Hepatic antioxidant status and hematological parameters in rainbow trout, Oncorhynchus mykiss, after chronic exposure to carbamazepine

Hepatic antioxidant status and hematological parameters in rainbow trout, Oncorhynchus mykiss, after chronic exposure to carbamazepine

Chemico-Biological Interactions 183 (2010) 98–104 Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage: www.el...

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Chemico-Biological Interactions 183 (2010) 98–104

Contents lists available at ScienceDirect

Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint

Hepatic antioxidant status and hematological parameters in rainbow trout, Oncorhynchus mykiss, after chronic exposure to carbamazepine Zhi-Hua Li a,∗ , Josef Velisek a , Vladimir Zlabek a , Roman Grabic a,b , Jana Machova a , Jitka Kolarova a , Tomas Randak a a University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, Research Institute of Fish Culture and Hydrobiology, Zatisi 728/II, 389 25 Vodnany, Czech Republic b Umea University, Department of Chemistry, SE-90187 Umea, Sweden

a r t i c l e

i n f o

Article history: Received 3 August 2009 Received in revised form 14 September 2009 Accepted 15 September 2009 Available online 22 September 2009 Keywords: Fish Liver Oxidative stress Blood indices Residual pharmaceutical

a b s t r a c t Recently, residual pharmaceuticals are generally recognized as relevant sources of aquatic environmental pollutants. However, the toxicological effects of these contaminants have not been adequately researched. In this study, the chronic toxic effect of carbamazepine (CBZ), an anticonvulsant drug commonly present in surface and ground water, on hepatic antioxidant status and hematological parameters of rainbow trout were investigated. Fish were exposed at sublethal concentrations of CBZ (1.0 ␮g/l, 0.2 mg/l and 2.0 mg/l) for 7, 21 and 42 days. Compared to the control group, fish exposed at higher concentration (0.2 mg/l or 2.0 mg/l) of CBZ showed significantly higher levels of hemoglobin, ammonia and glucose, and significantly higher plasma enzymes activities. During the exposure duration, erythrocyte count, hematocrit, mean erythrocyte hemoglobin, mean erythrocyte volume, mean color concentration and total protein content in all groups were not significantly different. At the highest test concentration (2.0 mg/l) of CBZ, oxidative stress was apparent as reflected by the significant higher lipid peroxidation and protein carbonyl levels in liver after 42 days exposure, associated with an inability to induce antioxidant enzymes activities including superoxide dismutase, glutathione peroxidase and glutathione reductase. After 42 days exposure, reduced glutathione level was significantly decreased in the fish exposed at 0.2 mg/l CBZ, compared with the control. In short, CBZ-induced physiological and biochemical responses in fish were reflected in the oxidant stress indices and hematological parameters. These results suggest that hepatic antioxidant responses and hematological parameter could be used as potential biomarkers for monitoring residual pharmaceuticals present in aquatic environment. © 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction In these days, a large volume of pharmaceuticals is used for the prevention, diagnosis and treatment of diseases in humans and animals. Most pharmaceuticals are not completely degraded after application [1]. As a result, the pharmaceutical metabolites and some unchanged forms of these compounds are excreted and subsequently enter the ecosystem [2]. In the last decade, researchers have detected a multitude of pharmaceuticals in the aquatic environment. Amongst these, carbamazepine (CBZ), an antiepileptic drug used to control seizures, has been one of the most frequently detected pharmaceutical residues in water bodies and has been proposed as an anthropogenic marker in water bodies [3]. However, its potential adverse ecological effects on aquatic organisms remain largely unknown, especially on fish [4].

