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Effects of Waterborne Lead Exposure in Mozambique Tilapia: Oxidative Stress, Osmoregulatory Responses, and Tissue Accumulation a

a

Hasan Kaya & Mehmet Akbulut a

Marine Science and Technology Faculty, Department of Basic Sciences, Çanakkale Onsekiz Mart University, 17100 Çanakkale, Turkey Published online: 07 May 2015.

Click for updates To cite this article: Hasan Kaya & Mehmet Akbulut (2015) Effects of Waterborne Lead Exposure in Mozambique Tilapia: Oxidative Stress, Osmoregulatory Responses, and Tissue Accumulation, Journal of Aquatic Animal Health, 27:2, 77-87, DOI: 10.1080/08997659.2014.1001533 To link to this article: http://dx.doi.org/10.1080/08997659.2014.1001533

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Journal of Aquatic Animal Health 27:77–87, 2015  C American Fisheries Society 2015 ISSN: 0899-7659 print / 1548-8667 online DOI: 10.1080/08997659.2014.1001533

ARTICLE

Effects of Waterborne Lead Exposure in Mozambique Tilapia: Oxidative Stress, Osmoregulatory Responses, and Tissue Accumulation Hasan Kaya* and Mehmet Akbulut

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Marine Science and Technology Faculty, Department of Basic Sciences, C¸anakkale Onsekiz Mart University, 17100 C ¸ anakkale, Turkey

Abstract We studied the oxidative stress and osmoregulatory damage as well as the accumulation of lead in Mozambique Tilapia Oreochromis mossambicus exposed to different sublethal concentrations—low, medium, and high (0.5, 2.5, and 5.0 mg/L)—of waterborne lead for 14 d in a semistatic condition. The accumulated levels of Na + ,K + -ATPase, glutathione (GSH), and thiobarbituric acid reactive substances (TBARS) were determined from samples of gill, liver, intestine, brain, kidney, and muscle tissues. At the end of the experiment, the GSH levels of most tissues were higher in the treated group than in the control group (especially in the liver and kidney) but lower in the intestine. The levels of TBARS in the gill and brain tissues of the fish exposed to high lead doses were significantly higher than those of fish in the control group. Na + ,K + -ATPase activity seemed to be significantly inhibited in the gill, intestine, and brain tissues across all treatment groups. At the end of the study, the total amount of lead that had accumulated within the various tissues ranked as follows: intestines > kidney > brain > gill > liver > muscle. Our findings suggest that sublethal concentrations of lead can disrupt the health of Mozambique Tilapia and cause oxidative stress and osmoregulatory damage.

Environmental problems are one of the leading threats to ecological balance as well as to human and animal health. Rapid and increasing population growth, industrialization, and the tendency to ignore environmental concerns are the primary reasons for the disruption of the balance between maintaining a stable ecosystem and meeting human needs. Toxic substances largely consist of metals and pesticides; they have different levels of importance depending on their ability to be transferred to aquatic ecosystems by natural or anthropogenic means, their resilience to environmental conditions, and their capacity for biomagnification through the food chain (Klassen et al. 1986). Lead is a heavy metal that is not required by living organisms but one that has been used throughout human history because of its unique physical and chemical properties (Eisler 1988). Lead is introduced into aquatic ecosystems as the result of ore processing activities and its use in battery and battery pack manufacturing as well as by some natural means, such as ero-

sion and atmospheric fallout caused by volcanoes (Nriagu and Pacyna 1988). Although the use of lead in paint and gasoline has now been restricted or prohibited, lead continues to infiltrate aquatic ecosystems via anthropogenic and natural pathways and is thus still a primary polluter (USEPA 2006). It is widely known that metal ions are good oxidative stress inductors (Lushchak 2011) and act to increase the number of free oxygen radicals (i.e., reactive oxygen species [ROS]), which induces oxidative stress. The increasing level of ROS, along with the inhibition of Na + ,K + -ATPase activity, causes functional disorders in osmoregulation as well as tissue damage (Atlı and Canlı 2007). This suggests that ROS, formed as the result of lead toxicity, inflict DNA damage and lead to the depletion of the antioxidant defense mechanisms (El-Ashmawy et al. 2006; Monteiro et al. 2011). Enzymatic and nonenzymatic antioxidants play a crucial role in the biological defense against oxidative stress caused by metals. Oxidative stress biomarkers are among

