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Responses of metallothionein and reduced glutathione in a freshwater fish Oreochromis niloticus following metal exposures
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Environmental Toxicology and Pharmacology 25 (2008) 33–38
Responses of metallothionein and reduced glutathione in a freshwater fish Oreochromis niloticus following metal exposures G¨ul¨uzar Atli ∗ , Mustafa Canli Department of Biology, Faculty of Sciences and Letters, University of C ¸ ukurova, 01330 Adana, Turkey Received 16 March 2007; received in revised form 16 August 2007; accepted 23 August 2007 Available online 30 August 2007
Abstract In this study, levels of reduced glutathione (GSH) and metallothionein (MT) which are known to be biomarker of metal exposures were measured in a freshwater fish Oreochromis niloticus following exposure to 0, 5, 10 and 20 M concentrations of Cu, Zn, Cd and Pb for 14 days. Metals and GSH were measured in the liver, gill, intestine, muscle and blood, and MT in the liver. Copper accumulation occurred only in the gill, while Zn accumulation occurred only in the muscle. Lead accumulated in the liver and gill, whereas Cd accumulated in all the tissues. Metal exposures did not alter GSH levels in the blood, muscle and gill, but its levels increased in the liver following Cd, Zn and Cu exposures. MT levels in the liver increased only in Cd-exposed fish. The results showed that there was no significant change in tissue GSH levels following metal exposures, except in the liver. The levels of liver GSH increased significantly by all the metals, except lead. Data indicated that only the liver may be suitable indicator tissue to determine the response of GSH and MTs to metal exposure in environmental monitoring studies. © 2007 Elsevier B.V. All rights reserved. Keywords: Reduced glutathione; Metallothionein; Metals; Fish; Oreochromis niloticus
1. Introduction Heavy metals in the aquatic environments have been seen as a potential threat for aquatic organism for several decades (Heath, 1987). Metals are known to inhibit the several biochemical and physiological mechanisms vital for fish metabolism (Hogstrand et al., 1999; Basha and Rani, 2003). The use of stress indices has been recently proposed to evaluate the effects of metals on aquatic organisms. Two major sources of cellular thiols reduced glutathione (GSH) and metallothioneins (MTs) are important biomarkers due to their critical function in maintaining appropriate redox potentials and viability in the cell (Schlenk and Rice, 1998; Atif et al., 2005). Glutathione (l-␥-glutamyl-cysteinyl-glycine) is a tripeptide that is mainly present in cells in its reduced form (GSH), which basically acts as an intracellular reductant and nucleophile. It functions in the synthesis of proteins and DNA, amino acid transport, maintenance of the thiol-disulfide status, free radi-
∗
Corresponding author. Fax: +90 322 3386070. E-mail address: [email protected] (G. Atli).
1382-6689/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2007.08.007
cal scavenging, signal transduction, as an essential cofactor of several enzymes, as a non-toxic storage form of cysteine, and as a defence against oxidizing molecules and potentially harmful xenobiotics such as metals (Pena-Llopis et al., 2001; Elia et al., 2003). It was suggested that GSH content showed both increases and decreases in fish tissues exposed to metals due to their organ-specific responses (Pena et al., 2000; Sayeed et al., 2003; Ali et al., 2004). MTs could be a sensitive indicator of heavy metal contamination in the aquatic environment and this has been suggested by several investigators, especially for cadmium contamination (Hogstrand and Haux, 1990; Canli et al., 1997; De Conto Cinier et al., 1998; Atli and Canli, 2003). MTs are low molecular weight (6000–7000 Da), cysteine-rich (33%), heat-resistant cytosolic proteins that bind metals such as cadmium, copper, zinc and mercury (Kagi and Schaffer, 1988; Roesijadi, 1992, Roesijadi and Robinson, 1994). MTs occur naturally in tissues and play significant role in the homeostasis of essential metals, particularly zinc and copper and also they bind non-essential metals for sequestration. Both essential and non-essential metals stimulate MT synthesis, though induction capacity may differ among metals (Hogstrand and Haux, 1990; Wu et al., 1999; Atli and Canli, 2003).
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G. Atli, M. Canli / Environmental Toxicology and Pharmacology 25 (2008) 33–38
2. Materials and methods
Pellet was resuspended in 150 L 0.25 M NaCl and 150 L 1N HCl containing 4 mM EDTA and 4.2 mL 0.43 mM DTNB buffered with 0.2 M Na-phosphate (pH 8.0) was added to the sample. The sample was then centrifuged at 4500 × g for 10 min and the absorbance was measured at 412 nm. MT concentration was estimated using reduced glutathione as a reference and expressed as g MT/mg protein. Total protein levels were determined following the method of Lowry et al. (1951) using the bovine serum albumin as a reference.