∗ Corresponding author. Tel.: +420 383 382 402; fax: +420 383 382 396. E-mail address: [email protected] (Z.-H. Li). 0009-2797/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2009.09.009

Antioxidant defenses, present in all aerobic organisms, include antioxidant enzymes and low molecular mass antioxidants whose function is to remove reactive oxygen species (ROS), thus protecting organisms from oxidative stress [5,6]. But when ROS generation exceeds the capacity of the cellular antioxidants, it will cause oxidative stress and oxidative damages [7]. In general, ROS is the considered to be a harmful byproduct of oxidative metabolism and play a critical role in initiating and catalyzing a variety of radical reactions in the presence of oxygen [6,8]. Marked blood biochemical responses often occur after aquatic organisms have been exposed to environmental organic contaminants [9]. Differential blood cell counts and plasmatic enzymes are an effective indicator of environmental stress and provide a general overview of the integrity of the immune system [10]. Therefore, the haematological analysis and biochemical parameters of blood plasma are useful to monitor the physiological status of fish and used as health indicators in aquatic environment even though they are not routinely used for fish diseases diagnosis [11,12]. To study CBZ-induced the responses of hepatic antioxidant status and blood biochemical parameters in fish, this

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paper chose rainbow trout (Oncorhynchus mykiss), a widely used model in aquatic toxicology, as test objective. Hematological and plasma biochemical parameters were analyzed, as well as the responses of the antioxidant defense system (SOD—superoxide dismutase, CAT—catalase, GPx—glutathione peroxidase, GR—glutathione reductase activities and GSH—reduced glutathione levels). Also, oxidative stress indices (CP—carbonyl protein and TBARS—thiobarbaturic acid levels) were measured in liver.

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2. Materials and methods

(PCV), mean erythrocyte hemoglobin (MCH), mean erythrocyte volume (MCV), mean color concentration (MCHC). The procedures were based on unified methods for the hematological examination of fish. Blood plasma obtained from cooled centrifuged blood samples (4 ◦ C, 837 × g) was stored at −80 ◦ C until use. Biochemical indices including ammonia (NH3 ), glucose (GLU), total proteins (TP), creatine kinase (CK), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and were determined using a VETTEST 8008 analyzer (IDEXX Laboratories Inc., U.S.A.).

2.1. Chemicals

2.5. Antioxidant indices

Carbamazepine and other chemicals were obtained from Sigma–Aldrich Corporation (USA). The CBZ was dissolved in dimethyl sulfoxide (DMSO) to make a stock solution at a concentration of 100 mg/ml.

2.5.1. Tissue samples and preparation of post-mitochondrial supernatant At the end of each exposure, three fish of every aquarium were killed randomly. The liver tissue was quickly removed, immediately frozen and stored at −80 ◦ C for analysis.Frozen tissue samples were weighed and homogenized (1:10, w/v) using an Ultra Turrax homogenizer (Ika, Germany) using 50 mM potassium phosphate buffer, pH 7.0, containing 0.5 mM EDTA. The homogenate was divided into two portions, one for measuring TBARS and CP, and a second centrifuged at 12,000 × g for 30 min at 4 ◦ C to obtain the post-mitochondrial supernatant for other antioxidant parameters analyses.

2.2. Fish Rainbow trout, weighing 264 ± 40 g (mean ± S.D.), were obtained from a local commercial hatchery (Husinec, Czech Republic). They were held in aquaria containing 250 l of freshwater continuously aerated to maintain dissolved oxygen values at 7.5–8.0 mg/l. Temperature was 15 ± 1 ◦ C and pH was 7.4 ± 0.2. Photoperiod was a 12:12 light–dark cycle. Fish were acclimatized for 14 days before the beginning of the experiment and were fed commercial fish food. The fish were starved for 24 h prior to sampling to avoid prandial effects during the assay. 2.3. Exposure to CBZ A 200 l semi-static system was used in which 11 rainbow trout were randomly distributed to each of ten aquaria. The nominal concentrations of CBZ used were 1.0 ␮g/l (E1 group, according to environmental concentration), 0.2 mg/l (E2 group, 1%, 96 h, LC 50, according to unpublished experimental results in our laboratory), and 2.0 mg/l (E3 group, 10%, 96 h, LC 50). Carbamazepine was dissolved in DMSO with a final concentration less than 0.05%. Two other groups were used as contrast groups, a control group exposed to clean freshwater and a DMSO group exposed to the volume of DMSO (v/v, 0.05%) used for the highest CBZ concentration. Each experimental condition was duplicated. The fish were fed daily with commercial fish pellets at 1% total body weight at a fixed time and the extra food was removed. Eighty percent of the exposed solution was renewed each day after 2 h of feeding to maintain the appropriate concentration of CBZ and DMSO and to maintain water quality. The test equipment was cleaned every 14 days. The test fish were exposed to CBZ for 7, 21 and 42 days. Experiments were carried out in accordance with the European Communities Council Directive (86/609/EEC) and were approved by a local ethics committee. To ensure agreement between nominal and actual compound concentrations in the aquaria, water samples were analyzed during the experimental period by LC–MS/MS. Water samples were collected from the test aquaria after 1 h and 24 h of renewing the test solutions. The mean concentration of CBZ in the water samples was always within 20% of the intended concentration. 2.4. Hematological and biochemical blood plasma parameters Blood samples were taken from each fish by caudal venipuncture using a syringe heparinized (Heparin inj., Leciva, Czech Republic) at a concentration of 5000 IU heparin sodium salt in 1 ml. An aqueous solution of heparin sodium salt at 0.01 ml/1 ml blood was used to stabilize the samples. The indices tested included hemoglobin concentration (Hb), erythrocyte count (Er), hematocrit