*Corresponding author: [email protected] Received March 30, 2014; accepted November 28, 2014

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the most important bioindicators used to reveal the effects of heavy metals on fish (Fırat 2007). Studies of the effects of lead on fish have so far determined that under high-toxicity conditions this metal can cause drowning due to respiratory failure resulting from excessive mucus production (Carpenter 1927; Westfall 1945); disrupt the Ca2 + and Na + homeostasis in concentrations often present in aquatic ecosystems (Rogers et al. 2003; Patel et al. 2006); and (under chronic conditions) lead to hematological (Hodson et al. 1978) and neurological (Davies et al. 1976) damage. On the other hand, no comprehensive study has examined the effect that lead may have on the oxidative stress mechanism in fish. In this study, we investigated the accumulation of sublethal lead concentrations within certain tissues (gill, liver, intestine, muscle, brain, and kidney) of a model fish species, Mozambique Tilapia Oreochromis mossambicus, as well as its impact on glutathione (GSH), thiobarbituric acid reactive substances (TBARS), and Na + ,K + -ATPase activity.

METHODS Experimental design.— The Mozambique Tilapia used in this study (n = 144) were obtained from C¸anakkale Onsekiz Mart University (Marine Sciences and Technology Faculty, Aquaculture Department), C¸anakkale, Turkey, and were adapted to ambient conditions in 12 stock aquariums, each with dimensions of 45 × 28 × 80 cm and containing 80 L of rested C¸anakkale city tap water, for 4 weeks. Fish weighing 45.2 ± 5 g (mean ± SE) were divided into 12 experimental aquariums, each containing 12 fish, and an experimental design with three replicates was established. Feeding was interrupted 24 h before the start of the experiments to help maintain water quality. During the experiment, the fish were fed twice a day with feed at about 1–2% of their body weight and their behavior was observed during each feeding. Care was also taken to ensure that all of the feed added to the tanks was eaten and that fecal waste was quickly removed from the tanks at every water change. In the experiment, fish were exposed to the following sublethal concentrations of lead: low, 0.5 mg/L; medium, 2.5 mg/L; and high, 5 mg/L. The control group was kept in freshwater only. Concentrations were determined as in Ay et al. (1999). The experiment had a semistatic design, and water was changed every day: a 75% change in the morning and a 25% change in the evening (modified from Smith et al. 2007). After each water change, lead nitrate solution was added to the aquarium water to maintain a constant lead concentration. The temperature and dissolved oxygen of the tap water were measured with a YSI MPS 556 probe, and pH was measured with a HANNA C 200 (Model HI 83200; Hanna Instruments) photometer every day. Total ammonia was determined by sampling water from the aquariums every 2 d and analysing it with a Thermo Scientific Aquamate VIS spectrophotometer; electrolyte analysis of the water was performed via Varian Liberty Sequential inductively coupled plasma optical spectrometry (ICP-OES). The measured

