Oxidative stress, neurotoxicity, and non-specific immune responses in juvenile red sea bream, Pagrus major, exposed to different waterborne selenium concentrations

Chemosphere 135 (2015) 46–52

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Oxidative stress, neurotoxicity, and non-specific immune responses in juvenile red sea bream, Pagrus major, exposed to different waterborne selenium concentrations Jun-Hwan Kim, Ju-Chan Kang ⇑ Department of Aquatic Life Medicine, Pukyong National University, Busan 608-737, Republic of Korea

h i g h l i g h t s  The antioxidant enzymes of Pagrus major were considerably increase by Se exposure.  The AChE activity was significantly inhibited by Se exposure.  The significant changes were observed in non-specific immune parameters.

a r t i c l e

i n f o

Article history: Received 14 May 2014 Received in revised form 24 March 2015 Accepted 26 March 2015

Handling Editor: Jim Lazorchak Keywords: Pagrus major Selenium Oxidative stress Neurotoxicity Non-specific immune response

a b s t r a c t Juvenile Pagrus major (mean length 15.8 ± 1.6 cm, and mean weight 90.4 ± 4.7 g) were exposed for 4 weeks with waterborne selenium concentration (0, 50, 100, 200, and 400 lg L1). In oxidative stress indicators, liver and gill superoxide dismutase (SOD) activity and glutathione S-transferase (GST) activity were markedly elevated after 4 weeks exposure. Similarly, glutathione (GSH) level in liver and gill was also increased in response to the highest Se exposure after 4 weeks exposure. In neurotoxicity, AChE activity was inhibited in brain and muscle tissues by waterborne Se exposure. In the non-specific immune responses, lysozyme activity of plasma and kidney was significantly increased by waterborne Se exposure. Peroxidase activity and anti-protease activity were decreased at high Se concentration. The results suggest that waterborne Se exposure can induce significant oxidative stress, inhibition of AChE activity, and immunological alterations. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Selenium is a naturally existent element which is found in different regions of the natural world such as the rocks and soils. But, the high level of selenium concentration in the aquatic environment can be reached by aquacultural drainwater, sewage sludge, coal-fired power plants, and metal ores. Selenium can be highly toxic to fish and wildlife at the higher concentration than a permissible amount, because it can rapidly accumulate and reach toxic level in the aquatic environment (Lemly, 2002). Selenium exposure at the high concentration to fish generates superoxide and oxidative stress (Spallholz and Hoffman, 2002), though moderate selenium concentrations for organisms help improve the immune system with the action to protect neutrophils from oxygen-derived radicals (Arthur et al., 2003). Oxidative stress has ⇑ Corresponding author. Tel.: +82 51 629 5944; fax: +82 51 629 5938. E-mail address: [email protected] (J.-C. Kang). http://dx.doi.org/10.1016/j.chemosphere.2015.03.062 0045-6535/Ó 2015 Elsevier Ltd. All rights reserved.

become a critical issue for aquatic toxicology (Orun et al., 2008). Oxidative stress is highly associated with toxic substances, and antioxidant activities occur against oxidative stress within intracellular space (Ates et al., 2008; Talas et al., 2008). Painter (1941) reported that the selenite toxicity is related to the oxidation of endogenous thiols. More recently, it has been suggested that selenium-mediated thiol oxidation cause reactive oxygen species (ROS) (Stewart et al., 1998; Spallholz et al., 2004; Chen et al., 2007; Plano et al., 2010), and oxidative stress can be a factor related to selenium-induced toxicity (Kitahara et al., 1993; Shen et al., 2001; Kim et al., 2007; Xiang et al., 2009). Aerobic organisms require mechanisms that prevent or limit cellular damage which is caused by reactive oxygen species (ROS), and cells have evolved an interdependent antioxidant defense system (Cavaletto et al., 2002). Superoxide dismutase (SOD) decomposes superoxide anion to hydrogen peroxide, and catalase decomposes H2O2 to molecular oxygen and water, and glutathione peroxidases reduce both H2O2 and lipid hydroperoxide (Almeida et al., 2007). Glutathione (GSH)