2.5.2. Indices of oxidative stress A 500 ␮l aliquot of homogenate was mixed with 1 ml of 30% (w/v) TCA and centrifuged for 10 min at 5000 × g. The supernatant was used for peroxidation of lipids (LPO) assays and the pellet used for CP assay. The TBARS method described by Lushchak et al. was used to evaluate hepatic LPO [13]. The TBARS concentration was calculated by the absorption at 535 nm and a molar extinction coefficient of 156 mM/cm. The value was expressed as nanomoles of TBARS/g wet weight tissue. Carbonyl derivatives of proteins were detected by reaction with 2,4-dinitrophenylhydrazine (DNPH) according to the method described by Lenz [14]. The amount of CP was measured spectrophotometrically at 370 nm using a molar extinction coefficient of 22 mM/cm. The values were expressed as nanomoles of CP/g of wet weight tissue. 2.5.3. Antioxidant enzymes assays Total superoxide dismutase (SOD; EC 1.15.1.1) activity was determined by the method of Marklund and Marklund [15]. This assay is based on the autoxidation of pyrogallol. Superoxide dismutase activity was assessed spectrophotometrically at 420 nm and expressed as the amount of enzyme/mg of protein. The catalase (CAT; EC 1.11.1.6) activity assay, using the spectrophotometric measurement of H2 O2 breakdown, measured at 240 nm, was performed following the method of Beers and Sizer [16]. Glutathione peroxidase (GPx; EC 1.11.1.9) activity was assayed following the rate of NADPH oxidation at 340 nm by the coupled reaction with glutathione reductase. The specific activity was determined using the extinction coefficient of 6.22 mM/cm [17]. Glutathione reductase (GR; EC 1.6.4.2) activity was determined spectrophotometrically, measuring NADPH oxidation at 340 nm [18]. One unit of CAT, GPx, or GR activity is defined as the amount of the enzyme that consumes 1 ␮mol of substrate or generates 1 ␮mol of product per min; activity was expressed in international units (or milliunits)/mg of protein. 2.5.4. Reduced glutathione (GSH) assay Reduced glutathione level was assayed using the methods of Sedlak and Lindsay (1968) with a modification by Ferrari et al.

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Table 1 Hematological parameters in rainbow trout affected by chronic exposure to CBZ. Control, DMSO and experimental (nominal concentrations of 1 ␮g/l, 0.2 mg/l and 2 mg/l) groups’ values are shown as Control, DMSO, E1, E2 and E3, respectively. Data are means ± S.D., n = 6. Data sharing the same superscript letter “a” indicate no significant differences (p > 0.05) compared with control value during the same experiment time (e.g. 7, 21 and 42 days, respectively). But data sharing the different superscript letter, such as Hb level sharing “b” in E3 group after 7 days of exposure, indicates significant differences (p < 0.05) compared with the control value after 7 days. Indices

