Fish Physiol Biochem (2008) 34:53–59 DOI 10.1007/s10695-007-9146-5
Effects of sodium selenite on some biochemical and hematological parameters of rainbow trout (Oncorhynchus mykiss Walbaum, 1792) exposed to Pb2+ and Cu2+ Burhan Ates Æ Ibrahim Orun Æ Zeliha Selamoglu Talas Æ Gokhan Durmaz Æ Ismet Yilmaz
Received: 16 March 2007 / Accepted: 13 June 2007 / Published online: 24 July 2007 Springer Science+Business Media B.V. 2007
Abstract This study was carried out to understand the preventive effect of selenium (Se4+) on heavy metal stress induced by lead and copper in rainbow trout (Oncorhynchus mykiss). Variation in glutathione peroxidase (Se-GSH-Px) and superoxide dismutase (SOD) activity, and in malondialdehyde (MDA) levels in liver, spleen, heart, and brain tissues of rainbow trout after 72 h of exposure to Pb2+ and Cu2+ were investigated in the presence and absence of Se4+. In the presence of Se4+, Se-GSH-Px activity and SOD activity were found to be higher and MDA levels were lower compared with in its absence. Hematological parameters were also determined and it has been observed that total leukocyte count (WBC), mean cell volume (MCV), and mean cell hemoglobin (MCH) were increased and erythrocyte
number (RBC), hemoglobin (Hb), and hematocrit value (Hct; P < 0.05) were decreased in fish exposed to heavy metals in the absence of selenium. Selenium presence recovered hematological parameters to normal levels. In the light of our findings, it could be stated that Pb2+ and Cu2+ lead to dramatic changes in biochemical and hematological parameters and selenium caused these parameters to converge to control levels when it was administered concurrently with these heavy metals. Keywords Heavy metals Sodium selenite (Se4+) Rainbow trout Oxidative stress Hematological parameters
Introduction B. Ates I. Yilmaz Department of Chemistry, Inonu University, Malatya 44280, Turkey I. Orun (&) Department of Biology, Faculty of Arts and Science, Aksaray University, Aksaray 68100, Turkey e-mail:
[email protected] Z. S. Talas Department of Biology, Nigde University, Nigde 51100, Turkey G. Durmaz Department of Food Engineering, Inonu University, Malatya 44280, Turkey
Fish have been largely used in the evaluation of the quality of aquatic systems as bioindicators for environmental pollutants (Barak and Mason 1990; Harrison and Klaverkamp 1990; Saiki 1990; Winger et al. 1990; Saiki et al. 1993; Kock et al. 1996). The potential utility of biomarkers for monitoring both environmental quality and the health of the organisms inhabiting polluted ecosystems has received increasing attention over the last few years. Heavy metals are essential trace elements required for maintaining cellular function and are an integral part of a number of heavy metals containing enzymes. However, high intracellular heavy metal
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levels can be toxic, including alterations in intracellular protein machinery, either directly via denaturation of enzymes or indirectly via generation of reactive oxygen species (ROS) (O’Brien and Pourahmad 2000; Droge 2002). Under normal physiological conditions, there is continuous production of ROS that induce oxidative stress and can result in damage to cell membranes, inactivation of enzymes, and damage to genetic material and other vital cell components. Furthermore, some diseases are associated with oxidative stress (Bomzon and Ljubuncic 2001). Copper and lead trace essential elements for cellular metabolism may become extremely toxic for aquatic animals as their concentration in water increases. At equilibrium, there are few free copper or lead ions in natural waters, since most copper and lead are associated with inorganic ions or organic substances. They are highly toxic to fish, so the concentrations required to control algae or pathogen agents must be below the toxicity threshold for fish (Carvalho and Fernandes 2006). Heavy metals enter the aquatic environment from farms and urban and industrial production sites, and cause long-term eco-toxicological effects (Strmack and Braunbeck 2000). Heavy metals prompt formation of reactive oxygen species by catalyzing free