSH level near normal (P < 0.001 group II vs.
Indicator Enzymes Mercury intoxication induced significant increase in the level of ALP, ALT, and AST enzymes (Table II), which are indicators of structural damage in organ system of animals.
Alkaline Phosphatase (ALP) Hg caused significant elevation in ALP activity as against the control group (P < 0.001 group I vs. II). In group III, ALP activity was significantly lower than group II (P < 0.001) group II vs. III). Although, ALP value was slightly higher in HgCl2 1 Na2SeO3 treated group, but the difference was statistically insignificant (P 5 NS group I vs. III) (Table II).
Alanine Transaminase (ALT) ALT activity which was significantly enhanced due to Hg (P < 0.001 group I vs. II) was maintained near normal in Hg 1 Se treated group (P 5 Ns group I vs. III) and was TABLE I. Reduced glutathione level Experimental Groups
GSH Level (mmol/gm)
Control (I) Exposed to 0.15 mg/L HgCl2 (II) Exposed to 0.15 mg/L, HgCl2 6 0.15 mg/L, Na2Seo3 (III) Fig. 4. Catalase activity in the Liver of different experimental groups of H. fossilis. Data are expressed as mean 1 SEM, n 5 3 for each group a: P < 0.001, b: P < 0.01, c: P < 0.05, dP 5 NS.
115.90 6 2.707 70.44 6 0.870a 113.95 6 2.571a,b
Expression levels of reduced glutathione (GSH) in different experimental groups in H. fossilis liver. All values are mean 6 SE (three replicates). a P < 0.00 5 I vs. II, II vs. III, b P 5 NS (not significant) I vs. III.
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TABLE II. Indicator enzymes level Experimental Groups
ALT Level (Mg Pyruvate/gm pro)
ALP Level (Mg Phenol/gm pro)
AST Level(lU/L)
64.66 6 1.76 146 6 2.40a 69.66 6 3.53a,b
28.33 6 0.33 112 6 3.50a 51.33 6 1.20a,b
42.33 6 2.02 115.33 6 1.76a 53.00 6 2.31a,b
Control (I) Exposed to 0.15 mg/L HgCl2 (II) Exposed to 0.15 mg/L, HgCl2 6 0.15 mg/L, Na2Seo3 (III)
Expression levels of indicator enzymes (ATL, ALP, and AST) observed in catfish liver. All values are mean 6 SE (three replicates). a P < 0.00 5 I vs. II, II vs. III. b P 5 NS (not significant) I vs. III.
lesser than Hg alone treated group (P < 0.001 group II vs. III) (Table II).
Aspartate Transaminase (AST) AST activity was significantly elevated in liver after exposure to Hg (P < 0.001group I vs. II). In HgCl2 1 Na2SeO3 treated fish AST level was maintained at a significantly lower level incomparison to group II (P < 0.001 group II vs. III). Although, normal enzyme value was not attained in presence of Se, which was still higher than control group, but the difference was not significant (P 5 NS group I vs. III) (Table II).
Metabolites Effect of mercuric chloride on protein and glucose contents and effectiveness of sodium selenite was estimated in the liver of the catfish.
Protein Content Significant decline in protein content was observed in mercury intoxicated liver due to cellular damage (P < 0.001 group I vs. II). In the presence of selenium protein content was maintained at a higher level, as against mercury intoxicated group (P < 0.001 group II vs. III). The difference in protein content between control and selenium treated group was insignificant (P 5 NS group I vs. III) (Table III).