Unit

Exposure time

Test groups Control

DMSO

E1

E2

E3

Hb

g/l

7 days 21 days 42 days

63.62 ± 10.63 59.21 ± 16.79a 40.82 ± 8.41a

64.52 ± 11.37 62.67 ± 9.13a 46.79 ± 8.35a

50.97 ± 13.79 63.32 ± 6.81a 47.98 ± 6.70a

78.48 ± 11.44 68.81 ± 8.15a 50.43 ± 9.40a*

83.56 ± 11.41b 71.68 ± 7.45a 51.92 ± 11.94a*

Er

T/l

7 days 21 days 42 days

1.02 ± 0.16a 0.82 ± 0.27a 0.66 ± 0.14a

0.88 ± 0.17a 0.82 ± 0.19a 0.74 ± 0.11a

0.87 ± 0.19a 0.90 ± 0.14a 0.67 ± 0.15a

1.16 ± 0.26a 0.93 ± 0.15a 0.86 ± 0.20a

0.96 ± 0.12a 0.95 ± 0.19a 0.81 ± 0.16a

PCV

l/l

7 days 21 days 42 days

0.34 ± 0.08a 0.33 ± 0.05a 0.26 ± 0.06a

0.32 ± 0.08a 0.30 ± 0.09a 0.27 ± 0.05a

0.24 ± 0.06a 0.29 ± 0.06a 0.30 ± 0.05a

0.39 ± 0.06a 0.34 ± 0.07a 0.31 ± 0.06a

0.40 ± 0.05a 0.30 ± 0.05a 0.26 ± 0.07a*

MCH

pg

7 days 21 days 42 days

62.19 ± 4.83a 73.89 ± 8.97a 62.45 ± 9.09a

74.06 ± 8.79a 79.65 ± 8.39a 62.96 ± 5.38a

58.19 ± 5.95a 71.11 ± 8.44a 74.26 ± 11.08a

68.85 ± 7.11a 74.89 ± 8.59a 59.47 ± 8.24a

67.08 ± 3.90a 77.79 ± 12.36a 64.51 ± 7.81a

MCV



7 days 21 days 42 days

331.14 ± 72.54a 446.50 ± 176.85a 395.90 ± 82.52a

362.20 ± 50.84a 376.50 ± 102.06a 367.37 ± 36.70a

243.30 ± 25.03a 326.98 ± 65.21a 461.99 ± 64.25a

343.30 ± 40.74a 384.94 ± 137.11a 365.33 ± 39.75a

424.08 ± 78.84a 333.42 ± 86.20a 334.75 ± 54.99a

MCHC

g/l

7 days 21 days 42 days

199.02 ± 54.36a 190.10 ± 68.49a 162.61 ± 28.29a

205.30 ± 11.32a 228.07 ± 72.75a 172.32 ± 15.49a

239.48 ± 10.24a 226.49 ± 53.63a 160.66 ± 8.80a

201.06 ± 10.06a 211.85 ± 50.78a 162.70 ± 12.88a

205.77 ± 13.31a 239.86 ± 30.75a 195.57 ± 26.44a

a

a

[19]. 5,5-Dithiobis-(2-nitrobenzoic acid) (DTNB) is a disulfide chromogen and turns to dark yellow by reducing with sulphydryl compounds. The absorbance of reduced chromogen was followed at 412 nm. GSH content (micrograms of GSH per milligram protein) was determined from a standard.

a

a

3. Results 3.1. Hematological and biochemical blood plasma parameters The hematological properties of rainbow trout exposed to CBZ are shown in Table 1. The Hb content following CBZ exposure was higher compared with the control, especially significantly increased (p < 0.05) in E3 group after 7 days exposure. The main hematological finding was that there was no significant change (p > 0.05) in Er count, PCV, MCV, MCH and MCHC in fish exposed to CBZ during the experimental period compared with the control. All indices for the DMSO group were comparable to the control group. The plasma biochemical parameters of the rainbow trout treated with CBZ are presented in Table 2. After 7 days, the NH3 and GLU

2.6. Protein estimation and statistical assays Protein levels were estimated spectrophotometrically by the method of Bradford using bovine serum albumin as a standard [20]. All values were expressed as mean ± S.D. and analyzed by SPSS for Win 13.0 software. One-way ANOVA following Tukey’s test was used to determine whether results of treatments were significantly different from the control group (p < 0.05).