2.1. Experimental protocol
2.4. Total metal analysis
O. niloticus were obtained from fish culturing pools of C ¸ ukurova University and transferred to the laboratory. They were acclimatized to the laboratory conditions for 1 month before the experiments. The laboratory was illuminated for 12 h with fluorescent lamps (daylight 65/80 W) and kept at 20 ± 1 ◦ C. The dechlorinated tap water used in the experiments had a pH value of 8.23 ± 0.15, total hardness of 324 ± 22.2 mg CaCO3 /L and an alkalinity of 142 ± 11.8 mg CaCO3 /L. The aquariums were aerated with air stones attached to an air compressor to saturate with oxygen (5.8 ± 0.97 mg O2 /L). The experiments were conducted on glass aquariums sized 40 cm × 40 cm × 50 cm, each containing eight fish in 55 L of contaminated test solution or tap water only for controls. Fish were exposed to 5, 10 and 20 M concentrations of Cu (CuCl2 ·2H2 O), Zn (ZnCl2 ), Cd (CdCl2 ·H2 O) and Pb (Pb(NO3 )2 ) for 14 days. Waters in aquariums were changed every 2 days to minimize metal loss just after feeding to reduce contamination of the environment with food remains. After 14 days of exposure period, all the fish were killed by transaction of the spinal cord. Total lengths (15.7 ± 1.21 cm) and weights (61.5 ± 12.8 g) of fish did not differ significantly (P > 0.05) among different exposure regimes and control. Liver, gill, intestine and muscle tissues were dissected using clean equipments and stored at −80 ◦ C until the enzyme analysis. Blood samples were taken from the caudal vein of each fish that is the best place to obtain fish blood (Congleton and La Voie, 2001). The tissues were homogenized (1:10, w/v) in 20 mM Tris buffer (pH 7.8) containing 0.25 M sucrose at 9500 rpm for 2 min. Homogenates were centrifuged at 10,000 × g (Hettich Universal 30 RF) for 30 min (+4 ◦ C) and supernatant was used for the analysis. 0.2 mL of whole blood is added to 2.0 mL of distilled water and this hemolysate was used for the determination of reduced glutathione. All chemical used in this study was obtained from Sigma or Merk (Germany). All the measurements were done on individual fish.
Metal concentrations in tissues were measured using a flame atomic absorption spectrophotometer (Perkin-Elmer AS 3100) with the method explained elsewhere (Canli, 1995). The detection limits of metals were 0.001, 0.002, 0.002 and 0.02 g/mL for Cd, Zn, Cu and Pb, respectively. Accuracy of the AAS and validity of measurements were tested with a reference material (TORT 1 lobster hepatopancreas, National Research Council, Canada). Mean values and standard deviations of the reference material were within 10% the ranges.
GSH and MT levels may provide a useful tool to assess the biological effects of metal contamination in aquatic environment. The aim of this study is to determine the GSH and MT levels in the tissues of O. niloticus exposed to different concentrations of Cd, Cu, Zn and Pb.
2.2. Glutathione determination
2.5. Statistical analysis Statistical analysis of data was carried out using SPSS statistical package programs. One-way ANOVA was used to compare variables among controls and treatments, and if significant (P < 0.05) differences were found, these data were reanalyzed by Duncan test to determine which individual groups were significantly different from control.
3. Results There were no significant changes in the levels of GSH in the gill (Fig. 1), blood (Fig. 2) and the muscle (Fig. 3) when compared to control values. Likewise, increases in GSH levels were also observed at 10 M Cu, 5 M and 20 M Cd and Zn exposures in the liver (Fig. 4). The highest increase in GSH levels was measured in the liver. MT levels increased significantly only in Cd-exposed fish (Fig. 5), highest increase being in the highest Cd exposure experiment. The other metal exposures did not cause significant change in MT levels when compared to control values.
Reduced glutathione was measured following the method of Beutler et al. (1963). First, 3.0 mL precipitating solution containing metaphosphoric acid, Na2 EDTA and NaCl was added to 2.0 mL of the sample. The mixture was centrifuged at 4500 × g for 10 min. 1.0 mL of supernatant was added to 4.0 mL of 0.3 M Na2 HPO4 solution and 0.5 mM DTNB (5,5 -dithiobis-2-nitrobenzoic acid) was then added to this solution. Reduced glutathione was measured as the difference in the absorbance values of samples in the presence and the absence of DTNB at 412 nm. GSH value was calculated as nmol GSH/mg protein in the tissues and mmol GSH/g Hb in whole blood using the reduced glutathione as a reference (hemoglobin levels were estimated in whole blood using the Drabkins’ solution).
2.3. MT determination MT was determined following the method of Viarengo et al. (1996). The supernatants were heated in water bath at 80 ◦ C for 10 min and centrifuged at 2500 × g for 10 min to remove the coagulated particules. Then, 1.05 mL of cold (−20 ◦ C) absolute ethanol and 80 L chloroform were added to 1 mL of supernatant. These samples were centrifuged at 6000 × g for 10 min at +4 ◦ C. Three volumes of cold ethanol were added to the supernatant and the mixture was maintained at −20 ◦ C for 1 h. The samples were then centrifuged at 6000 × g for 10 min at +4 ◦ C. The supernatant was decanted and pellet washed with 87% ethanol and 1% chloroform in homogenizing buffer and centrifuged at 6000 × g for 10 min (+4 ◦ C).
Fig. 1. Gill GSH level in O. niloticus exposed to metals for 14 days. Data are expressed as mean (n = 8) ± standard error. P values indicate the results of one-way ANOVA while asterisks indicate the results of LSD tests.
G. Atli, M. Canli / Environmental Toxicology and Pharmacology 25 (2008) 33–38
Fig. 2. Blood GSH level in O. niloticus exposed to metals for 14 days. See Fig. 1 for details.
Fig. 3. Muscle GSH level in O. niloticus exposed to metals for 14 days. See Fig. 1 for details.
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Fig. 4. Liver GSH level in O. niloticus exposed to metals for 14 days. See Fig. 1 for details.
Fig. 5. Liver MT level in O. niloticus exposed to metals for 14 days. See Fig. 1 for details.