values were as follows: temperature, 25.4 ± 0.3◦ C (mean ± SE); dissolved oxygen, 6.31 ± 0.11 mg/L; pH, 7.15 ± 0.04; hardness, 125.0 ± 6.2 mg/L CaCO3 ; total ammonia, 0.151 ± 0.02 mg/L. The electrolyte composition of the dechlorinated C¸anakkale tap water was measured as 0.310 ± 0.005, 0.049 ± 0.001, 0.534 ± 0.001, and 0.828 ± 0.006 mmol/L for Na + , K + , Mg + , and Ca2 + , respectively. Fish were randomly sampled on days 0, 7, and 14 for biochemistry analysis and the determination of lead accumulation in tissues (see below). The experiments were performed in accordance with the guidelines for fish research established by the Animal Ethics Committee at C¸anakkale Onsekiz Mart University. Preparation of the Pb(NO3 )2 solution and application.— The heavy metal salt, Pb(NO3 )2 (99.5% purity; Sigma-Aldrich, Steinheim, Germany), was used in the experiment. To obtain the needed concentrations, the main stock solution was prepared in ultradistilled water and appropriate dilutions made from it. Trisodium citrate dihydrate (C6 H5 Na3 O7 ·2H2 O) solution was used in the experiment to enable the Pb(NO3 )2 solution to be dispersed into the aquariums homogenously and to avoid its being precipitated to the floor of the aquarium. Biomarker analysis.—In the experiment, six fish on the first day (fish from the initial stock) and three fish from each aquarium on the 7th and 14th days were used for biomarker analysis. Fish were terminally anesthetized with ethyl 3-aminobenzoate methanesulfonate (MS-222) and dissected for tissue biomarker analysis (Smith et al. 2007). Gill, liver, intestine, brain, muscle, and kidney tissues were removed, immediately frozen in liquid nitrogen, and stored at −80◦ C until required. Tissues were homogenized (Stuart SHM1 Homogenisator) in five volumes (0.5 g of tissue in 2.5 mL) of ice-cold isotonic buffer (300 mmol/L sucrose, 0.1 mmol/L EDTA, and 20 mmol/L 4(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; adjusted to pH 7.8 with a few drops of Trizma base). Crude homogenates were stored in 0.5-mL aliquots at −80◦ C until required. Tissues were analyzed at the end of the experiment (day 14) for Na + ,K + -ATPase activity to determine the possible effects on osmoregulation and the presence of TBARS and GSH as indicators of oxidative stress. In the raw tissue homogenates, Na + ,K + -ATPase activity was determined according to Silva et al. (1977). The method is as follows: each sample (15 µL, in triplicate) was put into 400 mL of both K + (containing 10 mmol/L KCl in the buffer) and K− (free 10 mmol/L KCl in the buffer, plus 1.0 mmol/L ouabain) and then incubated at 37◦ C for 10 min. The reaction was stopped by adding 1 mL of ice-cold trichloroacetic acid (8.6%; weight : volume [w/v]), and 1 mL of a color reagent was added to each tube (9.6% [w/v] FeSO4 ·6H2 O and 1.15% [w/v] ammonium heptamolybdate dissolved in 0.66-M H2 SO4 ), and the color was allowed to develop for 15 min at room temperature. Absorbance was measured at 660 nm (Optizen POP UV/VIS spectrophotometer; Seoul, Korea) against 0–0.5 mmol/L phosphate standards (Bouskill et al. 2006).

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The TBARS assay was performed according to Camejo et al. (1998). Briefly, 40 µL of homogenate was added to one well of a 96-well microtiter plate (in triplicate), which contained 1 mol/L butylated hydroxytoluene (2,6-di-O-tert-butyl-4methylphenol), and the final volume was made up to 200 µL with 1 mmol/L phosphate-buffered saline (pH 7.4). Following this, 50% (w/v) trichloroacetic acid and 1.3% (w/v) thiobarbituric acid (dissolved in 0.3% [w/v] NaOH) were added, and the plate was incubated at 60◦ C for 60 min and then cooled on ice. Absorbances were read at 530 and 630 nm (Thermo Multiscan Go microplate reader), corrected for turbidity, and read against standards (0.5–25 nmol/mL 1,1,3,3-tetraethoxypropane) (Bouskill et al. 2006). Total GSH was determined according to Owens and Belcher (1965). To summarize, 40 µL of homogenate, blank, or standard were added in triplicate to a microplate well containing 20 µL of deoxynucleotide triphosphate, 260 µL of assay buffer (K2 HPO4 and EDTA; pH 7.5), and 20 µL of glutathione reductase. The reaction was started by the addition of 20 µL of NADPH, with changes in absorbance at 412 nm recorded over 10 min and total GSH (µmol/g wet weight of tissue) determined using the standard calibration curve. Tissue lead analysis.— At days 0, 7, and 14, fish (six fish at day 0 and three fish per treatment at days 7 and 14) were lethally anesthetized with MS-222 and dissected for lead ion analysis of the tissues. The tissues (gill, liver, intestine, muscle, brain, and kidney) were first rinsed and oven-dried to a constant weight, then digested in 5 mL of concentrated nitric acid, and finally diluted to 20 mL with deionized water for lead analysis. A blank digest was also carried out in the same way. The analysis was performed by ICP-OES (Jari´c et al. 2011). The wavelength of the ICP-OES analysis used for lead was 220.353. Spiny Dogfish Squalus acanthias muscle, a certified reference material for metal analysis (DORM-2), was used to calibrate the ICP-OES prior to analysis of fish tissue samples. The DORM-2 and American lobster Humarus americanus hepatopancreas reference material for metals (TORT-2) were purchased from the National Research Council of Canada. The concentrations found were within 90–115% of the certified values for all measured elements. Percentage tissue moisture content was calculated from wet and dry tissue weights. All heavy metal concentrations were expressed as micrograms per gram (µg/g) of dry weight. Statistical analysis.—Data sets were analyzed using Tukey’s post hoc test and Kruskal–Wallis one-way ANOVA by ranks followed by Dunn’s post hoc test (Logan 2010). For statistical analysis, SPSS version 17 (SPSS, Chicago, Illinois) was used, with a significance level of 0.05. RESULTS No deaths were observed in the control and medium-dose groups during the experiment; however, there were two fish mortalities in the group exposed to the high dose of lead. Feed intake started to decline from day 4 in the high-dose group and from day 9 in the medium-dose group. Escape and hiding movements, lethargy, sidestroke swimming, and mouths staying