Table 2 Biochemical indices of blood plasma in rainbow trout affected by chronic exposure to CBZ. Other information as in Table 1. Indices

Unit

Exposure time

Test groups Control

DMSO

GLU

mmol/l

TP

g/l

7 days 21 days 42 days 7 days 21 days 42 days

3.41 3.62 3.54 31.83 30.17 30.67

CK

␮kat/l 7 days

15.38 ± 0.26a 21 days 42 days

15.59 ± 0.18a 15.04 ± 0.41a 15.33 ± 0.43a

17.21 ± 0.16b 15.60 ± 0.45a 16.52 ± 0.36a

18.33 ± 0.28b 17.43 ± 0.61b 17.41 ± 0.43b

19.06 ± 0.18b 18.23 ± 0.58b 20.34 ± 0.66b*

19.14 ± 0.50b 22.51 ± 0.60b*

19.26 ± 0.15a 21 days 42 days

19.64 ± 0.47a 19.32 ± 1.03a 19.41 ± 0.34a

20.56 ± 0.34b 19.04 ± 0.90a 19.47 ± 0.28a

21.07 ± 0.24b 20.16 ± 0.47b 20.70 ± 0.57b

21.90 ± 0.26b 21.02 ± 0.44b 22.52 ± 0.64b*

21.63 ± 0.35b 24.52 ± 0.50b*

0.36 ± 0.02a 0.34 ± 0.11a 0.34 ± 0.07a

0.36 ± 0.02a 0.31 ± 0.11a 0.40 ± 0.20a

0.29 ± 0.03a 0.22 ± 0.09a 0.30 ± 0.17a

0.47 ± 0.07b 0.52 ± 0.06b 0.63 ± 0.13b

0.45 ± 0.29b 0.58 ± 0.11b 0.69 ± 0.09b

ALT

␮kat/l 7 days

␮kat/l

7 days 21 days 42 days

0.46a 0.40a 0.24a 1.07a 1.57a 0.75a

a

3.47 3.54 3.49 31.83 31.33 31.00

± ± ± ± ± ±

0.40a 0.43a 0.34a 1.07a 2.56a 0.58a

349.00 ± 24.21 359.67 ± 43.90a 372.50 ± 29.66a

E3

7 days 21 days 42 days

± ± ± ± ± ±

335.17 ± 23.98 344.33 ± 24.61a 339.83 ± 30.87a

E2

␮mol/l

LDH

346.67 ± 37.64 338.17 ± 26.98a 352.00 ± 28.41a

E1

NH3

a

a

3.56 4.50 5.56 32.00 32.17 30.83

± ± ± ± ± ±

0.28a 0.39b* 0.35b* 3.27a 2.41a 0.69a

422.83 ± 21.10 398.50 ± 16.83b 455.83 ± 35.30b b

4.53 4.61 6.10 35.33 29.50 30.67

± ± ± ± ± ±

0.26b 0.46b 0.19b* 3.40a 1.71a 1.37a

501.33 ± 12.09b 488.33 ± 57.60b 557.33 ± 31.56b 5.23 5.12 6.81 32.17 30.50 30.50

± ± ± ± ± ±

0.16b 0.52b 0.35b* 2.91a 1.26a 0.96a

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Fig. 1. Effect of CBZ on level of thiobarbituric acid reactive substances (TBARS) in liver of rainbow trout. Control, DMSO and experimental (nominal concentrations of 1 ␮g/l, 0.2 mg/l and 2 mg/l) groups’ values are shown as bars C, D, E1, E2 and E3, respectively. Data are means ± S.D., n = 6. Columns sharing the same superscript letter indicate no significant differences compared with control value during the same experiment time (p > 0.05). Columns with asterisk denote significant differences compared with control value in the same test group along time (p < 0.05).