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is the most plentiful intracellular thiol-based antioxidant, and function as a sulfhydryl buffer. It also has the function of detoxifying compounds via conjugation reactions catalyzed by glutathione S-transferases (Nordberg and Arnér, 2001). Glutathione S-transferase (GST) functions the detoxification enzyme existing all aerobic organisms, and catalyze the nucleophilic attack of the sulfur atom of the tripeptide glutathione (Kim et al., 2001). Selenium at high concentration can also lead to neuronal damage as neurotoxicity (Painter et al., 1996), whereas it has neuroprotective effects against the exposure to neurotoxic chemicals (Imam et al., 2001; Zafar et al., 2003). Acetylcholine plays a major role in both central and peripheral nervous system as one of the most important neurotransmitter. Acetylcholine functions as activator of muscles in the peripheral nervous system and has a major role in the enhancement of sensory perceptions in the central nervous system. Acetylcholinesterase (AChE) is a principal component of cholinergic system in fish and control the nervous impulse transmission in cholinergic synapses. Considerably high enzyme activity of AChE occurs in central nervous system of fish especially in brain. A significant reduction in acetylcholinesterase activity is commonly observed in fish exposed to various toxic substances such as organophosphorous compounds, metals, and chemicals (Modesto and Marinez, 2010). Therefore, the inhibition of acetylcholinesterase can be a biomarker for the neurotoxicity (Manzo et al., 1995). The fish immune parameters can be affected by aquatic toxicants such as pesticides, hydrocarbons, metals, and other chemicals. Many proofs of disrupted immune function in fish exposed to contaminated coastal waters have been reported by several field studies (Arkoosh et al., 1991; Hutchinson et al., 2003; Pulsford et al., 1995). In addition, excess selenium dose affects immune function in conjunction with oxidative stress (Fairbrother and Fowles, 1990; Fairbrother et al., 1994). Among non-specific immune response, lysozyme is a crucial factor in fish, which acts as a bacteriolytic agent against microbial invasion in fish (Yousif et al., 1994). Lysozyme activity in fish is altered by health condition, stress, sex, temperature, and aquatic toxicants (Balfry and Iwama, 2004; Saurabh and Sahoo, 2008). Especially, exposure to aquatic toxicants such as metals regulates lysozyme level. The peroxidase activity can be considered an immunological parameter as an indicator of leucocyte activation (Tapia-Paniagua et al., 2011). Anti-proteases are one of the elements of non-specific immunity of the vertebrates (Ellis, 2001). Therefore, the level of these parameters in fish is an appropriate indicator for measuring the potential influence of environmental hazards on fish innate immunity (Saurabh and Sahoo, 2008). Selenium has a narrative scope between its toxic and its beneficial effects (Frost and Olson, 1972; Shamberger, 1981; Rayman, 2008; Vinceti et al., 2009; Valdiglesias et al., 2010). But, the study about selenium toxicity has not been sufficiently conducted. Therefore, the aim of the present study was to evaluate the effect of waterborne selenium exposure to the Pagrus major on the oxidative stress, neurotoxicity, and non-specific immune responses.

2. Materials and methods 2.1. Experimental fish and conditions Red sea breams were obtained from a local fish farm in Tongyeong, Korea. The fish were acclimatized for 2 weeks under laboratory conditions was evaluated prior to selenium exposure. During the acclimation period, the fish were fed a commercial diet twice daily (Woosungfeed, Daejeon City, Korea) and maintained on a 12-h:12-h light/dark cycle and constant condition at all times

Table 1 The chemical components of seawater and experimental condition used in the experiments. Item

Value

Temperature (°C) pH Salinity (‰) Dissolved oxygen (mg L1) Chemical oxygen demand (mg L1) Ammonia (lg L1) Nitrite (lg L1) Nitrate (lg L1)

21.0 ± 1.0 8.1 ± 0.5 33.5 ± 0.6 7.1 ± 0.3 1.13 ± 0.1 12.5 ± 0.7 1.3 ± 0.3 11.48 ± 1.0

