Cytotoxicity of chromium ions may be connected with induction of oxidative stress

Chemosphere 80 (2010) 1044–1049

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Cytotoxicity of chromium ions may be connected with induction of oxidative stress Olena Yu. Vasylkiv a, Olha I. Kubrak a, Kenneth B. Storey b, Volodymyr I. Lushchak a,* a b

Department of Biochemistry, Precarpathian National University named after Vassyl Stefanyk, 57 Shevchenko Str., Ivano-Frankivsk, 76025, Ukraine Institute of Biochemistry, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario K1S 5B6, Canada

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Article history: Received 19 March 2010 Received in revised form 11 May 2010 Accepted 19 May 2010 Available online 14 June 2010 Keywords: Lactate dehydrogenase Catalase Carassius auratus Erythrocytes Plasma Hemoglobin

a b s t r a c t Chromium ions are frequently found in aquatic ecosystems and are known to be inducers of oxidative stress in fish solid tissues. The present study was designed to determine whether fish blood samples can be used to allow nonlethal diagnostic testing for chromium intoxication. First, we confirmed that 96 h exposures to water containing 10.0 mg L 1 chromium ions, either Cr3+ or Cr6+, induced oxidative stress in brain of goldfish (Carassius auratus). Multiple blood parameters were then evaluated. Cr6+ exposure triggered a 579% increase in the number of erythrocytes containing micronuclei, a frequently used marker of cellular toxicity. Leucocyte numbers were also perturbed by exposure to either Cr3+ or Cr6+ indicating that chromium ions could impair the immune system as well. The content of protein carbonyl groups, a marker of oxidative damage to proteins, was enhanced in fish plasma by exposure to either chromium ion and activities of catalase and lactate dehydrogenase also were affected. The data demonstrate that chromium ions induced oxidative stress in goldfish blood and were cytotoxic for erythrocytes. This indicates that analysis of plasma can be used as a good early nonlethal diagnostic marker of fish intoxication by transition metal ions. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Chromium ions are widely distributed in both terrestrial and aquatic environments, occurring predominantly in two valences: Cr6+ and Cr3+. Chromium chloride, niacin-bound chromium or chromium polynicotinate, and chromium picolynate are used as micronutrients and dietary supplements, whereas hexavalent chromium compounds are extensively used in diverse industries (Bagchi et al., 2002). As with other metals, chromium does not biodegrade, but remains in ecosystems. Hexavalent chromium may cause a number of pathologies, being toxic and carcinogenic to animals and humans. Chromium ions are rather toxic for all organisms, particularly for 1 fish. For example, for goldfish LD50 of Cr6+ (Riva 96 was 110 mg L et al., 1981). Both acute and chronic exposure to chromium substantially affects the histology of tissues and these effects also depend upon type of ion used. Although Cr6+ has received a lot of attention, some data on Cr3+ toxicity are also available. In Chinook salmon, Oncorhynchus tshawytscha, kidney was a target organ for Cr6+ exposure with both gross and microscopic lesions (e.g. necrosis of cells lining kidney tubules) and elevated lipid peroxidation seen (Farag et al., 2006). Pathological changes were also found in spleen and blood. In the teleost fish, Channa punctata, Cr6+ affected gills, kidney and liver (Mishra and Mohanty, 2009a). The gill lamellae became lifted, fused and showed oedema; trunk kidney demonstrated hypertrophy of the epithelial cells of renal tubules and reduction of * Corresponding author. Tel./fax: +380 342 714683. E-mail address: [email protected] (V.I. Lushchak). 0045-6535/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2010.05.023

tubular lumens; vacuolization and shrinkage of hepatocytes were found, and nuclear pyknosis and an increase of sinusoidal spaces were observed. In C. punctata exposure to hexavalent chromium ions also affected the pituitary-interrenal axis, kidney interrenal tissue and increased serum cortisol level (Mishra and Mohanty, 2009b). Over the last decade many studies have appeared and demonstrated that the reduction of Cr6+ in vivo increases the generation of free radicals (Pourahmad and O’Brien, 2001; Liu and Shi, 2001). It was also shown that fish mucus could reduce Cr6+ to Cr3+ which was seen as the mechanism for fish protection from exposure to Cr6+ (Arillo and Melodia, 1990). Detailed analyses of the biological effects of chromium ions in fish as they are related to free radical processes have been given in our previous studies (Lushchak et al., 2008, 2009a,b) and also summarized in a recent review article (Lushchak, 2008). In the present study we address the questions of whether Cr3+ and Cr6+ (the most broadly distributed in chromium ions in the environment) are cytotoxic, affect the immune system, or induce oxidative stress in goldfish blood, and therefore, whether fish blood can be used to allow nonlethal diagnostic testing for chromium intoxication.