radical chain reactions. Free radicals cause some disorders in bio-systems by disrupting the antioxidant/oxidant balance, causing DNA damage and malfunction of cell membranes. Organisms have their own defence mechanism against these defects, consisting of several antioxidant enzymes. These enzymes are inhibited by Pb2+ (Hsu and Guo 2002). As a sign of oxidative stress, assessment of biochemical and hematological parameters provides valuable information about physiological response to environmental changes. Sublethal doses administered to animals provide a clear understanding of the critical levels of certain pollutant chemicals. Fish in particular are commonly used to estimate the influence of environmental pollution due to the sensitivity of their biochemical and hematological parameters under such conditions (Lopes et al. 2001). Inorganic and/or organic selenium compounds are necessary for the development of the acquired immune system (Mruk et al. 2002). Biological importance of selenium is due to its being cofactor for glutathione peroxidase (GSH-Px), which plays a
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key role in the primary antioxidant defence system of the cell. GSH-Px prevents free radical formation by metabolizing peroxides formed in the cell. In recent years, there has been a great deal of studies carried out on selenium metabolism (Shi et al. 2004). In most of these studies the external selenium was given to experimental animals in the Se4+ form (Hintz 1999). The effect of Se4+ on biochemical and hematological parameters of rainbow trout exposed to Pb2+ and Cu2+ have been investigated. Se-GSH-Px activity, superoxide dismutase (SOD) and malondialdehyde (MDA) level, total leukocyte number (WBC), erythrocyte number (RBC), hemoglobin amount (Hb), hematocrit value (Hct), erythrocyte volume of erythrocyte index (MCV), average erythrocyte hemoglobin (MCH), and average erythrocyte hemoglobin concentrations (MCHC) were determined.
Materials and methods Experimental section The rainbow trout (O. mykiss) were purchased from Karakaya Dam Lake trout cultivating farm (Malatya, Turkey). Fish were fed for 15 days in a 8 · 5 · 1.5m stock pond to acclimatize them to the environment. After the adaptation period, ten fish were taken into a 52.82-gallon tank filled with natural spring water. The composition of the water in the ponds is shown in Table 1, temperatures were kept at 11 ± 0.4C, and pH was 7.52 ± 0.02. The fish used had an average weight of 112.05 ± 1.32 g and length of 17.38 ± 0.14 cm. Table 1 Some parameters of the water used in the experiment Criteria (ppm)
Before treatment
After treatment
Biological oxygen level
7.82 ± 0.22
7.62 ± 0.18
Chemical oxygen level
15.14 ± 0.17
16.25 ± 0.21
Suspended solids
36.75 ± 1.20
40.15 ± 1.74
Calcium
126.05 ± 1.54
114.07 ± 1.15
Sodium
22.40 ± 0.85
19.68 ± 0.71
Chlorine
27.45 ± 0.63
25.20 ± 0.52
Total nitrogen Hardness (CaCO3)
5.80 ± 0.22
6.74 ± 0.26
174.30 ± 3.14
168.20 ± 2.81
All data points are the average of n = 3 ± SD
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Preparation of Cu2+, Pb2+ , and Se4+
Lipid peroxidation assay
The stock solutions of Cu(NO3)2 3H2O (Merck), (Pb(NO3)2 (Merck), and Na2SeO3 5H2O (Merck) were used. The final concentration (2 ppm) was achieved by mixing stock solutions of heavy metals and sodium selenite into a 200-l aquarium. Before 72 h of heavy metal exposure, the fish were starved for 12 h.
The analysis of lipid peroxidation was carried out as described (Buege and Aust 1978) with minor modifications. The reaction mixture was prepared by adding 1 ml of homogenate to 4 ml of reaction solution (15% trichloroacetic acid: 0.375% thiobarbituric acid: 0.25 N HCl, 1:1:1, w/v); these were heated to 100C for 10 min. The mixture was cooled to room temperature, centrifuged (10,000·g for 10 min) and the absorbance of the supernatant was recorded at 532 nm. MDA results are expressed as nmol mg 1 of protein in the supernatant. The protein content of the supernatants for enzyme analysis and the MDA assay was determined using a colorimetric method (Lowry et al. 1951), with BSA as the standard. All analyses were performed in duplicate.