(Table 1). After acclimatization, 120 fishes (body length, 15.8 ± 1.6 cm; body weight, 90.4 ± 4.7 g) were selected for the study. Selenium exposure took place in 20 L glass tanks containing 6 fish per treatment group in triplicates. Sodium selenite (Sigma, St. Louis, MO, USA) solution was dissolved in the respective glass tanks. The selenium concentrations in the glass tanks were 0, 50, 100, 200, and 400 lg L1. An extremely high dose of 400 lg L1 is a highly improbable occurrence in a real environment, but it provided an opportunity to evaluate selenium toxicity in the experimental fish. The glass tank water was thoroughly exchanged once per two days, and made the same concentration in the respective glass tank. At the end of each period (at 2 and 4 weeks), fish were anesthetized in buffered 3-aminobenzoic acid ethyl ester methanesulfonate (Sigma Chemical, St. Louis, MO). 2.2. Antioxidant enzyme analysis Liver and gill tissues were excised and homogenized with 10 volumes of ice-cold homogenization buffer using Teflon-glass homogenizer (099CK4424, Glass-Col, Germany). The homogenate was centrifuged at 10,000 g for 30 min under refrigeration and the obtained supernatants were stored at 80 °C for analysis. Superoxide dismutase (SOD) activity was measured with 50% inhibitor rate about the reduction reaction of WST-1 using SOD Assay kit (Dojindo Molecular Technologies, Inc.). One unit of SOD is defined as the amount of the enzyme in 20 lL of sample solution that inhibits the reduction reaction of WST-1 with superoxide anion by 50%. SOD activity was expressed as unit mg protein1. * WST-1 = 2-(4-lodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt. Glutathione-S-transferase (GST) activity was measured according to the method of modified Habig (1974). The reaction mixture consisted of 0.2 M phosphate buffer (pH 6.5), 10 mM GSH (Sigma) and 10 mM 1-chloro-2,-dinitrobenzene, CDNB (Sigma). The change in absorbance at 25 °C was recorded at 340 nm and the enzyme activity was calculated as 340 nm and the enzyme activity was calculated as nmol min1 mg protein1. Reduced glutathione was measured following the method. Briefly, 0.2 ml fresh supernatant was added to 1.8 ml distilled water. Three ml of the precipitating solution (1.67 g metaphosphoric acid, 0.2 g EDTA and 30 g NaCl in 100 ml distilled water) was mixed with supernatants. The mixture was centrifuged at 4500 g for 10 min. 1.0 mL of supernatant was added to 4.0 ml of 0.3 M NaHPO4 solution and 0.5 mL DTNB (5,50 -dithiobis-2-nitrobenzoic acid) was then added to this solution. Reduced glutathione was measured as the difference in the absorbance values of samples in the presence and the absence of DTNB at 412 nm. GSH value was calculated as lmol mg protein1 in the tissues. 2.3. Inhibition of AChE activity AChE activity was determined brain (1:25) and muscle (1:10) homogenate in 0.1 M phosphate buffer, pH 8.0. The homogenate

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were centrifuged 10 000g for 20 min at 4 °C. The supernatant was removed and used to test AChE activity. AChE activity was determined according to the method of Ellman et al. (1961). AChE activity was normalized to protein content and expressed as nmol min1 mg protein1. Briefly, the activity on the homogenate was measured by determining the rate of hydrolysis of acetylthiocholine iodide (ACSCh, 0.88 mM) in final volume of 300 lL, with 33 lL of 0.1 M phosphate buffer, pH 7.5 and 2 mM DTNB. The reaction was started with the addition of the substrate acetylthiocholine, as soon as the substrate was added the hydrolysis and the formation of the dianion of DTNB were analyzed in 412 nm for 5 min (in intervals of 1 min) using a microplate reader. Protein concentration was determined using Bradford’s method (1976), with a bovine plasma albumin (Sigma, USA) as standard.