levels were significantly higher (p < 0.05) in E2 and E3 groups than those in the control. And there is a significantly higher (p < 0.05) GLU content in E1 groups after 21 days compared with the control. During the whole experimental period, there was no significant change (p > 0.05) in TP level in all groups. Compared with the control, there were significant higher (p < 0.05) CK and LDH activities levels in all CBZ treated groups after 7 days, but ALT activity was significantly increased (p < 0.05) only in E2 and E3 groups after the first week. For all plasma biochemical parameters, there were no significant differences between the control and DMSO groups.

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Fig. 3. Effect of CBZ on superoxide dismutase (SOD) activity in liver of rainbow trout. Other information as in Fig. 1.

3.2.2. Enzymatic antioxidants Superoxide dismutase activity changes in the present experiment were described in Fig. 3. After 7 days, SOD activities in all groups were increased slightly, but no significant change (p > 0.05). Compared with the control, a significant higher (p < 0.05) SOD activity was observed in E3 group after 21 days exposure and in E2 group after 42 days. Fig. 4 showed CAT activities in the liver of fish after different treatments. Similar to SOD, there was a similar increasing trend of CAT activities in E2 and E3 groups after 7 days, but slightly inhibited in E1 group. After 21 days exposure, there were significant higher (p < 0.05) CAT activities in E2 and E3 groups compared with the control. Glutathione reductase activities in fish liver in this study were described in Fig. 5. After the first period of 7 days, GR activities in E2 and E3 groups were little lower than that in the control, but they were not significantly different (p > 0.05). After 21days, GR activities in E2 and E3 groups were significantly induced, but after longer exposure (42 days), there was a significant higher

3.2. Antioxidant responses 3.2.1. Oxidative stress indices Levels of lipid peroxidation levels (LPO, as measured by tissue TBARS level) and carbonyl proteins levels (CP) in liver of all groups are summarized in Figs. 1 and 2, respectively. There was no significant change (p > 0.05) in LPO level in fish liver after 7 days exposure to CBZ when compared with the control. However, after a longer-term exposure (after 21 days) this biomarker was significantly evaluated (p < 0.05) in E2 and E3 groups. Although a slight hint for an increase of CP was observed in CBZ treated groups, there was no significant induction (p > 0.05) in CP formation in any group after 7 and 21 days of exposure. A significant higher (p < 0.05) CP level was observed in E3 group after 42 days exposure when compared with the control. However, both of the oxidative stress indices in other groups had no significant change (p > 0.05) during the whole experiment.

Fig. 4. Effect of CBZ on catalase (CAT) activity in liver of rainbow trout. Other information as in Fig. 1.

Fig. 2. Effect of CBZ on level of carbonyl proteins (CP) in liver of rainbow trout. Other information as in Fig. 1.

Fig. 5. Effect of CBZ on glutathione reductase (GR) activity in liver of rainbow trout. Other information as in Fig. 1.

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tory insufficiency and also related to increases in the concentration of circulating catecholamines or corticosteroids following stress [22,26]. Activities of plasma enzymes (CK, LDH and ALT) are also used as relevant stress indicators [27]. In this study, after long-term exposure to CBZ, CK, LDH and ALT levels were significantly (p < 0.05) higher than in control. A significant increase in activity of the above mentioned enzymes indicates the changes in the histological structure of the hepatic and extrahepatic tissues [12,28]. 4.2. Antioxidant responses

Fig. 6. Effect of CBZ on glutathione peroxidase (GPx) activity in liver of rainbow trout. Other information as in Fig. 1.