Table 1 Total metal concentrations (mean ± standard deviation g/g dry weight; n = 8) in the tissues of O. niloticus (asterisks indicate significant (P < 0.05) differences resulted from the Duncan tests between control and individual treatments) Tissue
Concentration (M)
Metal Cu
Zn
Cd
Pb
Gill
0 5 10 20
3.9 13.9 25.6 30.4
± ± ± ±
1.5 10.4* 2.4* 6.2*
177.4 182.7 173.1 196.1
± ± ± ±
96.4 0.01 81.3 45.8
<0.001 10.8 ± 2.3* 11.5 ± 1.3* 16.2 ± 3.4*
<0.02 8.2 ± 8.2 45.9 ± 34.4* 57.4 ± 26.1*
Liver
0 5 10 20
169.2 224.2 233.6 295.3
± ± ± ±
35.6 70.5 55.8 81.2
138.8 204.5 170.5 253.8
± ± ± ±
55.9 0.01 80.5 82.3
<0.001 27.7 ± 10.9* 64.8 ± 15.5* 81.9 ± 21.9*
<0.02 <0.02 14.3 ± 5.3* 10.2 ± 2.0*
Muscle
0 5 10 20
0.88 1.09 0.15 0.77
± ± ± ±
0.58 1.09 0.14 0.77
120.9 162.6 291.5 641.8
± ± ± ±
81.5 0.01 173.6 2.3*
<0.001 <0.001 1.5 ± 0.8* 2.18 ± 0.41*
<0.02 <0.02 <0.02 <0.02
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G. Atli, M. Canli / Environmental Toxicology and Pharmacology 25 (2008) 33–38
Total metal levels in the tissues of fish were shown in Table 1. There was no significant Zn accumulation in the gill, while the levels of the other metals increased significantly in this tissue. In the liver, Cd and Pb accumulated significantly, while the levels of Cu and Zn did not change significantly. In the muscle, Zn and Cd levels increased significantly, whereas there was no copper accumulation. However, Pb levels in the muscle were below the detection limit for the instrument. 4. Discussion Metal accumulation in fish tissues occur in relation to environmental concentrations, exposure duration and other characteristics of water such as ions, pH, hardness and temperature (Heath, 1987). The present results showed that metal exposures generally increased the total tissue concentrations of metals, though the increases of essential metals were not significant in some tissues, indicating their regulation in those tissues. The regulation of essential metals by fish was also demonstrated in previous studies. However, regulation of cadmium and lead was not evident for fish (Heath, 1987), as occurred in the present study. One of our previous studies revealed the natural occurrence of Cu and Zn MTs but not Cd, Pb and Fe MTs in the liver of O. niloticus (Atli and Canli, 2003). Exposure of fish to these metals in the same concentrations showed that the levels of Cu and Zn in MT fraction did not increase, whereas Fe and Pb were not associated with any of the proteins in both controls and metal exposed groups. However, Cd in MT fraction of Cd-exposed fish increased many fold. Likewise, the present study also showed that only significant increase in MT level was observed in the liver of fish exposed to Cd, whereas the other metal exposures did not cause significant increases in MT levels in the liver. Although significant Pb accumulation occurred in the liver, this was not reflected in MT increase in this tissue. This data agree with other data from the literature indicating the absence of Pb binding MT-like proteins (Reichert et al., 1979; Roesijadi and Robinson, 1994; Atli and Canli, 2003). No significant changes in the MT level in the liver of fish exposed to Cu and Zn concentrations may be related to the high basal levels of these metals in the liver. Linde et al. (2005) showed that MT in hepatic cytosols from Mugil cephalus contained the most Cu, though MT was not the major hepatic Cu-binding protein when total cytosolic Cu increased in the mullet. The present results are in accord with our previous data in the induction of MT-like protein, indicating Cd is the main inducer of MTs (Atli and Canli, 2003; Eroglu et al., 2005; Baykan et al., 2007). Some other studies carried out with tilapia sp. revealed that Cd levels increases in MT fraction in the liver of Cd-exposed fish (Ueng et al., 1996; Wu et al., 2002). It seems that Zn and Cu MTs cannot increase unless basal levels of Cu and Zn are increased considerably. GSH is synthesized in the liver and released to the blood for transferring to the other organs such as the kidney and muscle (Pena et al., 2000). Because metal exposures did not alter the levels of GSH in the blood, muscle and gill, it suggests that metals taken up from the gill were immediately transferred (via the blood) to the liver for the usage in the metabolism or sequestered.