2.4. Non-specific immune responses The plasma for analysis was separated from the blood sample. Kidney tissues were excised and homogenized with 10 volumes of ice-cold homogenization buffer (0.004 M phosphate buffer, pH 6.6) using Teflon-glass homogenizer (099CK4424, Glass-Col, Germany). The homogenate was centrifuged at 10 000g for 10 min under refrigeration and the obtained supernatant was stored at 70 °C (MDF-U53V, SANYO Electric Co. Ltd., Japan) for analysis. Protein content was determined by the Bio-Rad Protein Assay Kit (Bio-Rad Laboratories GmbH, Munich, Germany) based on the Bradford dye-binding procedure, using bovine serum albumin as standard. Lysozyme concentration was calculated through the measure of its enzyme activity. Lysozyme activity was determined by a turbidimetric method (Ellis, 1990) using Micrococcus lysodeikticus (Sigma) as substrate (0.2 mg mL1 0.05 M phosphate buffer, pH 6.6 for kidney sample and pH 7.4 for plasma). A standard curve was made with lyophilized hen egg white lysozyme (sigma) and the rate of change in turbidity was measured at 0.5-min and 4.5-min intervals at 530 nm. The results were expressed as lg mL1 and lg g1 equivalent of hen egg white lysozyme activity. The peroxidase activity was measured as an indicator of leukocyte activation by a colorimetric method (Quade MJ, Roth JA, 1997). Briefly, 5 lL of plasma were diluted with 50 lL of HBSS (Hank’s Balanced Salt Solution) in flat-bottomed 96-well plates. Plasma samples were mixed with the peroxidase substrate (80 lM 3,30 ,5,50 -tetramethylbenzidine hydrochloride (TMB; Sigma) and 2.5 mM H2O2). The color-change reaction was stopped after 2 min by adding 50 lL of 2 M sulphuric acid and the optical density was read at 450 nm in a plate reader (BMG, Fluoro Star Galaxy). Standard sample without plasma was used as blank. The peroxidase activity was determined defining as one unit the peroxidase that produces an absorbance change of 1 OD. A modification of the method described by Ellis (1990) was used (Magnadóttir et al., 1999) to investigate anti-protease activity. Briefly, 20 lL of plasma were incubated with the same volume of standard trypsin solution (Sigma T-7409, 1000–2000 BAEE) for 10 min at 22 °C. To this, 200 lL of 0.1 M phosphate buffer, pH 7.0 and 250 lL 2% azocasein (Sigma A-2765) were added and incubated for 1 h at 22 °C. Then, 500 lL of 10% trichloro acetic acid (TCA) was added and incubated for 30 min at 22 °C. The mixture was centrifuged at 6000g for 5 min. One hundred microliters of the supernatant was transferred to a 96 well non-absorbent microtray (Nunc) containing 100 lL well1 of 1 N NaOH. The OD was lead at 430 nm. The blank was phosphate buffer in place of plasma and trypsin and the reference sample was phosphate buffer in place of plasma. The percentage inhibition of trypsin activity compared to the reference sample was then calculated for each plasma sample.

2.5. Statistical analysis The experiment was conducted in two exposure periods (2 weeks and 4 weeks) and performed triplicate. Statistical analyses were performed using the SPSS/PC+ statistical package (SPSS Inc, Chicago, IL, USA). Significant differences between groups were identified using one-way ANOVA and Duncan’s test for multiple comparisons or Student’s t-test for two groups (Duncan, 1955). The significance level was set at P < 0.05.