DISCUSSION Findings of this investigation reveal that toxic effects of mercury (0.15 mg/L) induce noticeable structural damage in fish liver due to significant increase in lipid peroxidation. Changes in ALP, ALT, and AST levels are indicative of stress condition of fish. Mercury induced significant alterations were also noticed in antioxidant enzyme activities, whereas protein and glucose contents of liver were significantly declined. Mercury substantiates and significantly hampers histological architecture and biochemical parameters through reactive oxygen species mediated oxidative damage. Simultaneously treatment of HgCl2 exposed fishes with Na2Seo3 was able to maintain all these parameters near normal value. Results indicate that selenium plays protective role against mercury induced histological, biochemical and oxidative damage in catfish liver. Mercury caused histopathological changes in fish liver have been reported by several investigators. Kendall (1977) reported inflammation at the hepatic capsular surface, hydropic degeneration, cytoplasmic vacuoluation, and hepatic necrosis due to Hg intoxication. Hg induced other structural damages included degeneration of hepatic cells, elongation of blood vessels and swelling of hepatic nuclei
TABLE III. Protein and glucose level
Glucose Content Glucose content in liver of different experimental groups was variable. It significantly decreased in group II, as against control (P < 0.001 group I vs. II). The value of glucose in HgCl2 1 Na2SeO3 treated group was significantly higher than Hg alone treated group (P < 0.001 group II vs. III). The difference in glucose content between group I and II was non significant (Table III). Mercury adversely affects histology and other biochemical parameter in the liver of catfish, whereas selenium provided protection against all these toxic changes of mercury.
Environmental Toxicology DOI 10.1002/tox
Experimental Groups Control (I) Exposed to 0.15 mg/L HgCl2 (II) Exposed to 0.15 mg/L, HgCl2 6 0.15 mg/L, Na2Seo3 (III)
Protein (mg/gm Wet Weight)
Glucose (mg/dL)
47.66 6 2.02 21.00 6 1.15a
48.33 6 2.02 23.00 6 1.55a
47.33 6 3.21a,b
37.00 6 1.73a,b
Table showing glucose and protein contents in different experimental groups (I, II, and III) in liver of H. Fossilis. All values are mean 6 SE (three replicates). a P < 0.00 5 I vs. II, II vs. III. b P 5 NS (not significant) I vs. III.
SELENIUM PROTECTION AGAINST MERCURY TOXICITY
(Kumar and Kothari, 1990). Sinusoid damage, infiltration, and focal necrosis (Geed and Kothari, 1994) and necrosis of liver parenchyma and dilation of biliary passages (Kothari, 2008) are also on record. Histological damage noticed in catfish liver during this investigation is in accordance with these earlier reports. Co-administration of Se was able to maintain normal structure of hepatocytes in the catfish liver. At cellular level the plasmic membrane may be considered as a complex system of potential binding sites for Hg (Boudou et al., 1983). Higher concentration of Hg in fish liver has been reported by Benson et al. (2007) and Fatma and Nahed (2008). During this study a fair amount of Hg was noticed in cat fish liver, which was significantly reduced (P < 0.001 II vs. III group) when fish were simultaneously given Se exposure. Mercury induced significant increase in the level of lipid peroxidation as noticed during this study is also reported by few workers in fish tissues (Rana et al., 1995; Stohs and Bagchi, 1995; Monteiro et al., 2010). Catalase is an antioxidant enzyme produced naturally in the body and any reduction in this enzyme results in lipid peroxidation. Reduction in CAT activity under stress of heavy metals is a known phenomenon (Thirumavalavan, 2010; Rajkumar and Miton, 2011). Reduction in CAT activity as observed during this study is in accordance with the earlier reports. Increase in glutathione reductase (GR) activity under stress of heavy metals and other pollutants has been reported in fish liver and other fish tissues (Karakoc et al., 1997; Stephensen et al., 2000) as an adaptation to oxidative stress. Increase in the GR activity in catfish liver under Hg stress suggests that GR belongs to the defense system protecting the organism against oxidative stress, as also suggested by Tekman et al. (2008). Reduced Glutathione (GSH) is one of the most abundant and essential thiol for scavenging reactive oxygen species (Dekant et al., 1994; Dringen, 2000). Hg induced inhibition of GSH, as seen during this study in catfish has also been reported by other workers (Elia et al., 2000; Zhao et al., 2009; Mieiro et al., 2010; Patniaik et al., 2010; Bashandy et al., 2011). Metal induced decrease in GSH has been attributed to the direct binding of metal to GSH through its SH group or due to enhanced oxidation of this thiol (Elia et al., 2003). Findings of this study reveal that, while Hg induces the lipid peroxidation causing alterations in the CAT, GR, and GSH activities, concomitant administration of selenium maintains all these parameters near normal in fish liver. Little information is available on the preventive action of Se against Hg induced lipid peroxidation (Talas et al., 2008). This study supports the view that Se functions as an antioxidant against toxic action of Hg. Enzymes are biochemical molecules that control metabolic processes of organisms. Slight variation in enzyme activities would affect the organisms (Roy, 2002). Thus, by