Thus, GSH levels in the blood may be a good indicator to understand the degree of metal exposure, suggesting the fact that metal exposures in this study were in the tolerable range for O. niloticus for the studied period. Nevertheless, the results showed considerable differences in GSH levels among tissues, metals and their exposure concentrations. Similarly, variations of GSH levels in different fish species were also observed in other studies, indicating the tissue and metal specific characteristics of GSH induction (Pena et al., 2000; Elia et al., 2003; Ali et al., 2004). The induction of GSH in the liver of fish exposed to Cd, Zn and Cu is probably due to primary defence system which GSH involved in to protect the fish from the oxidative stress. Lima et al. (2006) indicated that increased GSH levels in O. niloticus exposed to a contaminated effluent appear to be an antioxidant adaptation to chronic exposure. On the other hand, the decreased GSH levels in the gill and kidney of Anguilla anguilla exposed to 1.0 and 2.5 M Cu were related to the increased use of GSH to stabilize Cu in its oxidative stress for preventing the redox cycling and free radicals regeneration (Ahmad et al., 2005). Recently, Zirong and Shijun (2007) showed that cadmium (3 mg/L) exposure for up to 40 days depleted the GSH levels in the liver of O. niloticus though GSSG–GSH ratio increased. They concluded that GSH first made a rapid protection against oxidative stress and resulted in a sharp decrease in its contents. Like metals, pesticides can affect GSH levels in fish tissues as they can also be an oxidative stress inducer (Pena et al., 2000; Sayeed et al., 2003). The induction of MT-like proteins occurred in the liver hepatocyte carcinoma cells of Poeciliopsis treated with 10 M Cd or 10 M Zn. A significant increase in GSH levels was observed at 10 M Cd exposure whereas Zn failed to change GSH content (Schlenk and Rice, 1998). They concluded that metal induced GSH and MT may provide better protection against oxidative damage caused by metals. Atif et al. (2005) determined that GSH level did not increase in the liver of Channa punctata exposed to 0.2 mg Cd/kg intraperitoneally, though MT-like protein was detected in the gel electrophoresis. The authors pointed the increase in GSH and MT levels as important protective mechanisms that fish adopt in the initial phases of exposure to aquatic pollutants. In addition, Sanchez et al. (2005) recorded that the biochemical responses were more rapid than metal accumulation, suggesting the involvement of differential mechanisms in metal metabolism involves metal intake by GSH and formation of GSH–metal complex, from which the metal is further transferred to MT apoproteins where it is stored. In the present study, metal exposures caused significant increases in GSH and MT concentrations, particularly in the liver of O. niloticus, though metal accumulation and induction of these biomarkers were not well correlated. It is well known that these molecules are major sources of thiols for preventing the interactions of metals with main cellular structures as a first line defence. Enhanced GSH level, induction of MT and increased Cd accumulation in the liver of fish exposed to Cd may be due to the possible Cd toxicity in this tissue that known as the main storage organ for Cd and place where biochemical parameters such as GSH and MT involved in pivotal detoxification mechanisms.
G. Atli, M. Canli / Environmental Toxicology and Pharmacology 25 (2008) 33–38
As a result, GSH and MT may be considered as sensitive biochemical indicators in ecotoxicology studies and response of these parameters could be useful indices of environmental quality. Nevertheless, the use of MTs or GSH as a sensitive biomarker for detection metal contamination may be limited when metal uptake rate cannot reach to a threshold level. This limit could be achieved much lower concentrations for non-essential metals (e.g. Cd), though the threshold levels for essential metals (e.g. Cu, Zn) are much higher. Therefore, it can be suggested that MTs or GSH could be a sensitive indicator of non-essential metals such as cadmium rather than essential metals (Lange et al., 2002; Atli and Canli, 2003; Lecoeur et al., 2004). Special attention should be given to the threshold levels of both essential and non-essential metal levels necessary to trigger MT and GSH induction in the aquatic environment. With this context, one can conclude that Cd MTs could be used as a sensitive tool to detect Cd contamination, though this may not be possible for Cu and Zn unless basal levels exceed. It seems that it is more complicated to use GSH as a sensitive biomarker for metal contamination in the aquatic environment because there is no considerable response to metal exposure in tissues of O niloticus, except in the liver. Additionally, the response of liver to metal exposures was not linear in relation to exposure concentrations, as there was no response at the highest metal exposures, except Cd exposure. Acknowledgement This study was supported by a grant FEF 2006 BAP23 from C ¸ ukurova University. References Ahmad, I., Oliveira, M., Pacheco, M., Santos, M.A., 2005. Anguilla anguilla L. oxidative stress biomarkers responses to copper exposure with or without -naphthoflavone pre-exposure. Chemosphere 61, 267–275. Ali, M., Parvez, S., Pandey, S., Atif, F., Kaur, M., Rehman, H., Raisuddin, S., 2004. Fly ash leachate induces oxidative stress in freshwater fish Channa punctata (Bloch). Environ. Int. 30, 933–938. Atif, F., Parvez, S., Pandey, S., Ali, M., Kaur, M., Rehman, H., Khan, H.A., Raisuddin, S., 2005. Modulatory effect of cadmium exposure on deltamethrin-induced oxidative stress in Channa punctata Bloch. Arch. Environ. Contam. Toxicol. 49, 371–377. Atli, G., Canli, M., 2003. Natural occurrence of metallothionein-like proteins in the liver of fish Oreochromis niloticus and effects of cadmium, lead, copper, zinc, and iron exposures on their profiles. Bull. Environ. Contam. Toxicol. 70, 619–627. Basha, P.S., Rani, A.U., 2003. Cadmium-induced antioxidant defense mechanism in freshwater teleost Oreochromis mossambicus (Tilapia). Ecotoxicol. Environ. Saf. 56, 218–221. Baykan, U., Atli, G., Canli, M., 2007. The effects of temperature and metal exposures on the profiles of metallothionein-like proteins in Oreochromis niloticus. Environ. Toxicol. Pharmacol. 23, 33–38. Beutler, E., Duron, O., Kelly, B.M., 1963. Improved method for the determination of blood glutathione. J. Lab. Clinic. Med. 61, 882–890. Canli, M., 1995. Natural occurrence of metallothionein-like proteins in the hepatopancreas of Norway lobster (Nephrops norvegicus L.) and effects of cadmium, copper and zinc exposures on levels of the metals bound on metallothioneins. Tr. J. Zool. 19, 313–321. Canli, M., Stagg, R.M., Rodger, G., 1997. The induction of metallothionein in tissues of the Norway lobster Nephrops norvegicus following exposure to cadmium, copper and zinc: the relationships between metallothionein and the metals. Environ. Pollut. 96, 343–350.