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open longer were observed in all of the groups exposed to lead, especially those exposed to the high dose. Oxidative Stress The GSH levels in the tilapia tissues are given in Figure 1. While the GSH level of the gill tissues was higher than that of the control group on day 7 for all treated groups, on day 14 only that of the low-dose (L) group was higher; those of the medium- (M) and high-dose (H) groups were significantly lower. Increases in the GSH level of the liver tissues were observed on day 14 in all treated groups. While the GSH level of the liver tissues was higher in all treated groups on day 14, that of the intestine tissues showed a significant decrease. While the GSH level of the muscle tissues in the L and M groups was slightly higher on day 7, it was slightly lower in the L group and much higher in the H group on day 14. On day 7, the GSH level in brain tissues was higher in all of the treated groups. By day 14, however, it was lower in both the L and M groups and only moderately higher in the H group. While the GSH level in kidney tissues was lower in the H group on day 7, on day 14 it was higher in all treated groups. The TBARS levels found in the tilapia tissues are given in Figure 2. While the level in gill tissues was significantly higher in the H group on days 7 and 14, there were no differences in the other groups. And although there were significant increases in the liver and intestine tissues of the H group on day 7, there were none for the other groups and by day 14 those for the H group had disappeared. The TBARS levels in the muscle tissues were statistically similar across all groups for the entire experiment. While there was an increase in the TBARS level in the brain for the H group on day 14, there were no significant differences between the L and M groups and the control. The TBARS level in the kidney tissues was lower in the L group on day 7, but all groups had comparable levels on day 14. Osmoregulatory Responses The Na + ,K + -ATPase activity in tilapia tissues is given in Figure 3. Na + ,K + -ATPase activity was significantly lower in the gill and brain tissues of all treated groups on both days 7 and 14 and, with the exception of the L group on day 7, in the intestines of all treated groups as well. There were no significant differences in Na + ,K + -ATPase activity in liver tissues at any time. While the Na + ,K + -ATPase activity in muscle tissues was higher in the H group on day 7, by day 14 it was higher in the M group but much lower in the H group. And while the Na + ,K + ATPase activity in kidney tissues was lower for the L and M groups on day 7, it was higher for the L and H groups on day 14 and equivalent for the M group. Lead Accumulation in Tissues The accumulation of lead in tilapia tissues is given in Figure 4. The accumulation in gill, liver, intestine, muscle, brain, and kidney tissues increased with respect to both dosage and time. At the end of the study, the rankings of lead accumulation

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FIGURE 1. Effect of sublethal exposure to lead on the level of GSH in Mozambique Tilapia tissues at 0, 7, and 14 d postexposure. Exposure groups are as follows: control (C; 0 mg/L), low (L; 0.5 mg/L), medium (M; 2.5 mg/L), and high (H; 5 mg/L). For given tissues and times, concentrations with different lowercase letters are significantly different (P < 0.05; one-way ANOVA followed by Dunn’s post hoc test).