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estimating the enzyme activities in an organism, we can easily identify disturbance in its metabolism. Changes in ALP, ALT, and AST activities in fish have been frequently used as stress biomarkers to assess histological, physiological and functional alterations of organ system and tissue damage (Bernet et al., 2001). Under stress of toxicants including heavy metals, elevated level of ALP (Oluah, 1999; Bernet et al., 2001; Jiraungkoorskul et al., 2003; Kothari, 2008), ALT (Yang et al., 2003; Oner et al., 2007) and AST (Sharma, 1999; Begum, 2004) are well documented. These enzymes may be released into blood plasma and serum level may increase due to cellular damage (Oluah, 1999; Bernet et al., 2001; Yang et al., 2003; Begum, 2004; Kothari, 2008). The involvement of ALP in active transport (Denielli, 1972), protein synthesis (Pilo et al., 1972) and glycogen metabolism (Gupta and Rao, 1974) is on record. Increased transaminase activity may be associated with rapid breakdown of carbohydrate and proteins to compensate the increased energy crisis due to Hg intoxication (Sharma, 1999; Kothari, 2008). Increased activity of indicator enzymes due to Hg poisoning was maintained near normal values in HgCl2 1 Na2SeO3 treated fish liver. Depletion of total protein content as noticed in this study may also be attributed to the destruction or necrosis of cellular structures and consequent impairment in protein synthesis machinery (David et al., 2004) and also due to either arrested metabolism in the liver or to use it to build up new cells or enzymes to reduce the stress (Sakr and Al Lail, 2005). Decrease in the glucose content in Hg exposed cat fish is in accordance with that reported by Martin and Arivoli et al. (2008). This decrease may be due to glucose utilization to meet excess energy demand imposed by severe anaerobic stress of mercury intoxication (Margarat et al., 1999). Depletion in glucose content may also be attributed to the reduction in normal food uptake due to Hg poisoning as noticed in catfish during this study, which is in agreement with earlier reports (Sharma, 1996; Martin and Arivoli, 2008). In HgCl2 1 Na2SeO3 treated group metal accumulation in liver was significantly low as compared to Hg alone treated group. Consequently in the presence of Se histological architecture, oxidative stress and activities of antioxidant enzymes, indicator enzymes and metabolites were maintained near normal values. It is well recognized that mercury and sulfur bind together to form complex. This binding property is the basis of chelating therapy used as a treatment in cases of acute Hg poisoning. Se has higher affinity to bind with Hg and form insoluble mercury-selenides (Moller-Madsen, 1990; MollerMadsen and Dancher, 1991), thus reducing the free concentration of the toxic ion, as suggested by Gasiewicz and Smith (1976). Selenoproteins (Selp) also have their anti-oxidative effect. Thus, by selenium treatment, fish may be able to constitute the selp, which helps in reducing the rate of formation of lipid radicals. which is another mechanism of protective
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action of selenium. Effectiveness of Se against increased LPO and also improved antioxidative enzyme activities induced by heavy metal toxicity have also been reported by earlier workers (Jin et al., 1999; Chen et al., 2006). Thus, the findings of this study reveal that application of selenium prevents Hg uptake on one hand and on the other; helps to maintain normal histoarchitecture and biochemical machine along with lipid peroxidation, glutathione content, and antioxidative enzyme activities in the liver of fresh water teleost H. fossilis. On the basis of this study it is concluded that, while Hg induces oxidative stress in liver as evidenced by an increase in lipid peroxidation, the antioxidants GSH and CAT were inhibited, whereas GR level was increased. Selenium not only maintained these antioxidants near normal but structural damage, indicator enzymes and metabolites activities were also comparable with those of control. Consequently normal histoarchitecture of catfish liver was maintained in the presence of Se. All the parameters taken together; suggest that Selenium effectively protects against various histophysiological disturbances caused in liver due to HgCl2.