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Environmental Toxicology and Pharmacology 25 (2008) 33–38
Responses of metallothionein and reduced glutathione in a freshwater fish Oreochromis niloticus following metal exposures G¨ul¨uzar Atli ∗ , Mustafa Canli Department of Biology, Faculty of Sciences and Letters, University of C ¸ ukurova, 01330 Adana, Turkey Received 16 March 2007; received in revised form 16 August 2007; accepted 23 August 2007 Available online 30 August 2007
Abstract In this study, levels of reduced glutathione (GSH) and metallothionein (MT) which are known to be biomarker of metal exposures were measured in a freshwater fish Oreochromis niloticus following exposure to 0, 5, 10 and 20 M concentrations of Cu, Zn, Cd and Pb for 14 days. Metals and GSH were measured in the liver, gill, intestine, muscle and blood, and MT in the liver. Copper accumulation occurred only in the gill, while Zn accumulation occurred only in the muscle. Lead accumulated in the liver and gill, whereas Cd accumulated in all the tissues. Metal exposures did not alter GSH levels in the blood, muscle and gill, but its levels increased in the liver following Cd, Zn and Cu exposures. MT levels in the liver increased only in Cd-exposed fish. The results showed that there was no significant change in tissue GSH levels following metal exposures, except in the liver. The levels of liver GSH increased significantly by all the metals, except lead. Data indicated that only the liver may be suitable indicator tissue to determine the response of GSH and MTs to metal exposure in environmental monitoring studies. © 2007 Elsevier B.V. All rights reserved. Keywords: Reduced glutathione; Metallothionein; Metals; Fish; Oreochromis niloticus
1. Introduction Heavy metals in the aquatic environments have been seen as a potential threat for aquatic organism for several decades (Heath, 1987). Metals are known to inhibit the several biochemical and physiological mechanisms vital for fish metabolism (Hogstrand et al., 1999; Basha and Rani, 2003). The use of stress indices has been recently proposed to evaluate the effects of metals on aquatic organisms. Two major sources of cellular thiols reduced glutathione (GSH) and metallothioneins (MTs) are important biomarkers due to their critical function in maintaining appropriate redox potentials and viability in the cell (Schlenk and Rice, 1998; Atif et al., 2005). Glutathione (l-␥-glutamyl-cysteinyl-glycine) is a tripeptide that is mainly present in cells in its reduced form (GSH), which basically acts as an intracellular reductant and nucleophile. It functions in the synthesis of proteins and DNA, amino acid transport, maintenance of the thiol-disulfide status, free radi-
∗
Corresponding author. Fax: +90 322 3386070. E-mail address: [email protected] (G. Atli).
1382-6689/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2007.08.007
cal scavenging, signal transduction, as an essential cofactor of several enzymes, as a non-toxic storage form of cysteine, and as a defence against oxidizing molecules and potentially harmful xenobiotics such as metals (Pena-Llopis et al., 2001; Elia et al., 2003). It was suggested that GSH content showed both increases and decreases in fish tissues exposed to metals due to their organ-specific responses (Pena et al., 2000; Sayeed et al., 2003; Ali et al., 2004). MTs could be a sensitive indicator of heavy metal contamination in the aquatic environment and this has been suggested by several investigators, especially for cadmium contamination (Hogstrand and Haux, 1990; Canli et al., 1997; De Conto Cinier et al., 1998; Atli and Canli, 2003). MTs are low molecular weight (6000–7000 Da), cysteine-rich (33%), heat-resistant cytosolic proteins that bind metals such as cadmium, copper, zinc and mercury (Kagi and Schaffer, 1988; Roesijadi, 1992, Roesijadi and Robinson, 1994). MTs occur naturally in tissues and play significant role in the homeostasis of essential metals, particularly zinc and copper and also they bind non-essential metals for sequestration. Both essential and non-essential metals stimulate MT synthesis, though induction capacity may differ among metals (Hogstrand and Haux, 1990; Wu et al., 1999; Atli and Canli, 2003).
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2. Materials and methods
Pellet was resuspended in 150 L 0.25 M NaCl and 150 L 1N HCl containing 4 mM EDTA and 4.2 mL 0.43 mM DTNB buffered with 0.2 M Na-phosphate (pH 8.0) was added to the sample. The sample was then centrifuged at 4500 × g for 10 min and the absorbance was measured at 412 nm. MT concentration was estimated using reduced glutathione as a reference and expressed as g MT/mg protein. Total protein levels were determined following the method of Lowry et al. (1951) using the bovine serum albumin as a reference.