3. Results 3.1. Antioxidant enzyme analysis Antioxidant enzyme analysis (SOD and GST) and GSH in liver and gill tissues of P. major is shown in Fig. 1. Liver and gill SOD activity of the P. major were substantially increased. The liver SOD activity was notably increased over 200 lg L1 of waterborne Se exposure at 2 weeks and over 100 lg L1 of waterborne Se exposure at 4 weeks. In case of gill SOD activity, a considerable increase was observed over 50 lg L1 of waterborne Se exposure at 2 weeks and over 100 lg L1 of waterborne Se exposure at 4 weeks. Liver and gill GST activity of the P. major were also considerably increased. The liver GST activity was notably increased at 400 lg L1 of waterborne Se exposure at 2 weeks and over 200 lg L1 of waterborne Se exposure at 4 weeks. The gill GST activity was increased over 200 lg L1 of waterborne Se exposure at 2 weeks and over 200 lg L1 of waterborne Se exposure at 4 weeks. Similarly, liver and gill GSH level were considerably increased. The liver GSH was increased at 400 lg L1 of waterborne Se exposure both 2 weeks and 4 weeks. In case of gill GSH, a notable increase was observed at 400 lg L1 of waterborne Se exposure at 2 weeks and 4 weeks. 3.2. Inhibition of AChE activity AChE activities of brain and muscle tissues exposed to waterborne selenium are shown in Fig. 2. AChE activity in brain tissue was noticeably inhibited in the concentration over 200 lg L1 at 2 weeks and 4 weeks, compared to control, 50, and 100 lg L1 groups. Brain AChE inhibition levels were 23% at 400 lg L1 after 2 weeks and 32% at 400 lg L1 after 4 weeks. In muscle tissue, AChE activity was inhibited at 400 lg L1 after 2 weeks and 4 weeks, whereas there was no considerable change between control and a concentration of 200 lg L1. Muscle AChE inhibition levels were 21% at 400 lg L1 after 2 weeks and 25% at 400 lg L1 after 4 weeks, respectively. 3.3. Non-specific immune response Plasma and kidney lysozyme concentrations of the P. major were markedly increased (Table 2). In plasma lysozyme activity, a significant increase was observed at 400 lg L1 of waterborne Se exposure after 2 weeks and at 100 and 400 lg L1 of waterborne Se exposure after 4 weeks. Kidney lysozyme activity was also significantly increased over 200 lg L1 of waterborne Se exposure at 2 weeks and over 100 lg L1 of waterborne Se exposure at 4 weeks. In contrast with lysozyme activity, peroxidase and antiprotease activity were decreased at high waterborne Se exposure (Table 3). Peroxidase was significantly decreased at 400 lg L1 of waterborne Se exposure after 2 weeks and 4 weeks. Anti-protease activity also considerably decreased at 400 lg L1 of waterborne Se exposure after 2 weeks and over 200 lg L1 of waterborne Se exposure after 4 weeks.

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500

Control 50µg/L 100µg/L 200µg/L 400µg/L

Liver bc c b

c

400

b

a a

300

a a

100

SOD activity (unit/ mg protein)

SOD activity ( unit/ mg protein)

600

a

200 100 0

80

Control 50µg/L 100µg/L 200µg/L 400µg/L

60

c c

40

20

2

4

12

c

b a

Liver

b

a

ab a ab

a a

10 8 6 4 2

GST activity (nmol/ min/ mg protein)

GST activity (nmol/ min/ mg protein)

14

0 2

18 16 14 12

Gill

Control 50µg/L 100µg/L 200µg/L 400µg/L

b

a

10

ab ab

a a

a

4 2 0 2

4

Weeks 2.5

Control 50µg/L 100µg/L 200µg/L 400µg/L

Liver b

b ab a a a

a a a a

2

1

0

GSH level (µmol GSH/ mg protein)

GSH level (µmol GSH/ mg protein)

b

6

4

5

3

b

b

8

Weeks

4

4

Weeks

18 Control 50µg/L 100µg/L 200µg/L 400µg/L

bc

a

a

Weeks

16

ab

b b bc

0 2

Gill

bc

2.0

1.5

Control 50µg/L 100µg/L 200µg/L 400µg/L

Gill b

b a

a a a

a

a ab a

1.0

0.5

0.0 2

4

Weeks

2

4

Weeks

Fig. 1. Changes of antioxidant enzyme (SOD activity, GST activity, and GSH level in liver and gill) of red sea bream, P. major exposed to the different concentration of selenium. Vertical bar denotes a standard error. Values with different superscript are significantly different (P < 0.05) as determined by Duncan’s multiple range test.

4. Discussion The hyper-accumulated selenium by high exposure of selenium generated reactive oxygen species (ROS) which was one of the important mechanisms for Se toxicity (Spallholz et al., 2004). Superoxide anions are generally dismutated by superoxide dismutase (SOD) to H2O2, which is the first defense mechanism against oxygen toxicity. In general, many studies reported that SOD activity is increased by the exposure to toxicant substances such as metals. Misra and Niyogi (2009) also reported the SOD activity increase of rainbow trout (Oncorhynchus mykiss) exposed to 50 and 100 lM of sodium selenite. In this study, SOD activity both in liver and gill of P. major was significantly increased by increasing selenium exposure concentration. Therefore, the notable increase in liver and gill of P. major may be a defense mechanism in

response to oxidative stress caused by waterborne Se. Glutathion S-transferases (GST) functions a main role in detoxification of deleterious electrophilic xenobiotics such as environmental toxicants (Keen and Jakoby, 1978). Mari (2001) suggested that oxidative stress can increase GST, which is a defense mechanism against oxidative stress or cellular damage. Basha and Rani (2003) observed a significant increase in GST of liver and kidney tissues of tilapia (Oreochromis mossambicus) exposed to waterborne Cd. Similarly, GST activity both in liver and gill of P. major was increased with the waterborne Se dose-dependent. The increase in GST activity of liver and gill should be expected due to organic xenobiotic detoxification. Glutathione (GSH) convert selenite into hydrogen selenide, which may then react with oxygen to generate ROS (Seko et al., 1989), and it induces oxidative damage when not eliminated by antioxidants (Miller, 2006; Misra