REFERENCES Aebi H. 1984. Catalase in vitro. Methods Enzymol 105:121–126. Agarwal R, Behari JR. 2007. Role of selenium in mercury intoxication in mice. Ind Health 45:388–395. Bashandy SA, Alhazza IM, El-desoky GE, Al-othman ZA. 2011. Hepato protective and hypolipidemic effects of spirulina platensis in rats administered mercuric chloride. Afr J Pharm Pharmacol 5:175–182. Begum G. 2004. Carboferan insecticide induced biochemical alteration in liver and muscle tissue of the fish Clarias batrachus (linn) and recorery response. Aquat Toxicol 66:83–92. Benson NU, Essien JP, Williams AB, Bassey DE. 2007. Mercury accumulation in fishes from tropical aquatic ecosystems in the niger delta of nigeria. Curr Sci 92:781–785. Bernet D, Schmidt H, Wahli T, Burhardt-Holm P. 2001. Effluent from a sewage treatment works causes changes in serum chemistry of brown trout (Salmo trutta L.). Ecotoxicol Environ Saf 48:140–147. Boudou A, Ribeyre F. 1983. Contamination of a aquatieciocenoses by mercury compounds: An experimental ecotoxicological approach. In: Nriagu JO, editor. Aquatic Toxicology. New York: Wiley. Chapter 3. pp 117–136. Carlberg I, Mannervik B. 1985. Glutathione reductase. Methods Enzymol 113:484–90. Chen C, Yu H, Zhao J, Li B, Qu L, Liu S, Zhang P, Chai Z. 2006. The roles of serum selenium and selenoproteins on mercury toxicity in environmental and occupational exposure. New evidence on variations of human body burden of methylmercury from fish consumption. Environ Health Perspect 114:297–301. Chen YW, Belzile N, Gurn JM. 2001. Antagonistic effect of selenium on mercury assimilation by fish population near sudbury metal smelters. J Limnol Oceanogr 46:1814–1818.
Environmental Toxicology DOI 10.1002/tox
Couch JA. 1975. Histopathological effects of pesticides and related chemicals on the liver of fishes. In: Ribelin WE, Magaki G, editors. Pathology of Fishes. Madison: University of Wisconsin Press. pp 559–584. David M, Mushigeri SB, Shivkumar R, Philip GH. 2004. Response of Cyprinus carpio (Linn) to sublethal concentration of cypermethrin: Alterations in protein metabolism profiles, Chemosphere 56:347–352. Dekant W, Vamvakas S, Anders MW. 1994. Formation and fate of nephrotoxic and cytotoxic glutathione-s-conjugates: Cysteine conjugate b-lyase pathway. In Anders WA, Dekant W, editors. Advances in Pharmacology, Vol. 27. San Diego, CA: Academic Press. pp 115–162. Denielli JF. 1972. Structural factors in cell permeability and secretion. Symp Soc Exp Biol 6:1–15. Dringen R. 2000. Metabolism and Functions of glutathione in brain. Prog Neurobiol 62:649–71. El-Demerdash FM. 2001. Effect of Selenium and mercury on the enzymatic activities and lipid peroxidation in brain, liver and blood of rats. J Environ Sci Health B 36:489–499. Elia AC, Dorr AJM, Mantilacci L, Taticchi MI, Galarini R. 2000. Effects of mercury on glutathione and glutathione-dependent enzymes in catfish (Ictalurus Melas R.) In Markert B, Friese K, editors. Trace Element—Their Distribution and Effects in the Environment: Trace Metals in the Environment. Amsderdam: Elsevier Science. pp 411–421. Elia AC, Galarini R, Taticchi MI, Dorr AJM, Mantilacci L. 2003. Antioxidant responses and bioaccumulation in Ictalurus melas under mercury exposure. Ecotoxicol Environ Saf 55:162–167. Ercal N, Gurer OH, Aykin-Burns N. 2001. Toxic metals and oxidative stress part I: Mechanisms involved in metal induced oxidative damage. Curr Top Med Chem 1:529–539. Fatma ASM, Nahed SG. 2008. Environmental