2.1. Experimental protocol
2.4. Total metal analysis
O. niloticus were obtained from fish culturing pools of C ¸ ukurova University and transferred to the laboratory. They were acclimatized to the laboratory conditions for 1 month before the experiments. The laboratory was illuminated for 12 h with fluorescent lamps (daylight 65/80 W) and kept at 20 ± 1 ◦ C. The dechlorinated tap water used in the experiments had a pH value of 8.23 ± 0.15, total hardness of 324 ± 22.2 mg CaCO3 /L and an alkalinity of 142 ± 11.8 mg CaCO3 /L. The aquariums were aerated with air stones attached to an air compressor to saturate with oxygen (5.8 ± 0.97 mg O2 /L). The experiments were conducted on glass aquariums sized 40 cm × 40 cm × 50 cm, each containing eight fish in 55 L of contaminated test solution or tap water only for controls. Fish were exposed to 5, 10 and 20 M concentrations of Cu (CuCl2 ·2H2 O), Zn (ZnCl2 ), Cd (CdCl2 ·H2 O) and Pb (Pb(NO3 )2 ) for 14 days. Waters in aquariums were changed every 2 days to minimize metal loss just after feeding to reduce contamination of the environment with food remains. After 14 days of exposure period, all the fish were killed by transaction of the spinal cord. Total lengths (15.7 ± 1.21 cm) and weights (61.5 ± 12.8 g) of fish did not differ significantly (P > 0.05) among different exposure regimes and control. Liver, gill, intestine and muscle tissues were dissected using clean equipments and stored at −80 ◦ C until the enzyme analysis. Blood samples were taken from the caudal vein of each fish that is the best place to obtain fish blood (Congleton and La Voie, 2001). The tissues were homogenized (1:10, w/v) in 20 mM Tris buffer (pH 7.8) containing 0.25 M sucrose at 9500 rpm for 2 min. Homogenates were centrifuged at 10,000 × g (Hettich Universal 30 RF) for 30 min (+4 ◦ C) and supernatant was used for the analysis. 0.2 mL of whole blood is added to 2.0 mL of distilled water and this hemolysate was used for the determination of reduced glutathione. All chemical used in this study was obtained from Sigma or Merk (Germany). All the measurements were done on individual fish.
Metal concentrations in tissues were measured using a flame atomic absorption spectrophotometer (Perkin-Elmer AS 3100) with the method explained elsewhere (Canli, 1995). The detection limits of metals were 0.001, 0.002, 0.002 and 0.02 g/mL for Cd, Zn, Cu and Pb, respectively. Accuracy of the AAS and validity of measurements were tested with a reference material (TORT 1 lobster hepatopancreas, National Research Council, Canada). Mean values and standard deviations of the reference material were within 10% the ranges.
GSH and MT levels may provide a useful tool to assess the biological effects of metal contamination in aquatic environment. The aim of this study is to determine the GSH and MT levels in the tissues of O. niloticus exposed to different concentrations of Cd, Cu, Zn and Pb.
2.2. Glutathione determination
2.5. Statistical analysis Statistical analysis of data was carried out using SPSS statistical package programs. One-way ANOVA was used to compare variables among controls and treatments, and if significant (P < 0.05) differences were found, these data were reanalyzed by Duncan test to determine which individual groups were significantly different from control.
3. Results There were no significant changes in the levels of GSH in the gill (Fig. 1), blood (Fig. 2) and the muscle (Fig. 3) when compared to control values. Likewise, increases in GSH levels were also observed at 10 M Cu, 5 M and 20 M Cd and Zn exposures in the liver (Fig. 4). The highest increase in GSH levels was measured in the liver. MT levels increased significantly only in Cd-exposed fish (Fig. 5), highest increase being in the highest Cd exposure experiment. The other metal exposures did not cause significant change in MT levels when compared to control values.
Reduced glutathione was measured following the method of Beutler et al. (1963). First, 3.0 mL precipitating solution containing metaphosphoric acid, Na2 EDTA and NaCl was added to 2.0 mL of the sample. The mixture was centrifuged at 4500 × g for 10 min. 1.0 mL of supernatant was added to 4.0 mL of 0.3 M Na2 HPO4 solution and 0.5 mM DTNB (5,5 -dithiobis-2-nitrobenzoic acid) was then added to this solution. Reduced glutathione was measured as the difference in the absorbance values of samples in the presence and the absence of DTNB at 412 nm. GSH value was calculated as nmol GSH/mg protein in the tissues and mmol GSH/g Hb in whole blood using the reduced glutathione as a reference (hemoglobin levels were estimated in whole blood using the Drabkins’ solution).
2.3. MT determination MT was determined following the method of Viarengo et al. (1996). The supernatants were heated in water bath at 80 ◦ C for 10 min and centrifuged at 2500 × g for 10 min to remove the coagulated particules. Then, 1.05 mL of cold (−20 ◦ C) absolute ethanol and 80 L chloroform were added to 1 mL of supernatant. These samples were centrifuged at 6000 × g for 10 min at +4 ◦ C. Three volumes of cold ethanol were added to the supernatant and the mixture was maintained at −20 ◦ C for 1 h. The samples were then centrifuged at 6000 × g for 10 min at +4 ◦ C. The supernatant was decanted and pellet washed with 87% ethanol and 1% chloroform in homogenizing buffer and centrifuged at 6000 × g for 10 min (+4 ◦ C).
Fig. 1. Gill GSH level in O. niloticus exposed to metals for 14 days. Data are expressed as mean (n = 8) ± standard error. P values indicate the results of one-way ANOVA while asterisks indicate the results of LSD tests.
G. Atli, M. Canli / Environmental Toxicology and Pharmacology 25 (2008) 33–38
Fig. 2. Blood GSH level in O. niloticus exposed to metals for 14 days. See Fig. 1 for details.
Fig. 3. Muscle GSH level in O. niloticus exposed to metals for 14 days. See Fig. 1 for details.
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Fig. 4. Liver GSH level in O. niloticus exposed to metals for 14 days. See Fig. 1 for details.
Fig. 5. Liver MT level in O. niloticus exposed to metals for 14 days. See Fig. 1 for details.