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FIGURE 2. details.

Effect of sublethal exposure to lead on levels of TBARS in Mozambique Tilapia tissues at 0, 7, and 14 d postexposure. See Figure 1 for additional

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FIGURE 3. Effect of sublethal exposure to lead on levels of Na + ,K + -ATPase activity in Mozambique Tilapia tissues at 0, 7, and 14 d postexposure. See Figure 1 for additional details.

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FIGURE 4.

Lead accumulation (dry weight) in Mozambique Tilapia tissues at 0, 7, and 14 d postexposure. See Figure 1 for additional details.

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in the various tissues in group L were as follows: kidney > gill > intestine > brain > liver > muscle; those for the M and H groups were intestine > kidney > brain > gill > liver > muscle.

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DISCUSSION Oxidative Stress Glutathione, which is an important antioxidant in tilapia, has a low molecular weight and a tripeptide form that contains thiol groups. By entering into a reaction with the ROS and peroxides, it protects cells against oxidative damage. The concentration of glutathione and its cellular oscillation are indicators of the oxidative stress that is caused by free-oxygen radicals (Halliwell 1999). The real mechanism behind the oxidative stress that results from lead toxicity is not yet fully known. One of the mechanisms that lead toxicity affects is GSH. By binding onto the sulfhydryl groups such as GSH, lead changes the oxidation–reduction reactions of the thiol groups, and as the result of the loss of thiols it disrupts cellular functions (G¨urer and Ercal 2000). The increases in the GSH levels observed in this study may be interpreted as an adaptive mechanism under moderate oxidative stress conditions. However, under high oxidative stress GSH levels may fall and the adaptive protection mechanism thus be lost (Zhang et al. 2004). Glutathione is the most important antioxidant tasked with scavenging the intracellular ROS. In the case of oxidative stress, increases in GSH synthesis are observed as increases in intracellular GSH levels. However, by disrupting the balance of oxidants and antioxidants in cells, lead may inflict DNA damage via lipid peroxidation and thus impair one of their antioxidant defense systems (Monteiro et al. 2011). Overrunning cellular defenses may lead to lipid peroxidation by oxidizing the membrane fatty acids, causing the proteins to change and leading to the nonsulfhydryl groups being oxidized. The unsaturated phospholipids and cholesterol that are found within the structure of the membranes easily enter into reactions with ROS and undergo lipid peroxidation (Porter 1984; Ahamed and Siddiqui 2007). Lipid membrane peroxidation causes the cellular functions to be altered by decreasing the cells’ fluidity and increasing their permeability (Hirosumi et al. 1988). In this study, the increasing TBARS levels in the brain, liver, kidney, intestine, and gill tissues of Mozambique Tilapia subjected to the 5-mg/L lead concentration indicate that ROS levels were raised. This resulted in oxidative stress that lowered the level of antioxidant enzymes and thus led to an increase in lipid peroxidation that damaged the structure of the membrane. Previous studies conducted on animals and humans also indicate that lead toxicity causes changes in lipid peroxidation and antioxidant defense mechanisms (Ercal et al. 1996; Pillai and Gupta 2005; Laxmiprya and Gupta 2006; Oruc¸ and Usta 2006 Oropesa et al. 2008). Laxmiprya and Gupta (2006) subjected Charles Foster rats to lead, cadmium, and a lead–cadmium combination for 15 d, both in vivo and in vitro, and found a decrease in GSH levels as well as an increase in lipid peroxidation