GR level only in E2 group compared with the control. Fig. 6 showed GPx activities in the liver of fish in all groups during the experiment. There were significantly increase (p < 0.05) of GPx activities in E2 and E3 groups after 21 days. Otherwise, GPx activities in E3 group were decreased and not significantly different from the control after 42 days. After 42 days, all antioxidant enzymes activities in E3 group were lower than those in E2 group, but still little higher than that in the control. There was no significant change (p > 0.05) in other groups during the experiment. 3.2.3. Hepatic GSH After 42 days exposure, reduced glutathione level was decreased slightly in E1 group and significantly in E2 group, but recovered to the comparative level in E3 group, compared with the control. 4. Discussion In recent years, awareness of residual pharmaceutically active compounds (PhACs) in the aquatic environment is growing as investigations into these pollutants increase and analytical detection techniques improve [2]. Fish exposed to environmental pollutants exhibit a variety of physiological responses, including blood balance disturbances and oxidative metabolism imbalances [9]. 4.1. Hematological and biochemical blood plasma parameters Hematological and biochemical profiles of blood can provide important information about the internal environment of the organism [21]. It is notable for Hb, Er and PCV levels of blood, due to their responsibilities for transportation of nutrition, oxygen and metabolic wastes [22]. In this study, the significant high percentage of Hb in E3 group after 7 days exposure was perhaps attributable to the erythropoiesis reactivation mechanism induced by the spleen and liver to compensate for the cerebral hypoxia induced by environmental stress [11]. There were no prominent changes in red blood cell indices (MCV, MCH and MCHC) of rainbow trout in all groups. It is apparent that no cubage change in erythrocyte occurred, indicating that CBZ-induced anemia in fish is characterized as normocytic anemia, in accordance with previous results [23,24]. In this study, CBZ showed elevated levels of plasma ammonia concentration in E2 and E3 groups after 7 days, indicating that detoxifying mechanisms were unable to convert the toxic ammonia to less harmful substances, in accordance with results for O. mykiss exposed to deltamethrin [25]. Moreover, in this study, significantly higher levels of blood GLU were observed in fish of all CBZ treated groups after 7 days than in controls, indicating metabolic stress. Increased plasma GLU levels maybe a response to respira-

Because of potential utility to provide biochemical biomarkers in environmental monitoring system, the antioxidant defense system is being increasingly studied [29–31]. Fish respond to exposure to pollutants by altering or adapting their metabolic functions. The efficiency of antioxidant defense system may be increased or inhibited under chemical stress depending on the intensity and the duration of the stress applied as well as the susceptibility of the exposed species [31]. It is not a general rule that an increase in xenobiotic concentrations induces antioxidant activities [32]. The results of this study show that long-term exposure of fish to CBZ resulted in oxidative stress and affected the enzymatic and nonenzymatic antioxidants in fish. The antioxidant enzymes can be induced by a low intensity oxidative stress; however, a relatively severe oxidative stress (E3 groups after 42 days) suppresses the activities of these enzymes due to oxidative damage and a loss in compensatory mechanisms. Lipid peroxidation (LPO) has been reported to be a major contributor to the loss of cell function under oxidative stress [33] and has usually been indicated by TBARS in fish [34]. Our results showed that long-term exposure to CBZ led to oxidative stress, with significantly higher LPO in E2 and E3 groups after 21 days, when compared to the control group. The higher hydroperoxide lipid production suggests that ROS-induced oxidative damage can be a major toxic effect of CBZ. It has been reported that LPO may be induced by a variety of environmental pollutants [8,35–37]. Reactive oxygen species directly attack protein and catalyzed the formation of carbonyl [38,39]. The formation of carbonyl proteins is non-reversible, causing conformational changes, decreased catalytic activity in enzymes and ultimately resulting, owing to increased susceptibility to protease action, in breakdown of proteins by proteases [40]. We observed that there were no significant changes in CP levels in all groups before 21 days, but a significantly higher CP levels in E3 group after 42 days, which maybe a result of over-accumulated ROS. Oxidative substances in cells may cause an elevation of antioxidant enzymes as a defense mechanism. SOD catalyzes the dismutation from superoxide radical anion to H2 O and H2 O2 [41,42]. CAT and GPx act cooperatively as scavengers of hydrogen peroxidase (CAT and GPx) and other hydroperoxides (GPx) [43]. Through inhibitory effects on oxyradical formation, the SOD–CAT system provides the first line of defense against oxygen toxicity and is used as a biomarker indicating ROS production [44,45]. Glutathione reductase catalysis the reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH) in a NADPH-dependent reaction [46,47]. In this study, although there were no significant changes, SOD, CAT and GPx activities in all groups were increased after 7 days, which indicates the generation of ROS. However, decreased GR activity in E2 and E3 groups after 7 days could be due to the availability of NADPH in the cell. After long-term exposure (after 21 days), all antioxidant enzymes (except SOD in E2 group, which was also induced, but not significantly) activities were significantly induced in E2 and E3 groups compared with the control, which are the adaptive responses to the oxidative stress. All enzymatic antioxidant activities of E3 group showed a decreasing trend