Preparation of tissues for biochemical analysis The liver, spleen and heart were dissected using the autopsy technique and stored in the deep freeze. In addition, brain tissue was obtained by opening the head–mouth axis. The tissues were rinsed with 0.9% NaCl and separated into two parts for determination of enzymatic activity and lipid peroxidation. The samples for enzyme analysis were homogenized in PBS buffer (pH 7.4) using PCV Kinematica Status Homogenizer. Homogenized samples were then sonicated for 1.5 min (30-s sonications interrupted by 30-s pause on ice). Samples were then centrifuged at 17,000 rpm for 15 min at +4C and supernatants, if not used for enzyme assays immediately, were kept in the deep freeze at 70C. The second parts of the tissue homogenate were used for lipid peroxidation analysis. Tissue was washed three times with ice-cold 0.9% NaCl solutions and homogenized in 1.15% KCl. The homogenates were assayed for MDA, the product of lipid peroxidation. Enzyme assays The activities of SOD and GSH-Px were determined spectrophotometrically. The SOD level, determined as nanograms of SOD mg 1 protein in the supernatant fraction, was measured through a standard curve generated by commercially obtained SOD (McCord and Fridovich 1969). GSH-Px, activity was determined at 37C in a coupled assay with glutathione reductase and by measuring the rate of nicotinamide adenine dinucleotide phosphate (NADPH) oxidation at 340 nm with hydrogen peroxide as the substrate (Lawrance and Burk 1976). Specific activity for this enzyme was given as micromoles of NADPH transformed min 1 mg 1 protein.
Hematological analysis Before collecting blood samples no anesthesia was applied to the fish as it may affect blood parameters and hemolyzed tissues (McKnight 1966). Blood samples were put into glass tubes containing anticoagulant (EDTA) and analyses were carried out immediately after sampling. The erythrocyte count (per mm3) in blood samples was carried out by pipetting and diluting (½00) the samples in the Hayem solution. One drop of hemolyzed blood was transferred onto Thoma lamella and examined under a light microscope (Soif, XZS-107B model) with a magnification of ·400 (Blaxhall and Daisley 1973). Leukocytes count were performed by transferring blood sample (diluted in Turck solution) with a leukocytes pipette onto counting lamella and examined as for erythrocytes (Blaxhall and Daisley 1973; Blaxhall 1981; De Wilde and Houston 1961). The amount of hemoglobin was determined according to the cyano-methemoglobin procedure (Kit 525A; Sigma Chemical, St. Louis, MO, USA) (Blaxhall and Daisley 1973). Nonclotted blood (20 ll) was diluted with 1 ml of Drabkin solution and left to stand for 10 min at room temperature. The absorbance of the mixture was read at 540 nm and the amount of hemoglobin was calculated according to the hemoglobin standard (Azizoglu and Cengizler 1996). The microhematocrit method was utilized in hematocrit determination (Wilhelm Filho et al. 1992;
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Jewet et al. 1991). Nonclotted blood was pipetted with a microhematocrit pipette, centrifuged at 12,500 rpm for 5 min and the ratio of blood components in the plasma was determined. Statistical analysis
Table 3 The effect of Se4+ added to or not added to Pb2+and Cu2+ on GSH-Px activity, SOD activity, and MDA levels in fish spleen GSH-Px (lmol/mg) Control 2+
Hematological data were analyzed using SPSS 9.0 for Windows using one-way analyses of variance (ANOVA). Differences between means were determined using Duncan’s multiple range test in which the significance level was defined as P < 0.05.