Table 1 Total metal concentrations (mean ± standard deviation g/g dry weight; n = 8) in the tissues of O. niloticus (asterisks indicate significant (P < 0.05) differences resulted from the Duncan tests between control and individual treatments) Tissue
Concentration (M)
Metal Cu
Zn
Cd
Pb
Gill
0 5 10 20
3.9 13.9 25.6 30.4
± ± ± ±
1.5 10.4* 2.4* 6.2*
177.4 182.7 173.1 196.1
± ± ± ±
96.4 0.01 81.3 45.8
<0.001 10.8 ± 2.3* 11.5 ± 1.3* 16.2 ± 3.4*
<0.02 8.2 ± 8.2 45.9 ± 34.4* 57.4 ± 26.1*
Liver
0 5 10 20
169.2 224.2 233.6 295.3
± ± ± ±
35.6 70.5 55.8 81.2
138.8 204.5 170.5 253.8
± ± ± ±
55.9 0.01 80.5 82.3
<0.001 27.7 ± 10.9* 64.8 ± 15.5* 81.9 ± 21.9*
<0.02 <0.02 14.3 ± 5.3* 10.2 ± 2.0*
Muscle
0 5 10 20
0.88 1.09 0.15 0.77
± ± ± ±
0.58 1.09 0.14 0.77
120.9 162.6 291.5 641.8
± ± ± ±
81.5 0.01 173.6 2.3*
<0.001 <0.001 1.5 ± 0.8* 2.18 ± 0.41*
<0.02 <0.02 <0.02 <0.02
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G. Atli, M. Canli / Environmental Toxicology and Pharmacology 25 (2008) 33–38
Total metal levels in the tissues of fish were shown in Table 1. There was no significant Zn accumulation in the gill, while the levels of the other metals increased significantly in this tissue. In the liver, Cd and Pb accumulated significantly, while the levels of Cu and Zn did not change significantly. In the muscle, Zn and Cd levels increased significantly, whereas there was no copper accumulation. However, Pb levels in the muscle were below the detection limit for the instrument. 4. Discussion Metal accumulation in fish tissues occur in relation to environmental concentrations, exposure duration and other characteristics of water such as ions, pH, hardness and temperature (Heath, 1987). The present results showed that metal exposures generally increased the total tissue concentrations of metals, though the increases of essential metals were not significant in some tissues, indicating their regulation in those tissues. The regulation of essential metals by fish was also demonstrated in previous studies. However, regulation of cadmium and lead was not evident for fish (Heath, 1987), as occurred in the present study. One of our previous studies revealed the natural occurrence of Cu and Zn MTs but not Cd, Pb and Fe MTs in the liver of O. niloticus (Atli and Canli, 2003). Exposure of fish to these metals in the same concentrations showed that the levels of Cu and Zn in MT fraction did not increase, whereas Fe and Pb were not associated with any of the proteins in both controls and metal exposed groups. However, Cd in MT fraction of Cd-exposed fish increased many fold. Likewise, the present study also showed that only significant increase in MT level was observed in the liver of fish exposed to Cd, whereas the other metal exposures did not cause significant increases in MT levels in the liver. Although significant Pb accumulation occurred in the liver, this was not reflected in MT increase in this tissue. This data agree with other data from the literature indicating the absence of Pb binding MT-like proteins (Reichert et al., 1979; Roesijadi and Robinson, 1994; Atli and Canli, 2003). No significant changes in the MT level in the liver of fish exposed to Cu and Zn concentrations may be related to the high basal levels of these metals in the liver. Linde et al. (2005) showed that MT in hepatic cytosols from Mugil cephalus contained the most Cu, though MT was not the major hepatic Cu-binding protein when total cytosolic Cu increased in the mullet. The present results are in accord with our previous data in the induction of MT-like protein, indicating Cd is the main inducer of MTs (Atli and Canli, 2003; Eroglu et al., 2005; Baykan et al., 2007). Some other studies carried out with tilapia sp. revealed that Cd levels increases in MT fraction in the liver of Cd-exposed fish (Ueng et al., 1996; Wu et al., 2002). It seems that Zn and Cu MTs cannot increase unless basal levels of Cu and Zn are increased considerably. GSH is synthesized in the liver and released to the blood for transferring to the other organs such as the kidney and muscle (Pena et al., 2000). Because metal exposures did not alter the levels of GSH in the blood, muscle and gill, it suggests that metals taken up from the gill were immediately transferred (via the blood) to the liver for the usage in the metabolism or sequestered.