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after 42 days exposure. However, they were still higher than control group, which indicate the accumulation of ROS but not enough to make enzymes poisoned. Reduced glutathione is the major cytosolic low molecular weight sulfhydryl compound that acts as a cellular reducing and a protective reagent against numerous toxic substances [48]. Hence, GSH is often the first line of defense against oxidative stress. Under oxidative stress conditions, ROS are reduced by conjugation with GSH directly or by means of GSH-related enzymes, which decrease GSH levels [5,6]. In this study, long-term (42 days) exposure to CBZinduced a decrease in hepatic GSH, especially significant decrease in E2 group compared with the control, probably by the result of impaired GSH synthesis. 5. Conclusion In summary, chronic exposure to CBZ caused both physiological and biochemical parameters effects in rainbow trout. In this study, there was no significant change of hepatic antioxidant status and most of hematological parameters in fish exposed to CBZ at environmental concentration (E1 group), indicating the adaptive responses to environmental stress. Based on the obtained data, the trout fish, O. mykiss, has enough tolerances to CBZ-induced changes in surrounding condition. With increasing CBZ concentration (E2, E3 groups), hematological profiles and blood biochemical indices were affected, especially hemoglobin, ammonia and glucose contents and plasmatic enzymes activities. Meanwhile, enzymatic (SOD, CAT, GR and GPx) and non-enzymatic (GSH) antioxidants in fish liver showed their protective functions significantly when oxidative stress occurred. According to results of this present study, the hematological indices and antioxidant responses could provide useful parameters for evaluating the physiological effects on rainbow trout, but the application of these findings will need more detailed laboratory study before they can be established as special biomarkers for monitoring aquatic environment. Besides, other classical morphologic indices (e.g. condition factor and hepatosomatic index) in fish could provide useful information for evaluating environmental stress, which should be given more attention in the future. Furthermore, it is not clear that whether these CBZ-induced responses in fish were related with the level of stress hormone (especially catecholamines and cortisol), which also need to be further investigated. Conflicts of Interest None. Acknowledgements This study was supported by the USB RIFCH no. MSM 6007665809 and the Ministry of the Environment of the Czech Republic SP/2e7/229/07. References [1] J.P. Bound, N. Voulvoulis, Pharmaceuticals in the aquatic environment-A comparison of risk assessment strategies, Chemosphere 56 (2004) 1143–1155. [2] K. Fent, A.A. Weston, D. Caminada, Ecotoxicology of human pharmaceuticals, Aquat. Toxicol. 76 (2006) 122–159. [3] M. Clara, B. Strenn, N. Kreuzinger, Carbamazepine as a possible anthropogenic marker in the aquatic environment: investigations on the behaviour of carbamazepine in wastewater treatment and during groundwater infiltration, Water Res. 38 (2004) 947–954. [4] H. Sanderson, R.A. Brain, D.J. Johnson, C.J. Wilson, K.R. Solomon, Toxicity classification and evaluation of four pharmaceuticals classes: antibiotics, antineoplastics, cardiovascular, and sex hormones, Toxicology 203 (2004) 27–40. [5] J.F. Zhang, H. Shen, X.R. Wang, J.C. Wu, Y.Q. Xue, Effects of chronic exposure of 2,4-dichlorophenol on the antioxidant system in liver of freshwater fish Carassius auratus, Chemosphere 55 (2004) 167–174.

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