pollution-induced biochemical changes in tissues of Tilapia Zillii, Solea vulgaris and Mugil capito from Lake Qarun, Egypt. Global Veterinaria 2:327–336. Foster EP, Drake DL, Didomenico G. 2000. Seasonal changes and tissue distribution of mercury in largemouth bass (Micropterus salmoides) from Dorena Reservoir, Oregon. Arch Environ Contam Toxicol 72:733–740. Gasiewicz TA, Smith JC. 1976. Interaction of cadmium and selenium in rat plasma in vivo and in vitro. J Biochem Biophys Acta 428:113–122. Gluth G, Freitag D, Hank W, Korte F. 1985. Accumulation of pollutant in fish. Comp Biochem Physiol 81:273–277. Gupta V, Rao G. 1974. Histological studies on the chloride plexes of the goat embryos II. Histological distribution of acid and alkaline phosphatase. Acta Histochem 49:60–63. Havelkova M, Dusek L, Nemethova D, Poleszczuk G, Svobodova Z. 2008. Comparison of mercury distribution between liver and musc—A biomonitoring of fish from lightly and heavily contaminated localities. Sensors (Basel) 8:4095–4109. Hedayati A, Safahieh A, Savari A, Marammazi JG. 2010. Assessment of aminotransferase enzymes in yellowfin sea Bream (Acanthopagrus latus) under experimental condition as
SELENIUM PROTECTION AGAINST MERCURY TOXICITY
935
biomarkers of mercury pollution. World J Fish Marine Sci 2: 186–192.
mullet (Liza aurata) as an effect of mercury exposure. Ecotoxicology 19:1034–1045.
Jin L, Guo P, Xu X. 1999. Effect of selenium on mercury methylation in facultative lake sediments. J Toxical Environ Chem 69: 255–261.
Moller-Madsen B, Danscher G. 1991. Localization of mercury in CNS of the rat IV. The effect of selenium on orally administered organic and inorganic mercury. Toxicol Appl Pharmacol 108:457–473.
Jiraungkoorskul W, Upatham ES, Kruatrachue M, Shaphong S, Vichasri-Grams S, Pokethityook P. 2003. Biochemical and histopahtoloical effects of glyphosate herbicide on. Nile tilapia (Oreochromis niloticus). Environ Toxicol 18:260–267. Jollow DJ, Mitchell JR, Zampaglione N, Gillette JR. 1974. Bromobenzene-induced liver necrosis. Protective role of glutathione and evidence for 3,4-bromobenzene oxide as the hepatotoxic metabolite. Pharmacology 11:151–169. Karakoc FT, Hewer A, Phillips DH, Gaines AF, Yuregir G. 1997. Biomarkers of marine pollution observed in species of mullet living in two eastern mediteranean harbours. Biomarkers 2:303–309. Kendall MW. 1977. Acute effects of methylemercury toxicity on Channel Catfish (Ictalurus punctatus) liver. Bull Environ Contam Toxicol 18:143–151. Kim JH, Birks E, Heisinger JF. 1977. Protective action of selenium against mercury in northern creek chubs. J Bull Environ Contain Toxicol 17;132–136. Kohler A. 1990. Identification of contaminant induced cellular and sub cellular lesions in the liver of flounder (Platychthys fleus L.) caught at differently polluted estuaries. Aquat Toxicol 16:271–294. Kothari S, Bhalerao S, Jat D. 2003. Protection by herbal compound against toxic effects of mercuric chloride in fish intestine. J Ecophysiol Occup Health 3:51–58. Kothari S, Bhalerao S, Sharma SK. 1999. Effect of an herbal compound on mercury induced biochemical changes in the liver of Catfish. Indian J Environ and Toxicol 9:30–32. Kothari S. 2008. Protective action of essential phospholipids accumulation and toxicity of mercuric chloride in fish liver. In: Mishra PK, Maudar KK, editors. Status of Medical Biotechnology Research in the State of Madhya Pradesh: Past Accomplishments, Current Trends and Challenges Ahead. Bhopal, India: MEDBIO 216–230. Kumar MR, Kothari SC. 1990. Histological and histochemical studies in the liver of Heteropneustes fossilis (Bloch) treated with mercuric chloride. Environmental Series. 