2. Materials and methods 2.1. Reagents Phenylmethylsulfonyl fluoride (PMSF), 2,4-dinitrophenylhydrazine (DNPH), potassium phosphate monobasic, pyruvate and

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ethylenediamine-tetraacetic acid (EDTA) were purchased from Sigma–Aldrich Corporation (USA). NADH was obtained from Reanal (Hungary). All other reagents were of analytical grade from local industries (Ukraine, Russia). 2.2. Animals and chromium exposure Goldfish (Carassius auratus L.) weighing 50–100 g were obtained commercially in June 2009 and held in a 1000 L open tank (no more than 5 kg total mass of fish) for 7 weeks before experimentation. Holding conditions included natural photoperiod, constant air bubbling, and dechlorinated tap water at 27.0–27.5 °C, pH 7.00–7.50 and oxygen concentration of 7.5–8.5 mg L 1. About 50% of the tank water volume was replaced each third day in order to prevent its contamination. Fish were fed with commercial food during acclimation to laboratory conditions, but were not fed during experimentation. For experiments, groups of six fish were transferred into 120 L glass aquaria (containing 100 L of water), in a static mode, under the same environmental conditions. The most critical parameters of water condition were monitored every 24 h during fish treatment. On the basis of these measurements, we can state that fish in the three investigated groups (control, Cr3+ and Cr6+-treated) were maintained in the same environmental conditions during 96 h exposure: water temperature 27.0–27.5 °C, pH in the range 7.00–7.65 and oxygen concentration of 7.5–8.5 mg L 1. Fish were exposed to nominal Cr3+ or Cr6+ concentrations of 10.0 mg L 1 (calculated for chromium ion) added as CrCl3
6H2O or K2Cr2O7, respectively, for 96 h. Salts were dissolved in the experimental tanks 24 h before fish were transferred into the aquaria. Fish in control groups were treated in the same manner, but chromium salts were omitted. Water was not changed over the course of the experiment in order to avoid stressing the animals. After exposure, blood was quickly sampled from caudal vessels using 50 mM EDTA as an anticoagulant. Fish were then sacrificed by transspinal transsection and brain was dissected out, rinsed in ice-cold 0.9% w:v NaCl solution and placed in pre-chilled homogenization buffer.

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2.4. Preparation of smears for microscopic examination Drops of whole blood from fish caudal vessels were directly smeared on slides (n = 4 per fish) and air-dried. Smears were fixed for 40 min with eosin–methylene alcohol solution followed by asure-eosine water solution staining for 30 min (Ivanova, 1983). Cytological analysis was conducted by scoring at a 1000 magnification using an Olympus CX-300 microscope. Different types of leucocytes were identified according to the Fish Blood Cell Atlas (Ivanova, 1983). A total of 200 leucocyte cells were counted per smear, assigned to different leucocyte categories and then the percentage in each category was calculated. Microphotos were taken using an integrated photonozzle Olympus SP-500 UZ and Quick PHOTO MICRO 2.3 soft for Windows. The frequency of micronuclei was evaluated per 1000 cells. For the scoring of micronuclei, the following criteria were adopted from Fenech et al. (2003): (a) diameters of micronuclei are less than one-third of the main nucleus, (b) micronuclei are separated from or marginally overlap with the main nucleus but show clear identification of the nuclear boundary, and (c) micronuclei show similar staining as the main nucleus. 2.5. Assay of enzyme activities Brain samples were homogenized (1:10 w:v) using a Potter– Elvejhem glass homogenizer in pre-chilled 50 mM potassium phosphate (KPi) buffer, pH 7.0, containing 0.5 mM EDTA; a few crystals of phenylmethylsulfonyl fluoride (PMSF) were added prior to homogenization. Homogenates were centrifuged (15000g, 4 °C, 15 min) in an Eppendorf 5415 R centrifuge (Germany). Supernatants were removed and used for enzyme activity assays. The activities of catalase in brain supernatants, plasma and blood cell hemolysates were measured as described previously (Lushchak et al., 2005). The activity of lactate dehydrogenase (LDH) was assayed specrophometrically using a Specol 211 spectrophotometer (Germany) by monitoring the change in NADH absorbance at 340 nm (Lushchak et al., 2001). One unit of enzyme activity is defined as the amount of enzyme consuming 1 lmol of substrate per minute. Activities are expressed as international units per milligram protein.

2.3. Hemoglobin determination and hemolysate preparation Total hemoglobin concentration and relative content of its derivatives were determined in whole blood by a ‘‘multiwavelength” spectrophotometric method for the simultaneous determination of five hemoglobin derivatives (Zwart et al., 1981) with some modifications according to the method of Bilyi et al. (2000). Aliquots (0.02 mL) of blood were added into 3 mM sodium–potassium phosphate buffer (pH 6.36) for a total sample volume of 2 mL. After 2–3 min of incubation, allowing erythrocyte hemolysis, samples absorbance was measured at six wavelengths 554.8, 557.4, 569.5, 620.3, 500 and 524.1 nm, representing the absorbance maxima for deoxyhemoglobin (RHb), oxyhemoglobin (HbO2), carboxyhemoglobin (HbCO), sulphhemoglobin (SHb), methemoglobin (MetHb) and total hemoglobin, respectively. Total hemoglobin concentration (g L 1) and relative content (%) of ligand forms was calculated from these values using the known of molar extinction coefficients at the six analytical wavelengths, as described by Bilyi et al. (2000). The rest of the blood was used to obtain plasma and erythrocytes. After the removal of plasma by centrifugation (1500g, 15 min, 4 °C) erythrocytes were washed thoroughly three times in 0.9% w:v NaCl at 4 °C and then disruptured osmotically by the addition of five volumes of ice-cold distilled water (Shipkova et al., 2006). Hemolysates were centrifuged (8000g, 15 min, 4 °C) to remove the ghosts. Obtained plasma and hemolysates were used for enzyme activity assays.