levels. Similarly, Gerber et al. (1978) and Shafiq-ur-Rehman (1995) determined that lead toxicity caused lipid peroxidation in the brains of rats. In addition, Shafiq-ur-Rehman et al. (1995) indicated that the lipid peroxidation occurring in different regions of the brain is consistent with the accumulation of the lead that he observed. Ahmad et al. (2005), on the other hand, found that the most susceptible organ in European Eels Anguilla anguilla that were subjected to wastewater pollution was the gill. In that study, Ahmad et al. (2005) also detected increased levels of TBARS as well as inhibition of gill-based antioxidant enzyme activities. Ercal et al. (1996) observed a decrease in the GSH activities of the liver tissues of Chinese hamsters Cricetulus griseus subjected to lead toxicity and an increase in lipid peroxidation. It is known that the liver is the pivotal organ for detoxification activities (Glusczak et al. 2007), and antioxidants decrease the sensitivity of the kidneys in the oxidative battle (Seth et al. 2001). In our study, we found that lead changed GSH levels in the liver, kidney, and intestines and led to an increase in TBARS levels. Given that metals are metabolized in the liver and secreted via the intestines and kidneys, it may be that lead has the potential to cause oxidative stress and inflict damage to the liver, intestine, and kidney tissues of Mozambique Tilapia.

Osmoregulatory Responses The enzyme Na + ,K + -ATPase is important (especially to the osmoregulatory organs such as the gills, kidneys, and intestines) in both saltwater and freshwater bony fishes and is basically tasked with the intracellular protection of homeostasis (Atlı 2010). Na + ,K + -ATPase is found in all animal tissues and undertakes the transfer of Na + and K + across the cell membrane. Many studies have been conducted regarding the impact of heavy metals on Na + ,K + -ATPase (Canlı and Stagg 1996; Ay et al. 1999; De La Torre et al. 2005; Atlı and Canlı 2011). In acute and chronic applications, the reaction of Na + ,K + -ATPase to heavy metals varies, depending on the duration of exposure, the concentration, and the type of the metal and tissue (Atlı and Canlı 2011). In our study, although significant inhibition of Na + ,K + -ATPase activity was observed in the gill, intestine, and brain tissues of treated Mozambique Tilapia, increases in such activity were found in the muscle and kidney tissues of treated fish, the degree of these increases depending on exposure time and concentration. In addition, no changes in Na + ,K + -ATPase activity were observed in liver tissues across all concentrations and durations. In accordance with these results, the inhibition of Na + ,K + ATPase activity observed (especially in the gill, intestine, and brain) is thought to have occurred because the enzyme molecule is very susceptible to the metals from sulfhydryl groups. Lead degrades phosphorylation activity by bonding with the carboxyl group in the active region of the enzyme as a result of the damage to the membrane or irregularities occurring in ion hemostasis (Atlı and Canlı 2011). Other studies also suggest that other

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heavy metals also inhibit Na + ,K + -ATPase activity (Ay et al. 1999; Handy et al. 2002; Hoyle et al. 2007; Atlı and Canlı 2011). Ay et al. (1999) exposed Redbelly Tilapia Tilapia zilli to copper and lead concentrations of 0.5, 1.2, and 4.0 mg/L for 14 d and reported that the gill Na + ,K + -ATPase activity was significantly inhibited. Similarly, Rogers et al. (2003) detected a 40% reduction in gill Na + ,K + -ATPase activity in Rainbow Trout Oncorhynchus mykiss exposed to a 1-mg/L lead concentration after 2 d. In our study, increases were observed in Na + ,K + -ATPase activity in kidney and muscle tissues of Mozambique Tilapia. Such increases may be attempts to reduce the loss of homeostatic organization and to return the inhibited enzymes to normal levels. Such recovery mechanisms are probably initiated by increasing the enzyme count in some tissues or by engaging the antioxidant mechanisms (Ay et al. 1999). Stagg and Shuttleworth (1982) examined the effects of 200 µg/L copper in European Flounder Platichthys flesus in vivo and found inhibition in Na + ,K + -ATPase activity in the gill. However, these authors also pointed out that recovery mechanisms were initiated for the enzyme to return to its control levels. Lauren and McDonald (1987) subjected Rainbow Trout to a copper concentration of 55 µg/L for 28 d, and after 24 h they detected an inhibition rate of 33% in Na + ,K + -ATPase activity in the gill. However, they also reported that this inhibition had ended by day 14. Tissue Accumulation Fish have the ability to obtain metals from water by both active and passive processes and to accumulate them in their bodies (Heath 1987; Langston 1990). Metal accumulation in the tissues of organisms that live in aquatic ecosystems generally varies with the duration of exposure to the metal, the type of metal, the metabolic activity in the tissues, and the physical and chemical properties of the water (such as the pH, temperature, salinity, and hardness). Similarly, metal accumulation in fish tissues also depends on the uptake of the metal, its distribution and deposition in the tissues, and excretion (Heath 1987; Langston 1990). In our study, the levels of lead that accumulated in the tissues of Mozambique Tilapia varied depending on the time of exposure, concentration, and tissue type. At the end of the study, the various tissues ranked as follows with respect to lead accumulation (from highest to lowest): intestine, kidney, brain, gill, liver, and muscle. However, other studies have indicated that the liver accumulates metals to a greater extent than do other organs (Tulasi et al. 1992; Allen 1994; Roesijadi and Robinson 1994). The liver plays vital roles in chemical alteration, excretion, and the detoxification of metals (C¸o˘gun 2008). Heavy metals taken up from the aquatic environment are excreted by being bound to metal-binding proteins such as metallothionein (MT), which is subsequently excreted (Heath 1987; Canlı et al. 1997; Ay et al. 1999). The principal places for the production of the metalbinding proteins like MT are the intestines, kidneys, and liver. Therefore, heavy metals are found at high levels in such tissues (Thomas et al. 1983; Allen 1994; Ay et al. 1999).