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40

Control 50µg/L 100µg/L 200µg/L 400µg/L

a

30

Brain a

a

ab

AChE (nmol/ min/ mg protein)

AChE (nmol/ min/ mg protein)

50

abab

b b

bc

30

c

20

10

25 20

a a

a a a

Muscle a

ab

ab b

b

15 10 5

0 2

Control 50µg/L 100µg/L 200µg/L 400µg/L

0

4

2

Weeks

4

Weeks

Fig. 2. Changes of AChE inhibition in brain and muscle of red sea bream, P. major exposed to the different concentration of selenium. Vertical bar denotes a standard error. Values with different superscript are significantly different (P < 0.05) as determined by Duncan’s multiple range test.

Table 2 Changes of lysozyme concentrations (plasma and kidney) in red sea bream, P. major exposed to selenium for 4 weeks. Parameters

Period (week)

Selenium concentration (lg L1) 0

50

100

200

Plasma (lg mL1)

2 4

4.21 ± 0.46a 4.26 ± 0.79a

4.53 ± 0.58a 5.15 ± 0.83ab

5.53 ± 0.75ab 5.61 ± 0.67b

5.69 ± 0.58ab 5.33 ± 0.49ab

Kidney (lg g1)

2 4

56.6 ± 6.2a 55.3 ± 9.7a

62.7 ± 5.3a 65.5 ± 8.5ab

68.5 ± 7.4a 73.5 ± 5.6b

85.8 ± 8.1b 90.5 ± 10.6c

400 6.78 ± 0.64b 6.94 ± 0.52c 94.5 ± 6.2b 102.5 ± 8.4c

Values are mean ± S.E. Values with different superscript are significantly different at 2 weeks and 4 weeks (P < 0.05) as determined by Duncan’s multiple range test.

Table 3 Changes of peroxidase activity and Anti-protease activity in red sea bream, P. major exposed to selenium for 4 weeks. Parameters

Period (week)

Selenium concentration (lg L1) 0

50

100

200

Peroxidase activity (U mL1)

2 4

5.85 ± 0.38a 5.71 ± 0.56a

5.49 ± 0.57a 5.39 ± 0.36a

5.53 ± 0.40a 5.54 ± 0.33a

5.38 ± 0.56a 5.11 ± 0.45a

Anti-protease activity (% inhibition)

2 4

36.6 ± 1.7a 36.7 ± 2.0a

36.1 ± 2.2a 35.9 ± 1.5a

35.8 ± 1.5a 36.2 ± 1.1a

34.5 ± 2.3ab 32.1 ± 2.1b

400 4.42 ± 0.44b 4.24 ± 0.38b 31.52 ± 1.2b 30.9 ± 1.8b

Values are mean ± S.E. Values with different superscript are significantly different at 2 weeks and 4 weeks (P < 0.05) as determined by Duncan’s multiple range test.

and Niyogi, 2009). GSH induces ROS production by selenite, oxidative damage by Se toxicity causes both depleted GSH and elevated GSH levels (Shen et al., 2000). Miller et al. (2007) also reported the depleted GSH in juvenile rainbow trout exposed to waterborne selenite. In contrast, a significant increase in GSH was reported in medake (Oryzias latipes) exposed to waterborne Nano-Se of 100 lg Se/L (Li et al., 2008). GSH level both in liver and gill of P. major was increased by increasing selenium exposure concentration. The alterations in GSH level of P. major may result in oxidative damage by waterborne Se. These results indicate that waterborne Se exposure to P. major significantly affects antioxidant enzymes as oxidative stress. The increase of ROS induces apoptotic cell death (Risso-de Faverney et al., 2001; Krumschnabel et al., 2005), and it may cause neurodegenerative and immune disorders in human (Franco et al., 2009). Acetylcholine is one of the most important neurotransmitter in either central or peripheral nervous system, and the inhibition of acetylcholinesterase has been proposed as a biomarker of the neurotoxicity (Manzo et al., 1995). A significant reduction in acetylcholinesterase activity is commonly observed in fish exposed to toxic substances (Modesto and Marinez, 2010), and the accumulation of acetylcholine according to inhibition of AChE activity may influence the fleeing and reproductive behavior of fish