2.6. Protein carbonyl determination A 50 lL aliquot of plasma was mixed 1:1 v/v with 40% w:v trichloroacetic acid and then centrifuged for 5 min at 5000g. Protein carbonyl (CP) content was measured in the resulting pellets, by reaction with 2,4-dinitrophenylhydrazine (DNPH), as described previously (Lushchak et al., 2005). The content of carbonyl protein groups was evaluated spectrophotometrically at 370 nm. Data are expressed as nanomoles CP per milligram total protein. 2.7. Protein measurements and statistics Protein concentration was measured by the Bradford method with Coomassie Brilliant Blue G-250 (Bradford, 1976) using bovine serum albumin as a standard. Data are presented as means ± SEM Statistical analysis was performed using the Student’s t-test. 3. Results 3.1. Erythrocyte parameters The number of erythrocytes containing micronuclei in control and chromium-exposed goldfish was investigated and clearly demonstrated the cytotoxicity of the two chromium ions toward

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erythrocytes. Fish exposed to 10 mg L 1 of Cr6+ for 96 h showed a significant 579% increase in the number of micronucleus-containing erythrocytes. However, in fish treated with Cr3+ only a tendency to increase micronuclei numbers was seen (Fig. 1). Total hemoglobin concentration in goldfish blood increased by 63% after exposure of fish to Cr3+, whereas exposure to Cr6+ had no significant effect (just a tendency to increase) (Table 1). When we turned our attention to the different forms of hemoglobin, no significant changes were found in either oxygenated hemoglobin or methemoglobin in response to the two chromium ion treatments.

3.2. Leucocyte formula Exposure of goldfish to chromium affected the relative amounts of different leucocytes (Table 2). Among mature leucocyte forms, eosynophils and basophils were not found in either control or chromium-treated groups. The amount of stab (band) neutrophils decreased by 55% in fish treated with Cr6+, whereas Cr3+ did not affect the number of these cells. The amount of segmented neutrophils was strongly reduced in goldfish after incubation with either Cr3+ or Cr6+, by 61% and 77%, respectively. The level of monocytes did not change at all, but the amount of lymphocytes increased by 8.2% and 13.3% under Cr3+ and Cr6+ treatments, respectively. Of the blast forms, hemocytoblasts were not found in any of the three fish groups. Myeloblasts were found in the control group, but not in chromium-treated groups. Promyelocyte numbers did not change significantly in fish treated with Cr3+ (although a tendency to decrease was seen), but they were not found in the Cr6+-treated fish. The level of myelocytes decreased by one-half after each chromium exposure, whereas the amount of metamyelocytes was reduced by 56% and 71% after fish treatment with Cr3+ and Cr6+, respectively.

Table 1 Effects of 10 mg L 1 Cr3+ and Cr6+ exposure for 96 h on the total hemoglobin level and content of its ligand forms in goldfish blood. Parameter

Fish group Control

Total hemoglobin (g L HbO2 (%) MetHb (%)

1

)

41.1 ± 4.8 85.0 ± 5.9 13.8 ± 4.6

Cr3+

Cr6+

67.1 ± 4.1 85.0 ± 4.1 12.1 ± 4.0

*

51.3 ± 9.5 78.4 ± 6.9 18.0 ± 9.4

Data are expressed as means ± SEM, n = 5–6. Significantly different from the control values, P < 0.05.

*

Table 2 Effects of 10 mg L 1 Cr3+ and Cr6+ exposure for 96 h on the relative content of different leucocytes (percentage) in goldfish peripheral blood. Leucocytes type

Fish group Control

Lymphocytes Stab neutrophils Segmented neutrophils Eosinophils Basophils Monocytes Hemocytoblasts Myeloblasts Promyelocytes Myelocytes Metamyelocytes

80.9 ± 1.7 4.9 ± 0.7 3.1 ± 0.6 NF NF 3.9 ± 0.4 NF 0.3 ± 0.2 1.4 ± 0.4 2.1 ± 0.3 3.4 ± 0.7

Cr3+

Cr6+ *

87.5 ± 0.9 5.0 ± 0.5 1.2 ± 0.3* NF NF 4.8 ± 0.8 NF NF 0.7 ± 0.3 1.0 ± 0.0* 1.5 ± 0.2*

91.7 ± 1.5* 2.2 ± 0.6* 0.7 ± 0.2* NF NF 3.5 ± 0.6 NF NF NF 1.0 ± 0.4* 1.0 ± 0.4*

Data are presented as percentages of total leucocytes number, evaluated per 200 leucocytes cells. NF – not found. Data are expressed as means ± SEM, n = 6. * Significantly different from the control values, P < 0.05.