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In our study, however, the liver was found to accumulate lead the least, surpassing only muscle tissue. The reason may be that metal-binding proteins such as MT do not actually bind lead, and there is no real evidence that they do (Reichert et al. 1979; Roesijadi and Robinson 1994). Linde et al. (1999) studied metal accumulation and the synthesis of MT in Brown Trout Salmo trutta and European Eels and found that copper binds to MT but lead does not cause the synthesis of such proteins. The findings from our study show similarities with the data from previous studies (Ay et al. 1999; Linde et al. 1999; Atlı and Canlı 2007; C¸o˘gun 2008), which determined that kidney tissues accumulated the most lead, followed by gill and liver tissues (Ay et al. 1999; Linde et al. 1999; Atlı and Canlı 2007; C¸o˘gun 2008). We also studied the accumulation of lead in intestine and brain tissues of Mozambique Tilapia. While intestine tissue had the highest level of lead accumulation, the brain was among the organs showing the greatest lead accumulation after the kidneys. The intestines in fish are tasked with digestion, the absorption of nutrients, excretion of digested nutrients, and the osmoregulatory process. The kidneys are important organs, as they take part in the excretion of the heavy metals. The accumulation of heavy metals in those organs in high concentrations is thought to be a result of the functions of those organs (Cicik 2003). In fish, muscle tissue does not play a highly active role in the accumulation of metals. In our study, the lowest rate of lead accumulation was in muscle tissue, which is the edible part of the fish. The same result has been found in other studies (Ay et al. 1999; Atlı and Canlı 2007; C¸o˘gun 2008). Conclusions Our findings suggest that sublethal concentrations of lead result in oxidative stress and osmoregulatory damage in Mozambique Tilapia, as well as in the accumulation of lead in some of their tissues. Given that aquatic ecosystems are under the constant threat of heavy metal pollution, determining the changes that heavy metals induce in fish is of the utmost importance to the sustainability of those ecosystems. However, there is a deficiency of studies of lead toxicity in marine fish. It is possible that the mechanisms of such toxicity are different in marine fishes than in freshwater fishes, and new studies should be conducted in this regard. In addition, because the solubility and toxicity of heavy metals such as lead may increase in salt water as a result of global warming, conducting studies on this issue is crucial. ACKNOWLEDGMENTS This study was supported by the C¸anakkale Onsekiz Mart University Scientific Foundation (project 2010/26). REFERENCES Ahamed, M., and M. K. J. Siddiqui. 2007. Low-level lead exposure and oxidative stress: current opinions. Clinica Chimica Acta 383:57–64. Ahmad, I., M. Oliveira, M. Pacheco, and M. A. Santos. 2005. Anguilla anguilla L. oxidative stress biomarker responses to copper exposure with or without naphthoflavone pre-exposure. Chemosphere 61:267–275.

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