(Bretaud et al., 2000). Jebali et al. (2006) reported significant AChE decrease of Seriola dumerilli exposed to cadmium. Similarly, Devi and Fingerman (1995) observed that significant inhibition of AChE activity in red swamp crayfish, Procambarus clarkii occur by the exposure of the waterborne mercury, cadmium, and lead. In this study, the inhibition of considerable AChE activity in brain and muscle of P. major exposed to waterborne Se was observed. Higher brain AChE activity was also observed compared to that of muscle, which is similar with previous study (Straus and Chambers, 1995). Considering the notable inhibition of AChE activity, waterborne Se exposure may substantially affect P. major as neurotoxicity. The immune system can provide potential biomarkers that may be sensitive enough to assess environmental contamination (Bainy and Marques, 2003). Among non-specific immune parameters, lysozyme is an important parameter in the immune defense. The level of lysozyme in fish is a useful parameter to monitor the potential influence of environmental hazards on fish innate immunity (Saurabh and Sahoo, 2008). Exposure to toxic substances such as heavy metals modulates lysozyme levels causing alterations in immuno-regulatory functions (Bols et al., 2001). The bactericidal ability of lysozyme has obviously illustrated its function of defense system in fish (Grinde et al., 1988). Therefore, a higher lysozyme

J.-H. Kim, J.-C. Kang / Chemosphere 135 (2015) 46–52

activity has been commonly observed in diseased fish (Studnicka and Siwicki, 1990). Low and Sin (1998) reported increased kidney lysozyme activity exposed to waterborne mercury in blue gourami, Trichogaster trichopterus. Secombes et al. (1997) also observed elevated lysozyme levels in plaice exposed to sewage sludge. Similarly, waterborne Se exposure significantly increased the plasma and kidney lysozyme activity of P. major. The considerable increases of lysozyme activity may result from an immunological stimulation by waterborne Se exposure to P. major. The peroxidase is a major enzyme which uses oxidative radicals to produce hypochlorous acid to kill pathogens. Pipe et al. (1999) reported that reduction in peroxidase activity by the waterborne copper exposure of 0.5 ppm to marine mussel, Mytilus edulis. The peroxidase activity exposed to waterborne Se of P. major was notably decreased at the high Se exposure concentration, which implies that waterborne Se may affect the immunomodulation of P. major. Anti-proteases are one of important innate defense parameter in the non-specific immunity components of the vertebrates. Fish plasma contains a number of protease inhibitors which may play an important role in inhibiting the ability of bacteria to invade and grow (Ellis, 2001). Protease inhibitors play a role in delaying or inhibiting pathogen that produce toxic proteases (Magnadóttir et al., 1999). Thilagam et al. (2009) reported the decreased level of protease inhibitors in Japanese sea bass exposed to 17b-estradiol. The waterborne Se exposure at high concentrations considerably affected the experimental fish, P. major. The notable decrease at high concentrations demonstrated that waterborne Se exposure may affect the regulation ability to hydrolyze protein in vivo of the immune defense system. In conclusion, the results of the present study showed that the waterborne Se to P. major induces the considerable increase in antioxidant enzymes (SOD and GST) and GSH. The waterborne Se exposure also inhibited AChE activity in brain and muscle tissues of P. major. In addition, our results demonstrated that waterborne Se exposure in excess of permissible Se concentration considerably modulates the immune responses of P. major such as lysozyme, peroxidase, and anti-protease activity, because the majority of the parameters reflecting humoral responses were affected. Considering the results of the notable alterations in antioxidant enzymes, inhibition of AChE activity, and non-specific immune parameters, the selenium exposure should adversely influence the experimental fish, P. major. References

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