3.3. Catalase and lactate dehydrogenase activities in brain Goldfish exposure to 10 mg L 1 Cr3+ for 96 h decreased the catalase activity in brain by 41% (Fig. 2), which corresponds well with our previous data (Lushchak et al., 2009a). However, animal exposure to 10 mg L 1 of Cr6+ for the same length of time increased catalase activity by 60% (Fig. 2). LDH activity in brain decreased under both chromium exposures by 20% and 24% for Cr3+ and Cr6+ treatments, respectively.

Fig. 2. Effects of 10 mg L 1 Cr3+ and Cr6+ exposure for 96 h on the activities of catalase and lactate dehydrogenase (LDH) in goldfish brain. Data are expressed as means ± SEM, n = 5–6. *Significantly different from the corresponding control group, P < 0.05.

3.4. Protein carbonyls in plasma Fig. 3 demonstrates the effects of chromium exposure on the level of an oxidative stress marker – protein carbonyl groups (CP) – in goldfish plasma and erythrocytes. Exposure to either chromium ion increased CP content in plasma by approximately the same amount, 104% and 95% for Cr3+ and Cr6+, respectively.

Fig. 1. Effects of 10 mg L 1 Cr3+ and Cr6+ exposure for 96 h on the formation of micronuclei in goldfish erythrocytes. The frequency of micronuclei is expressed per 1000 erythrocytes. Data are expressed as means ± SEM for six individual fish in the group and an average of four smears for each fish. *Significantly different from the control group, P < 0.05.

3.5. Activities of catalase and LDH in plasma and erythrocytes Goldfish exposure to Cr3+ had no effect on catalase activity in plasma, whereas Cr6+ exposure elevated enzyme activity significantly by 274% (Fig. 4). A similar 106% increase in catalase activity

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Fig. 3. Effects of 10 mg L 1 Cr3+ and Cr6+ exposure for 96 h on concentrations of protein carbonyls in goldfish plasma. Other information as in Fig. 2.

Fig. 4. Effects of 10 mg L 1 Cr3+ and Cr6+ exposure for 96 h on the activities of catalase in goldfish plasma and erythrocytes. Other information as in Fig. 2.

was seen in erythrocytes from Cr6+-exposed fish. But, unlike the results for plasma, fish given Cr3+ exposure showed a strong 70% reduction in catalase activity in erythrocytes (Fig. 4). Fish treatment with either chromium ion decreased LDH activity in plasma to the same extent, 34% of the control value (Fig. 5). Unexpectedly, fish exposure to Cr6+ enhanced LDH activity in erythrocytes by 127%, whereas Cr3+ exposure resulted in only a tendency for LDH increase (Fig. 5).

4. Discussion The cytotoxicity of transition metal ions and other toxicants for fish has been evaluated for several species by using the micronucleus assay in erythrocytes (Udroiu, 2006). For example, in C. punctata mercury, arsenic and copper increased micronucleus frequency (Yadav and Trivedi, 2009). Significant increases in micronucleated erythrocytes were also found in fathead minnows, Pimephales promelos, after exposure to Cr6+ (de Lemos et al., 2007a) and exposure to the herbicide glyphosate enhanced the percentage of micronucleated cells in goldfish erythrocytes (Cavasß and Könen, 2007). This approach was also effective for evaluating the genotoxicity of petrochemical effluents on P. promelas (de Lemos et al., 2007b) and Hose with colleagues (1984) to access the effects benzo(a)pyrene-treated rainbow trout alevins. Al-Sabti et al. (1994) evaluated the frequency of micronuclei induction in Prussian carp (Carassius auratus gibelio) after exposure to various sublethal chromium concentrations and found that both

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Fig. 5. Effects of 10 mg L 1 Cr3+ and Cr6+ exposure for 96 h on the activities of LDH in goldfish plasma and erythrocytes. Other information as in Fig. 2.

tri- and hexavalent chromuin ions enhanced this parameter. Therefore, we applied the micronucleus test to evaluate the genotoxicity of chromium on goldfish erythrocytes. Cr6+ strongly enhanced the incidence of micronuclei in goldfish erythrocytes (Fig. 1), clearly demonstrating that this form of chromium is cytotoxic for goldfish. The results for Cr3+ exposure were not significant, but showed a trend towards elevated micronuclei numbers which could suggest that higher concentrations or longer exposure times to Cr3+ could also lead to significantly enhanced cytotoxicity. Fish exposure to chromium ions could affect gill morphology (Mishra and Mohanty, 2009a) and that might impair respiration and oxygen supplementation to tissues. Due to that, we checked whether fish exposure to Cr3+ and Cr6+ affected total hemoglobin content, oxyhemoglobin and methemoglobin levels. Goldfish treatment with Cr3+ significantly enhanced total hemoglobin level, whereas Cr6+ exposure resulted in only a tendency to increase (Table 1). At the same time, neither ion significantly changed the amounts of ligand hemoglobin forms. These data show that fish may compensate for damage to gill tissue by enhancing the capacity of blood to transport oxygen via an increase in the total amount of hemoglobin, probably due to increased numbers of red blood cells released into circulation. The immune system is very sensitive to environmental challenges and it is well known that chromium ions can affect immune function in fish (Khangarot et al., 1999), although specific information on goldfish was not available. Therefore, we investigated the effects of Cr3+ and Cr6+ exposure on the relative content of different forms of leucocytes in the peripheral blood of goldfish (Table 2). Both chromium treatments significantly enhanced the total number of lymphocytes. Eosinophils and basophils were not found in either control, or chromium-exposed fish, and the amount of stab neutrophils decreased with Cr6+ exposure, whereas segmented neutrophils were suppressed by both chromium treatments (Table 2). The amount of blast leucocyte forms, such as myelocytes and metamyelocytes, decreased significantly with exposure to both chromium forms. These data are in good agreement with previous work where the effects of metal ions on fish leucocyte numbers were studied. For example, copper ion exposure increased lymphocyte number in Mozambique tilapia, Oreochromis mossambicus (Nussey et al., 1995) and cadmium exposure decreased the amount of blast cells in goldfish blood (Murad and Houston, 1988). Our data add to the collective body of literature to demonstrate that chromium ions, like other metal ions, affect the immune system of fish. Given the above results on chromium ion genotoxicity and effects on the immune system, we wondered about the mechanisms