Pb
2+
Pb
plus Se
Cu
4+
plus Se
MDA (nmol/mg)
7.31 ± 0.63a
1.04 ± 0.28a
b
0.21 ± 0.07
b
10.43 ± 1.00a
0.42 ± 0.06
b
6.01 ± 0.48b
3.97 ± 0.23b
0.17 ± 0.08b
4.45 ± 0.65bc
ab
b
2.89 ± 0.34cd
3.16 ± 0.12 4+
Cu2+ 2+
SOD (ng/mg)
4.81 ± 1.32 5.68 ± 0.97
ab
0.42 ± 0.07
1.99 ± 0.44d
All data points are the average of n = 8 ± SD Different superscript letters indicate statistically significant differences (P < 0.05)
Results Changes in Se-GSH-Px and SOD activity, and the MDA level in the liver, spleen, heart, and brain tissues of rainbow trouts are shown on Tables 2–5. A statistically significant decrease (P < 0.05) in SeGSH-Px and SOD activity was observed in the tissues mentioned except the brain of fish exposed to only heavy metals (Pb2+, Cu2+) in comparison with the control group. However, there were a statistically significant increase in MDA levels in the tissues of rainbow trout exposed to Pb2+ and Cu2+ (P < 0.05). In groups treated with heavy metals plus Se4+, enzyme activities and MDA level converged to control group values. Cu2+ administration caused a statistically significant increase (P < 0.05) in the level of WBC, MCV, MCH compared with the control group. However, a decrease in the levels of RBC, Hb, and Hct has been Table 2 The effect of Se4+ added to or not added to Pb2+and Cu2+ on glutathione peroxidase (GSH-Px) activity, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) levels in fish liver
Control
MDA (nmol/mg)
6.96 ± 1.48a
0.80 ± 0.25a
0.30 ± 0.12c
2+
Pb
a
4+
plus Se
2+
Cu
SOD (ng/mg)
MDA (nmol/mg)
4.17 ± 0.86a
0.71 ± 0.16ab
2.49 ± 0.19c
2.24 ± 0.16b
0.36 ± 0.08b
10.78 ± 0.72a
ab
ab
6.04 ± 0.56b
b
8.25 ± 0.93a
a
5.78 ± 0.81b
3.46 ± 0.43
b
Cu
2+
GSH-Px (lmol/mg)
2.56 ± 0.13 4+
plus Se
a
5.37 ± 1.28
0.50 ± 0.14
0.24 ± 0.06
0.78 ± 0.06
All data points are the average of n = 8 ± SD Different superscript letters indicate statistically significant differences (P < 0.05) Table 5 The effect of Se4+ added to or not added to Pb2+and Cu2+ on GSH-Px activity, SOD activity, and MDA levels in fish brain
Control 2+
Pb
2+
Pb
4+
plus Se
Cu2+ 4+
plus Se
GSH-Px (lmol/mg)
SOD (ng/mg)
MDA (nmol/mg)
6.22 ± 0.96
1.06 ± 0.27a
2.33 ± 0.30c
5.15 ± 0.72
b
9.20 ± 0.78a
6.27 ± 0.65
a
1.02 ± 0.17
6.64 ± 0.50b
5.61 ± 0.44
0.33 ± 0.05b
5.78 ± 0.43b
7.10 ± 1.21
a
3.48 ± 0.39c
0.69 ± 0.06
0.96 ± 0.34
All data points are the average of n = 8 ± SD
2+
Pb Pb2+ plus Se4+
b
2.66 ± 0.20 4.32 ± 0.92ab
ab
0.47 ± 0.09 0.49 ± 0.10ab
4.18 ± 0.54 2.21 ± 0.09b
Cu2+
2.88 ± 0.25b
0.14 ± 0.04b
2.13 ± 0.27b
Cu2+ plus Se4+
6.39 ± 1.35a
0.79 ± 0.14a
1.52 ± 0.16b
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Pb2+
Cu
SOD (ng/mg)
Different superscript letters indicate statistically significant differences (P < 0.05)
Control
2+
GSH-Px (lmol/mg)
All data points are the average of n = 8 ± SD
Table 4 The effect of Se4+ added to or not added to Pb2+and Cu2+on GSH-Px activity, SOD activity, and MDA levels in fish heart
Different superscript letters indicate statistically significant differences (P < 0.05)
observed in this group (P < 0.05). On the other hand, relatively lower (P < 0.05) values were obtained from the Cu2+ plus Se4+ group compared with the group in which only lead was given. However, there were no significant changes in MCHC values in any of the group (Tables 6, 7). In the case of lead
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Table 6 Changes in hematological parameters in rainbow trout (Oncorhynchus mykiss) during prolonged exposure to Pb2+ and Pb2+plus Se4+ Control
Pb2+
Pb2+plus Se4+
Total leukocyte count (104/mm3)
4.26 ± 0.71b
7.32 ± 0.58a
5.04 ± 0.71b
Erythrocyte count (106/mm3)
1.27 ± 0.05a
0.72 ± 0.04b
1.09 ± 0.08b
Hemoglobin (g/100 ml)
8.40 ± 0.35a
6.90 ± 0.23b
7.70 ± 0.44a
Hematocrit (%)
30.20 ± 1.81a
25.20 ± 0.84b
26.70 ± 0.96b
Erythrocyte indexes MCV (l3)
237.79 ± 2.41b 262.50 ± 6.70a 244.95 ± 3.95b
MCH (lg)
66.14 ± 3.29b
95.83 ± 5.82a
70.64 ± 3.71b
MCHC (%)
27.81 ± 0.73
27.38 ± 0.82
28.83 ± 1.43