Thus, GSH levels in the blood may be a good indicator to understand the degree of metal exposure, suggesting the fact that metal exposures in this study were in the tolerable range for O. niloticus for the studied period. Nevertheless, the results showed considerable differences in GSH levels among tissues, metals and their exposure concentrations. Similarly, variations of GSH levels in different fish species were also observed in other studies, indicating the tissue and metal specific characteristics of GSH induction (Pena et al., 2000; Elia et al., 2003; Ali et al., 2004). The induction of GSH in the liver of fish exposed to Cd, Zn and Cu is probably due to primary defence system which GSH involved in to protect the fish from the oxidative stress. Lima et al. (2006) indicated that increased GSH levels in O. niloticus exposed to a contaminated effluent appear to be an antioxidant adaptation to chronic exposure. On the other hand, the decreased GSH levels in the gill and kidney of Anguilla anguilla exposed to 1.0 and 2.5 M Cu were related to the increased use of GSH to stabilize Cu in its oxidative stress for preventing the redox cycling and free radicals regeneration (Ahmad et al., 2005). Recently, Zirong and Shijun (2007) showed that cadmium (3 mg/L) exposure for up to 40 days depleted the GSH levels in the liver of O. niloticus though GSSG–GSH ratio increased. They concluded that GSH first made a rapid protection against oxidative stress and resulted in a sharp decrease in its contents. Like metals, pesticides can affect GSH levels in fish tissues as they can also be an oxidative stress inducer (Pena et al., 2000; Sayeed et al., 2003). The induction of MT-like proteins occurred in the liver hepatocyte carcinoma cells of Poeciliopsis treated with 10 M Cd or 10 M Zn. A significant increase in GSH levels was observed at 10 M Cd exposure whereas Zn failed to change GSH content (Schlenk and Rice, 1998). They concluded that metal induced GSH and MT may provide better protection against oxidative damage caused by metals. Atif et al. (2005) determined that GSH level did not increase in the liver of Channa punctata exposed to 0.2 mg Cd/kg intraperitoneally, though MT-like protein was detected in the gel electrophoresis. The authors pointed the increase in GSH and MT levels as important protective mechanisms that fish adopt in the initial phases of exposure to aquatic pollutants. In addition, Sanchez et al. (2005) recorded that the biochemical responses were more rapid than metal accumulation, suggesting the involvement of differential mechanisms in metal metabolism involves metal intake by GSH and formation of GSH–metal complex, from which the metal is further transferred to MT apoproteins where it is stored. In the present study, metal exposures caused significant increases in GSH and MT concentrations, particularly in the liver of O. niloticus, though metal accumulation and induction of these biomarkers were not well correlated. It is well known that these molecules are major sources of thiols for preventing the interactions of metals with main cellular structures as a first line defence. Enhanced GSH level, induction of MT and increased Cd accumulation in the liver of fish exposed to Cd may be due to the possible Cd toxicity in this tissue that known as the main storage organ for Cd and place where biochemical parameters such as GSH and MT involved in pivotal detoxification mechanisms.
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As a result, GSH and MT may be considered as sensitive biochemical indicators in ecotoxicology studies and response of these parameters could be useful indices of environmental quality. Nevertheless, the use of MTs or GSH as a sensitive biomarker for detection metal contamination may be limited when metal uptake rate cannot reach to a threshold level. This limit could be achieved much lower concentrations for non-essential metals (e.g. Cd), though the threshold levels for essential metals (e.g. Cu, Zn) are much higher. Therefore, it can be suggested that MTs or GSH could be a sensitive indicator of non-essential metals such as cadmium rather than essential metals (Lange et al., 2002; Atli and Canli, 2003; Lecoeur et al., 2004). Special attention should be given to the threshold levels of both essential and non-essential metal levels necessary to trigger MT and GSH induction in the aquatic environment. With this context, one can conclude that Cd MTs could be used as a sensitive tool to detect Cd contamination, though this may not be possible for Cu and Zn unless basal levels exceed. It seems that it is more complicated to use GSH as a sensitive biomarker for metal contamination in the aquatic environment because there is no considerable response to metal exposure in tissues of O niloticus, except in the liver. Additionally, the response of liver to metal exposures was not linear in relation to exposure concentrations, as there was no response at the highest metal exposures, except Cd exposure. Acknowledgement This study was supported by a grant FEF 2006 BAP23 from C ¸ ukurova University. References Ahmad, I., Oliveira, M., Pacheco, M., Santos, M.A., 2005. Anguilla anguilla L. oxidative stress biomarkers responses to copper exposure with or without -naphthoflavone pre-exposure. Chemosphere 61, 267–275. Ali, M., Parvez, S., Pandey, S., Atif, F., Kaur, M., Rehman, H., Raisuddin, S., 2004. Fly ash leachate induces oxidative stress in freshwater fish Channa punctata (Bloch). Environ. Int. 30, 933–938. Atif, F., Parvez, S., Pandey, S., Ali, M., Kaur, M., Rehman, H., Khan, H.A., Raisuddin, S., 2005. Modulatory effect of cadmium exposure on deltamethrin-induced oxidative stress in Channa punctata Bloch. Arch. Environ. Contam. Toxicol. 49, 371–377. Atli, G., Canli, M., 2003. Natural occurrence of metallothionein-like proteins in the liver of fish Oreochromis niloticus and effects of cadmium, lead, copper, zinc, and iron exposures on their profiles. Bull. Environ. Contam. Toxicol. 70, 619–627. Basha, P.S., Rani, A.U., 2003. Cadmium-induced antioxidant defense mechanism in freshwater teleost Oreochromis mossambicus (Tilapia). Ecotoxicol. Environ. Saf. 56, 218–221. Baykan, U., Atli, G., Canli, M., 2007. The effects of temperature and metal exposures on the profiles of metallothionein-like proteins in Oreochromis niloticus. Environ. Toxicol. Pharmacol. 23, 33–38. Beutler, E., Duron, O., Kelly, B.M., 1963. Improved method for the determination of blood glutathione. J. Lab. Clinic. Med. 61, 882–890. Canli, M., 1995. Natural occurrence of metallothionein-like proteins in the hepatopancreas of Norway lobster (Nephrops norvegicus L.) and effects of cadmium, copper and zinc exposures on levels of the metals bound on metallothioneins. Tr. J. Zool. 19, 313–321. Canli, M., Stagg, R.M., Rodger, G., 1997. The induction of metallothionein in tissues of the Norway lobster Nephrops norvegicus following exposure to cadmium, copper and zinc: the relationships between metallothionein and the metals. Environ. Pollut. 96, 343–350.
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