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of these changes. One of the possible mechanisms of chromium-induced intoxication could be an induction of oxidative stress by these ions. We (Lushchak et al., 2008, 2009a,b) and others (Krumschnabel and Nawaz, 2004; Roling et al., 2006; Ahmad et al., 2006) have demonstrated that both Cr3+ and Cr6+ induced oxidative stress in fish tissues. This effect was explained by the ability of chromium ions to enter redox processes due to donation/reception of electrons. Unfortunately, there are no direct data to confirm which chromium ion is predominant in fish tissues, but there are rather strong indications that in vivo Cr6+ may be reduced to Cr3+ (Das and Chandra, 1990; Cheung and Gu, 2003; Ekenberg et al., 2005). The involvement of glutathione (GSH) and glutathione-related enzymes was implicated in this case (Lalaouni et al., 2007; Lushchak et al., 2008; Raghunathan et al., 2009). In order to clarify the issue of the specificity of effects of the chromium ions commonly found in the environment, Cr3+ and Cr6+, we recently compared their effects on free radical processes in goldfish tissues (Lushchak et al., 2008, 2009a,b). That data clearly showed that although both ions were able to induce oxidative stress, some tissue and ion specificities were found. In order to confirm the approach used in the present work, we first checked to determine whether chromium ions induced similar changes as were seen in our previous work. For that, we investigated the activity of catalase in goldfish brain and found that Cr3+ decreased catalase activity (Fig. 2) by virtually the same extent as it did earlier (Lushchak et al., 2009a). Earlier it was found that LDH is rather insensitive to inactivation by free radicals in vitro (Vasylkiv and Lushchak, 2010), but enzymes may respond to ROS differently in vitro and in vivo. Therefore, we decided to determine whether the activity of LDH could be affected under conditions of chromium-induced oxidative stress. Surprisingly, we found a small, but significant decrease in LDH activity in goldfish brain under these conditions (Fig. 2). Several factors may contribute to the decrease in the LDH activity under chromium treatment and inactivation by ROS may be one of them. Decreased activities of several other enzymes under oxidative stress conditions have also been linked with inactivation by ROS (Peña-Llopis et al., 2003; Kochhann et al., 2009; Misra and Niyogi, 2009). The small change in LDH activity might not result in a substantial modification of glycolytic capacity, but it demonstrates that glycolytic and associated enzymes could be involved in the response to chromium exposure. Earlier we studied the induction by chromium ions of oxidative stress in goldfish brain, liver and kidney (Lushchak et al., 2008, 2009a,b), but chromium effects on blood were not investigated. Therefore, we decided to determine whether these ions were able to induce oxidative stress in goldfish blood. It is broadly accepted that blood is a good system with which to monitor the effects of environmental or toxic exposures because it can often be sampled nonlethally. Interestingly, both Cr3+ and Cr6+ elevated the content of protein carbonyl groups (CP) in plasma by about 2-fold (Fig. 3), confirming our previous findings of the induction of oxidative stress by chromium in goldfish tissues (Lushchak et al., 2008, 2009a,b). The activities of antioxidant enzymes are frequently used as markers of oxidative stress (Hermes-Lima and Storey, 1995, Hermes-Lima and Storey, 1998; Falfushynska et al., 2008; Falfushynska and Stolyar, 2009; Grinevicius et al., 2009). However, the effectiveness of an enzyme such as catalase as a marker is a rather complicated issue because activity may change due to at least two processes. Firstly, catalase may be inactivated by ROS, particularly superoxide anion and hydrogen peroxide (Lardinois, 1995). Secondly, catalase activity may be enhanced via de novo synthesis as a part of an adaptive response to oxidative stress. The two processes undoubtedly take place simultaneously, as was demonstrated in a yeast model (Bayliak et al., 2006). Therefore, the net effect depends on many circumstances, but despite