All data points are the average of n = 8 ± SD Different superscript letters indicate statistically significant differences (P < 0.05) Table 7 Changes in hematological parameters in rainbow trout (Oncorhynchus mykiss) during prolonged exposure to Cu2+ and Cu2+plus Se4+ Control
Cu2+
Cu2+ plus Se4+
Total leukocyte count (104/mm3)
4.26 ± 0.71b
6.32 ± 0.18a
5.04 ± 0.64b
Erythrocyte count (106/mm3)
1.27 ± 0.05a
0.92 ± 0.02b
1.15 ± 0.04a
Hemoglobin (g/100 ml)
8.40 ± 0.35a
7.20 ± 0.28b
7.80 ± 0.26a
Hematocrit (%)
30.20 ± 1.81a
26.40 ± 0.74b
28.00 ± 0.62a
Erythrocyte indexes MCV (l3)
237.79 ± 2.41b 286.95 ± 7.65a 243.47 ± 2.85b
MCH (lg)
66.14 ± 3.29b
78.26 ± 4.22a
67.82 ± 3.44b
MCHC (%)
27.81 ± 0.73
27.27 ± 0.54
27.85 ± 0.96
All data points are the average of n = 8 ± SD Different superscript letters indicate statistically significant differences (P < 0.05)
administration, mostly parallel results were found with Cu2+, but RBC and Hct values in the Pb2+ plus Se4+ group were not increased significantly compared with those in the Pb2+ group.
Discussion To predict the impact of environmental pollution on living systems there has been an increasing trend toward using pollutants in controlled medium to monitor the biological changes occurring in organisms. Certain doses and periods are being used and target tissues are examined for required parameters in such studies (Wachowicz et al. 2001; Lemly 2002; Raymond 1989). In particular, the level of antioxidant enzyme is a good indicator for the impacts of pollutants like heavy metals. The heavy metal damage is an important factor in many pathological and toxicological processes. One of the most important biochemical parameters for those toxicological effects is the SOD level of tissues. SOD is called the first line of the cell against ROS due to the superoxide radical being a precursor to several other highly reactive species (Fridovich 1997). GSH-Px, on the other hand, scavenges H2O2, which is a precursor of ROS in addition to its protective role against lipid peroxidation of cell membrane. Lipid peroxidation, a complex, self-propagating and highly destructive process, increases the rigidity (decreases the fluidity) of cellular membranes. In the case of oxidative stress, the MDA level was regarded as a well-suited indicator for the extent of lipid peroxidation (Shi et al. 2004). Hematological parameters are determined as an index of their health status as well (Perottoni et al. 2004). Pb2+ and Cu2+ administration altered all parameters for all tissues except for GSH-Px activity of brain tissue (P < 0.05). This finding is not surprising due to the brain being the most shielded part of the body and the lipophilic structure of the brain does not allow metals and chelates to pass over cell membranes. On the contrary, spleen and liver are the primary organs that face heavy metals and sharp changes in biochemical parameters are normal for these tissues. The change in MDA level was more dramatic (P < 0.05) than changes in enzyme values because oxidative stress originating from heavy metals directly affects the MDA level. As an important indicator for oxidative damage, the MDA level decreased in all tissues of fish exposed to Pb2+, Cu2+, and Se4+ (Tables 2–5). These results were in negative correlation with SOD and GSH-Px activity in which selenium acts as a cofactor. Se4+ can contribute to the antioxidative defence system at a
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concentration of 2 ppm for rainbow trouts. This tolerance can be explained by the cofactor nature of selenium for GSH-Px. Fish exposed to Pb2+ and Cu2+ display a tendency toward decreased antioxidant enzyme activities RBC, HGB, and HCT levels. The toxic effects of Pb2+ and Cu2+ on the inhibition of erythropoiesis in the hematopoietic organism and the inhibition of antioxidant enzyme activity have prevented this reaching a statistically significant level. But in the case of Se4+ plus these metals, this might be related to the fact that the toxic effects of heavy metals are decreased. Decreases in erythrocyte, hemoglobin, and hematocrit values can be an indicator of anemia with the subsequent result of inhibition of