3:127–132. Linden A, Gulden M, Martin HJ, Maser E, Sibert H. 2008. Peroxide induced cell death and lipid peroxidation in glioma cells Toxicol In Vitro 22:1371–75. Lowry OW, Rosenbrough NJ, Farr AK, Randall RJ. 1951. Protein measurement with the folin phenol reagent. J Biochem 193: 265–275. Margarat A, Jagadeesan G. 1999. Abtodotal recovery ion carbohydrate metabolism of mercury in toxicated mice musculus(L). Uttarpradesh J Zool 19:196–198. Martin PDP and Arivoli S. 2008. Biochemical study of freshwater fish Catla-Catla with reference to mercury chloride. Iran J Environ Health Sci Eng 5:109–116. Mieiro CL, Ahmad I, Pereira ME, Duarte AC, Pacheco M. 2010. Antioxidant system breakdown in brain of feral guldan grey
Moller-Madsen B. 1990. Localization of mercury in CNS of the rat intraperitoneal injection of methylmercuric chloride (CH3HgCl2) and mercuric chloride (HgCl2). Toxicol Appl Pharmacol 103:303–323. Monteiro DA, Rantin FT, Kalinin AL. 2010. Inorganic mercury exposure: Toxicological effects, oxidative stress biomarkes and bioaccumulation in the tropical freshwater fish matrinxa, Brycon amazonicus (Spix and agassiz, 1829). Ecotoxicology 19: 105–123. Oluah NS. 1999. Plasma asparate aminotransferase activity in the Catfish Clarias albopuntatus exposed to sublethal Zinc and mercury. Bull Environ Contam Toxicol 63:343–349. Oner M, Atli G, Canli M. 2007. Changes in serum Biochemical parameters of fresh water fish Oreochromis Niloticus following prolonged Metal (Ag Cd Cr Cu Zn) Exposures. Environ Toxicol Chem 27:360–366. Parihar MS, Choudhary M, Shetty R, Hemnanai T. 2004. Susceptibility of hippocampus and cerebral cortex to oxidative damage in streptozotocin treated mice prevention by extracts of withania somnifera and aloe vera. J Clin Neurosci 11:397–402. Patnaik BB, Roy A, Agarwal S, Bhattacharya S. 2010. Induction of oxidative stress by non-lethal dose of mercury in rat liver: Possible relationships between apoptosis and necrosis. J Environ Bio 31:413–416. Pentreath RJ. 1976. The accumulation of mercury from food by the plaice Pleuronectes platessa L. J Exp Marine Biol 25:51–65. Pilo B, Ansari MV, Shah RV. 1972. Studies of wound healing and repair in pigeon liver III. Histochemical studies on acid and alkaline phosphatase activities during the process. J Anim Morphol Physiol 9:205–212. Prohaska JR, Ganther HE. 1977. Interactions between Selenium and methylmercury in rat brain. Chem, Biol Interact 16:155–167. Rajkumar JSI, John Milton MC. 2011. Studied biochemical markers of oxidative stress in Mughil cephalus exposed to cadmium, copper, lead and zinc. Int J Pharma Biosci 2:41–49. Rana SVS, Rekha S, Seema V. 1995. Mercury induced lipid peroxidaiton in kidney, brain and gills of a fresh water fish, Channa punctuatus, Japanese. J Ichthyol 42:255–259. Reitman S, Frankel SA. 1957. Colorimetric method for determination of serum glutamic oxaloacetic and glumatic pyruvic transminase. Am J Clin Pathol 28:56–63. Roy SS. 2002. Some toxicological aspects of chlorpyrifos to the inter tidal fish Boleopthlmus dissumeieri. PhD Thesis. India: University of Mumbai. pp 52–71. Sakr SA, Al Lail JSM. 2005. Fenvalerate induced histopathological and histochemical changes in the liver of the catfish Clarias Gariepinus. J Appl Sci Res 1:263–267.