that, catalase activity is a commonly analyzed component in investigations of oxidative stress effects on organisms. It should be remembered that, as opposed to mammalian mature erythrocytes that lack a nucleus and protein biosynthesis machinery, fish erythrocytes contain nuclei and are competent in protein biosynthesis. Therefore, the increased activity of catalase in erythrocytes of goldfish exposed to Cr6+ (Fig. 4) could be connected with biosynthesis de novo. One more issue should be mentioned. Under stress conditions tissue depots of erythrocytes, such as the spleen, are emptied resulting in their release into the bloodstream. Moreover, not only mature erythrocyte forms are released, but also juvenile forms. It is well known that the activities of antioxidant enzymes in old cells are usually lower that those in young ones (Arabuli et al., 2005; Goncharova et al., 2008; Nagababu et al., 2010). Hence, the increase in erythrocyte catalase activity as a result of Cr6+ exposure could also be influenced by a change in the proportion of newer versus older erythrocytes in the circulation. This possibility needs further investigation. The decrease of catalase activity in erythrocytes of Cr3+-exposed fish could be a result of its inactivation by ROS, particularly superoxide anion (Lardinois, 1995), whereas enhanced activity in plasma of Cr6+-treated fish (Fig. 4) could result from its release from tissues damaged by ROS. Because we suggested the possible inactivation of LDH by ROS in goldfish brain exposed to chromium ions, we further measured the activity of this enzyme in plasma and erythrocytes. We were able to demonstrate that chromium exposure of fish substantially reduced LDH activity in plasma (Fig. 5). This shows that plasma LDH activity in combination with CP content may be markers of oxidative stress exposure, markers that can potentially be evaluated by nonlethal sampling techniques. Interestingly, in many cases, various stresses have been demonstrated to enhance plasma LDH activity (Váczi et al., 2009; Ozer Sehirli et al., 2009) and this was typically explained as leakage from surrounding tissues due their injury. It should be noted also, that LDH activity in erythrocytes increased during fish exposure to Cr6+. The de novo synthesis of LDH in circulating erythrocytes, as well as by an increased portion of young erythrocytes which entered the bloodstream from storage depots could be implicated here. In the present study, we confirmed that chromium ions, namely Cr3+ and Cr6+, are inducers of oxidative stress in goldfish tissues and that blood is also subjected to the stress. Moreover, the activity of LDH was shown to be a potential marker of this stress. The development of oxidative stress induced by chromium ions enhanced the number of erythrocytes possessing micronuclei. Therefore, this shows that goldfish exposure to chromium ions is cytotoxic. The data on leukocytes also demonstrate that white blood cells were affected by this treatment. Overall, the results show that the parameters evaluated here, i.e. oxidative stress markers, number of micronucleated erythrocytes and leukocyte count, can be good early nonlethal diagnostic markers of fish intoxication by chromium ions.

Acknowledgements We are grateful to I. Z. Drahomyretska for technical assistance and to J. M. Storey for critical reading of the manuscript. The research received partial support from the Ministry of Education and Science of Ukraine to VIL (#0106U002245) and from a discovery grant from the Natural Sciences and Engineering Research Council of Canada to KBS. References Ahmad, I., Maria, V.L., Oliveira, M., Pacheco, M., Santos, M.A., 2006. Oxidative stress and genotoxic effects in gill and kidney of Anguilla anguilla L. Exposed to