erythropoiesis in the hemopoietic organism. Furthermore, increases in MCV values showed that anemia was of the macrocytic type, because there was no change in the MCH and MHCH values. In addition, high leukocyte values depending on metal stress were the result of the stimulatory effect of the toxic agent on the immune system. There were decreases in blood parameters such as erythrocyte number, hemoglobin level, and hematocrit value; however, no change was observed in leukocyte number. There was an important decrease in hematocrit value in the fish samples. Our results are in good accordance with previously reported results. Catalase (CAT) inhibition may be related to the accompanied direct binding of metal ions to –SH groups on the enzyme molecule, and increased hydrogen peroxide and superoxide radical due to oxidative stress. It was indicated that rapid inactivation of CAT at a high hydrogen peroxide concentration was due to the converting of active enzyme compounds to inactive compounds (Wong and Whitaker 2002). In addition, stimulation of CAT activity can be caused by an effective antioxidant defence system acting against oxidative stress and/or compensating for the decrease in other antioxidant enzymes such as SOD and GSH-Px. On the other hand, no significant change in CAT activity may be attributed to the increase in other antioxidant enzymes such as GSH-Px and/or non-enzymatic mechanisms such as GSH and metallothioneins. In vitro the general inhibition of CAT activity in all tissues of O. Niloticus may be due to the direct effect of metals. Roche and Boge (1993) demonstrated that the modification of CAT activity in sea bass erythrocytes in response to metallic ions in vitro that
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Se4+ activated, while Pb2+, Cu2+ inhibited the activity, is highly dependent on the nature of the metal. They also suggest that deterioration of the protective defence system might occur due to the formation of oxyradicals caused by metals, whereas enhancement of CAT activity might compensate for the decreased activity of other antioxidant enzymes. Increasing evidence indicates that multifactorial mechanisms might be involved in metal-induced toxicity and that one of the well-known mechanisms is a metalinduced ROS. Fenton-like reactions appear to play a major role in the oxidative stress observed in redoxactive metal (Pb2+, Cu2+) toxicity. Conclusions The response of GSH-Px and SOD activities, MDA level, and hematological parameters in different tissues of O. mykiss exposed to Pb2+ and Cu2+ in the presence and absence of Se4+ were studied. Pb2+ and Cu2+ exposure alone altered these parameters significantly and Se4+ showed recovering effects when administered along with Pb2+ or Cu2+. Accumulation of metals in organisms in contaminated water is an important aspect of environmental awareness, because it may affect all members of the food chain, including fish. Biochemical and hematological parameters can be accepted as sensitive biomarkers for biomonitoring the aquatic environment before the detrimental effects occur for aquatic species. In further studies the effect of other metals on some other physiological parameters of different aquatic species could be investigated in controlled polluted waters. References Azizoglu A, Cengizler I_ (1996) an investigation on determination of some hematologic parameters in healthy Oreochromis niloticus (L.). Turk J Vet Anim Sci 20:425–431 Barak DA, Mason C (1990) A survey of heavy metals levels in eels (Anguilla anguilla) from some rivers in East Anglia, England: the use of eels as pollution indicators. Int Revue Ges Hydrobiol 75:827–833 Blaxhall PC (1981) A comparison of methods used for the separation of fish lymphocytes. J Fish Biol 18:177–181 Blaxhall PC, Daisley KW (1973) Routine hematological methods for use with fish blood. J Fish Biol 5:771–781 Bomzon A, Ljubuncic P (2001) Oxidative stress and vascular smooth muscle cell function in liver disease. Pharmacol Ther 89:295–308
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