Environmental Toxicology DOI 10.1002/tox
936
KOTHARI AND CHOUGHULE
Scerbo R, Ristori T, Stefanini B, De Ranieri S, Barghiani C. 2005. Mercury assessment and evaluation of its impact on fish in the Ceciana River basin. J Environ Pollut 134:179–186. Sharma B. 1999. Effect of carbaryl on some biochemical constituents in the blood and liver of C batrachus a freshwater teleost. J Toxicol Sci 24:157–164. Sharma R. 1996. Toxicological effects of HgCl2 on the enzymes of kidney and ovary of Channa punctatus. J Nacton 8:53–56. Siegel BZ, Siegel SM, Correa T, Dagan C, Galvez G, Leeloy L, Padua A, Yaeger E. 1991. The protection of invertebrates, fish and vascular plants against inorganic mercury poisoning by sulfur and Selenium derivatives. Arch Environ Contam Toxicol 20:241–246. Stephensen E, Svavarsson J, Sturve J, Ericson G, Adolfsson-Erici M, Forlin L. 2000. Biochemical indicators of pollution exposure in shorthorn sculpin (Myxocephalus scorpius), caught in four harbours on the southwest coast of iceland. Aquat Toxicol 48:431–442. Stohs SJ, Bagchi D. 1995. Oxidative mechanisms in the toxicity of metals ions. Free Radic Biol Med 2:321–336. Sunderlaland EM, Chmura GL. 2000. An inventory of historical mercury emission of Matritime Canada: Implication for present and future contamination. Sci Tot Environ 256:39–57.
Environmental Toxicology DOI 10.1002/tox
Talas ZS, Orun I, ozdemir I, Erdogan K, Aldan A, Yilmaz I. 2008. Studied Antioxidative role of selenium against the toxic effect of heavy metals (Cdp2, Crp3) on liver of rainbow trout (Oncorhynchus Mykiss Walbaum 1972). J Fish Physiol Biochem 34: 217–222. Tekman B, Ozdemir H, Senturk M, Ciftci M. 2008. Purification and characterization of glutathione reductase from rainbow trout (Oncorhynchus mykiss) liver and inhibition effects of metal ions on enzyme activity. Comp Biochem Physiol C 148: 117–121. Thirumavalavan R. 2010. Effect of Mercury on lipid peroxidation and antioxidants in fresh water fish labeo rohita. J Int J Curr Res 8:108–112. Trinder P. 1969. Determination of glucose in serum, plasma and CSF, GOD/POD method. Ann Clin Biochem 6:24. Yang JH, Chen H. 2003. Serum metabolic enzyme activities and hepatocyte ultrastructure of common carp after Gallium exposure. Zool Stud 42:455–461. Zhao JQ, Wen YF, Bhadauria M, Nirala SK, Sharma A, Shrivastava S, Shukla S, Agrawal OP, Mathur R. 2009. Protective effects of propolis on inorganic mercury induced oxidative stress in mice. Indian J Exp Biol 47:264–269.