O.Yu. Vasylkiv et al. / Chemosphere 80 (2010) 1044–1049 chromium with or without pre-exposure to beta-naphthoflavone. Mutat. Res. 608, 16–28. Al-Sabti, K., Franko, M., Andrijanic, B., Knez, S., Stegnar, P., 1994. Chromium-induced micronuclei in fish. J. Appl. Toxicol. 14, 333–336. Arabuli, M.B., Khetsuriani, R.G., Dalakishvili, I.M., Tkhilava, G., Sanikidze, T.V., 2005. Age related changes of physical–chemical parameters of erythrocytes. Georgian Med. News 129, 113–116. Arillo, A., Melodia, F., 1990. Protective effect of fish mucus against Cr(VI) pollution. Chemosphere 20, 397–402. Bagchi, D., Stohs, S.J., Downs, B.W., Bagchi, M., Preuss, H.G., 2002. Cytotoxicity and oxidative mechanisms of different forms of chromium. Toxicology 180, 5–22. Bayliak, M., Semchyshyn, H., Lushchak, V., 2006. Effect of hydrogen peroxide on antioxidant enzyme activities in Saccharomyces cerevisiae is strain-specific. Biochemistry (Moscow) 71, 1013–1020. Bilyi, O.I., Velikyi, M.M., Dudok, K.P., 2000. Original method of concurrent determination of hemoglobin derivatives. Proc. SPIE, Int. Soc. Opt. Eng. 3926, 223–228. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Cavasß, T., Könen, S., 2007. Detection of cytogenetic and DNA damage in peripheral erythrocytes of goldfish (Carassius auratus) exposed to a glyphosate formulation using the micronucleus test and the comet assay. Mutagenesis 22, 263–268. Cheung, K.H., Gu, J.D., 2003. Reduction of chromate (CrO24 ) by an enrichment consortium and an isolate of marine sulfate-reducing bacteria. Chemosphere 52, 1523–1529. Das, S., Chandra, A.L., 1990. Chromate reduction in Streptomyces. Experientia 46, 731–733. de Lemos, C.T., Rödel, P.M., Terra, N.R., Erdtmann, B., 2007a. Evaluation of basal micronucleus frequency and hexavalent chromium effects in fish erythrocytes. Environ. Toxicol. Chem. 20, 1320–1324. de Lemos, C.T., Rödel, P.M., Terra, N.R., D’Avila de Oliveira, N.C., Erdtmann, B., 2007b. River water genotoxicity evaluation using micronucleus assay in fish erythrocytes. Ecotoxicol. Environ. Saf. 66, 391–401. Ekenberg, M., Martander, H., Welander, T., 2005. Biological reduction of hexavalent chromium a field study. Water Environ. Res. 77, 425–428. Falfushynska, H.I., Stolyar, O.B., 2009. Responses of biochemical markers in carp Cyprinus carpio from two field sites in Western Ukraine. Ecotoxicol. Environ. Saf. 72, 729–736. Falfushynska, H.I., Romanchuk, L.D., Stolyar, O.B., 2008. Different responses of biochemical markers in frogs (Rana ridibunda) from urban and rural wetlands to the effect of carbamate fungicide. Comp. Biochem. Physiol. Part C 148, 223–229. Farag, A.M., May, T., Marty, G.D., Easton, M., Harper, D.D., Little, E.E., Cleveland, L., 2006. The effect of chronic chromium exposure on the health of Chinook salmon (Oncorhynchus tshawytscha). Aquat. Toxicol. 76, 246–257. Fenech, M., Chang, W.P., Kirsch-Volders, M., Holland, N., Bonassi, S., Zeiger, E., 2003. Human micronucleus project. HUMAN project: detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures. Mutat. Res. 534, 65–75. Goncharova, N.D., Marenin, V.Y., Bogatyrenko, T.N., 2008. Stress, aging and reliability of antioxidant enzyme defense. Curr. Aging Sci. 1, 22–29. Grinevicius, V.M., Geremias, R., Laus, R., Bettega, K.F., Laranjeiras, M.C., Fávere, V.T., Wilhelm Filho, D., Pedrosa, R.C., 2009. Textile effluents induce biomarkers of acute toxicity, oxidative stress, and genotoxicity. Arch. Environ. Contam. Toxicol. 57, 307–314. Hermes-Lima, M., Storey, K.B., 1995. Antioxidant defenses and metabolic depression in a pulmonate land snail. Am. J. Physiol. 268, 1386–1393. Hermes-Lima, M., Storey, K.B., 1998. Role of antioxidant defenses in the tolerance of severe dehydration by anurans. The case of the leopard frog Rana pipiens. Mol. Cell. Biochem. 189, 79–89. Hose, J.E., Hannah, J.B., Puffer, H.W., Landolt, M.L., 1984. Histologic and skeletal abnormalities in benzo(a)pyrene-treated rainbow trout alevins. Arch. Environ. Contam. Toxicol. 13, 675–684. Ivanova, N.T., 1983. Fish Blood Cells Atlas. Comparative Morphology and Classification of Fish Blood Corpuscles, Moscow. Khangarot, B.S., Rathore, R.S., Tripathi, D.M., 1999. Effects of chromium on humoral and cell-mediated immune responses and host resistance to disease in a freshwater catfish, Saccobranchus fossilis (Bloch). Ecotoxicol. Environ. Saf. 43, 11–20. Kochhann, D., Pavanato, M.A., Llesuy, S.F., Correa, L.M., Konzen Riffel, A.P., Loro, V.L., Mesko, M.F., Flores, E.M., Dressler, V.L., Baldisserotto, B., 2009. Bioaccumulation and oxidative stress parameters in silver catfish (Rhamdia quelen) exposed to different thorium concentrations. Chemosphere 77, 384–391. Krumschnabel, G., Nawaz, M., 2004. Acute toxicity of hexavalent chromium in isolated teleost hepatocytes. Aquat. Toxicol. 70, 159–167.

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Lalaouni, A., Henderson, C., Kupper, C., Grant, M.H., 2007. The interaction of chromium (VI) with macrophages: depletion of glutathione and inhibition of glutathione reductase. Toxicology 236, 76–81. Lardinois, O.M., 1995. Reactions of bovine liver catalase with superoxide radicals and hydrogen peroxide. Free Radical Res. 22, 251–274. Liu, K.J., Shi, X., 2001. In vivo reduction of chromium(VI) and its related free radical generation. Mol. Cell. Biochem. 222, 41–47. Lushchak, V.I., 2008. Oxidative stress as a component of transition metal toxicity in fish. In: Svensson, E.P. (Ed.), Aquatic Toxicology Research Focus. Nova Science Publishers Inc., Hauppauge, NY, pp. 1–29. Lushchak, V.I., Bagnyukova, T.V., Storey, J.M., Storey, K.B., 2001. Influence of exercise on the activity and distribution between free and bound forms of glycolytic and associated enzymes of horse mackerel. Braz. J. Med. Biol. Res. 34, 1055–1064. Lushchak, V.I., Bagnyukova, T.V., Husak, V.V., Luzhna, L.I., Lushchak, O.V., Storey, K.B., 2005. Hyperoxia results in transient oxidative stress and an adaptive response by antioxidant enzymes in goldfish tissues. Int. J. Biochem. Cell Biol. 37, 1670–1680. Lushchak, O.V., Kubrak, O.I., Nykorak, M.Z., Storey, K.B., Lushchak, V.I., 2008. The effect of potassium dichromate on free radical processes in goldfish: possible protective role of glutathione. Aquat. Toxicol. 87, 108–114. Lushchak, O.V., Kubrak, O.I., Torous, I.M., Nazarchuk, T.Yu., Storey, K.B., Lushchak, V.I., 2009a. Trivalent chromium induces oxidative stress in goldfish brain. Chemosphere 75, 56–62. Lushchak, O.V., Kubrak, O.I., Lozinsky, O.V., Storey, J.M., Storey, K.B., Lushchak, V.I., 2009b. Chromium(III) induces oxidative stress in goldfish liver and kidney. Aquat. Toxicol. 93, 45–52. Mishra, A.K., Mohanty, B., 2009a. Chronic exposure to sublethal hexavalent chromium affects organ histopathology and serum cortisol profile of a teleost, Channa punctatus (Bloch). Sci. Total Environ. 407, 5031–5038. Mishra, A.K., Mohanty, B., 2009b. Effect of hexavalent chromium exposure on the pituitary – interrenal axis of a teleost, Channa punctatus (Bloch). Chemosphere 76, 982–988. Misra, S., Niyogi, S., 2009. Selenite causes cytotoxicity in rainbow trout (Oncorhynchus mykiss) hepatocytes by inducing oxidative stress. Toxicol. In vitro 23, 1249–1258. Murad, A., Houston, A.H., 1988. Leucocytes and leucopoietic capacity in goldfish, Carassius auratus, exposed to sublethal levels of cadmium. Aquat. Toxicol. 13, 141–154. Nagababu, E., Mohanty, J.G., Bhamidipaty, S., Ostera, G.R., Rifkind, J.M., 2010. Role of the membrane in the formation of heme degradation products in red blood cells. Life Sci. J. 86, 133–138. Nussey, G., van Vuren, J.H.J., du Preez, H.H., 1995. Effect of copper on the differential white blood cell counts of the Mozambique tilapia (Oreochromis mossambicus). Comp. Biochem. Physiol. Part C 111, 381–388. Ozer Sehirli, A., Sener, G., Ercan, F., 2009. Protective effects of pycnogenol against ischemia reperfusion-induced oxidative renal injury in rats. Renal Fail. 31, 690– 697. Peña-Llopis, S., Ferrando, M.D., Peña, J.B., 2003. Fish tolerance to organophosphateinduced oxidative stress is dependent on the glutathione metabolism and enhanced by N-acetylcysteine. Aquat. Toxicol. 65, 337–360. Pourahmad, J., O’Brien, P.J., 2001. Biological reactive intermediates that mediate chromium(VI) toxicity. Adv. Exp. Med. Biol. 500, 203–207. Raghunathan, V.K., Ellis, E.M., Grant, M.H., 2009. Response to chronic exposure to hexavalent chromium in human monocytes. Toxicol. In vitro 23, 647–652. Riva, M.C., Flos, R., Crespi, M., Balasch, J., 1981. Lethal potassium dichromate and whitening (blankophor) exposure of goldfish (Carassius auratus): chromium levels in gills. Comp. Biochem. Physiol. Part C 68, 161–165. Roling, J.A., Bain, L.J., Gardea-Torresdey, J., Bader, J., Baldwin, W.S., 2006. Hexavalent chromium reduces larval growth and alters gene expression in mummichog (Fundulus heteroclitus). Environ. Toxicol. Chem. 25, 2725–2733. Shipkova, M., Lorenz, K., Oellerich, M., 2006. Measurement of erythrocyte inosine triphosphate pyrophosphohydrolase (ITPA) activity by HPLC and correlation of ITPA genotype–phenotype in a Caucasian population. Clin. Chem. 52, 240–247. Udroiu, I., 2006. The micronucleus test in piscine erythrocytes. Aquat. Toxicol. 79, 201–204. Váczi, M., Costa, A., Rácz, L., Tihanyi, J., 2009. Effects of consecutive eccentric training at different range of motion on muscle damage and recovery. Acta Physiol. Hung. 96, 459–468. Vasylkiv, O.Yu., Lushchak, V.I., 2010. Comparative characteristics and inactivation by free radicals of partially purified lactate dehydrogenase from white muscle and liver of goldfish (Carassius auratus L.), Ukr. Biochem. J. 82, 27–33. Yadav, K.V., Trivedi, S.P., 2009. Sublethal exposure of heavy metals induces micronuclei in fish, Channa punctata. Chemosphere 77, 1495–1500. Zwart, A., Buursma, A., Van Kampen, E.J., Oeseburg, B., Zijlstra, W.G., 1981. A multi ‘‘wavelength” spectrophotometric method for the simultaneous determination of five hemoglobin derivatives. J. Clin. Chem. Clin. Biochem. 9, 459–463.