Ecotoxicology and Environmental Safety 114 (2015) 44–51

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The detoxification process, bioaccumulation and damage effect in juvenile white shrimp Litopenaeus vannamei exposed to chrysene Xianyun Ren, Luqing Pan n, Lin Wang Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao 266003, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 21 September 2014 Received in revised form 31 December 2014 Accepted 7 January 2015

This study aimed to evaluate the effect of chrysene (CHR) on detoxification enzymes, bioaccumulation and effect of CHR on biomolecule damage in different organs of the juvenile white shrimp Litopenaeus vannamei. In this study, juvenile white shrimp L. vannamei were exposed to CHR for 21 days at four different concentrations as 0, 0.3, 2.1 and 14.7 μg/L. Results showed that CHR bioaccumulation increased rapidly at first then reached a plateau. The activities of aryl hydrocarbon hydroxylase (AHH), 7-ethoxyresorufin O-deethylase (EROD), epoxide hydrolase (EH), glutathione-S-transferase (GST), sulfotransferase (SULT) and uridinediphosphate glucuronyltransferase (UGT) were induced and then became stable gradually. Moreover, 2.1 and 14.7 μg/L CHR treatments increased activity of superoxide dismutase (SOD) in gills and hepatopancreas, while total antioxidant capacity (T-AOC) and GSH/GSSG were suppressed after CHR exposure. Additionally, lipid peroxidation (LPO) levels, protein carbonyl (PC) contents and DNA damage were induced throughout the exposure period, and different trends were detected with time of exposure. Overall, these novel findings of CHR bioaccumulation and resulted toxicity demonstrate that CHR could affect the physical status of L. vannamei. This study will form a solid basis for a realistic extrapolation scientific data for aquaculture water monitoring and food security. & 2015 Elsevier Inc. All rights reserved.

Keywords: Detoxification enzymes Litopenaeus vannamei Chrysene Damage effect Bioaccumalation

1. Introduction Polycyclic Aromatic Hydrocarbons (PAHs) are persistent hydrophobic organic pollutants ubiquitously found in the environment and they originate mainly from incomplete combustion of organic materials and fossil fuels. As a kind of global persistent organic pollutants (POPs), PAHs have also been included in the group of persistent toxic substances (PTS), and 16 unsubstituted PAHs have been listed as “Priority Pollutants” by the United States Environment Protection Agency (US EPA), due to their well-known carcinogenic and mutagenic properties (Chung et al., 2007). In 2008, the European Food Safety Authority (EFSA) introduced a system of four specific PAHs, namely, benzo[a]anthracene (BaA), benzo[a]pyrene (BaP), benzo[b]fluoranthene (BbF) and chrysene (CHR), assessing that the sum of the four PAH compounds was the most suitable indicator for PAHs in food (EFSA, 2008). In recent years, PAH pollution has been much aggravated by the development of offshore oil and marine traffic, making the concentrations of CHR in the marine environment increasingly higher (Xiu et al., 2014) According to a survey, the CHR contents in surface sediment n Correspondence to: Fisheries College, Ocean University of China, Yushan Road 5, Qingdao 266003, China. Fax: þ 86 532 82032963. E-mail address: [email protected] (L. Pan).

http://dx.doi.org/10.1016/j.ecoenv.2015.01.004 0147-6513/& 2015 Elsevier Inc. All rights reserved.

from Zhanjiang Bay, China, was up to 39.65 ng/g dry weight (Huang et al., 2012). To the best of our knowledge, the mechanisms of PAHs toxicity to crustaceans have been rarely studied. while the mechanisms of PAHs toxicity concentrated in fishes and bivalves in aquatic animals (Pan et al., 2006; Yin et al., 2007; Liu et al., 2014). The biotransformation of PAHs is a complex process that involves xenobiotic-metabolizing phase I and phase II enzymes. In phase I, PAHs is introduced a functional group (e.g. –OH, –COOH, –NO2) by the multienzymatic system cytochrome P450 (CYP450) (Dam et al., 2008), of which the induction of phase I P450 enzyme, measured as an increase in ethoxyresorufin-O-deethylase (EROD) activities have been widely used as biomarkers (Rewitz et al., 2006). In phase II, the PAHs metabolites conjugated with polar endogenous constituents such as glutathione-S-transferase (GST), to produce water-soluble conjugates that are easily excreted (Rey–Salgueiro et al., 2011). PAHs exerted their toxic effects by either the parent compound or subsequently, during metabolism resulting in production of reactive oxygen species (ROS) (Parrilla–Taylor et al., 2013). Although ROS plays an important role in host defense, overproduction and residuals can cause cellular damage. Most cells have protective mechanisms to balance ROS production and avoid oxidative stress, namely antioxidants. Antioxidant enzyme systems are a well-developed regulatory mechanism scavenging ROS, including non-enzymatic small antioxidant molecules (such as

X. Ren et al. / Ecotoxicology and Environmental Safety 114 (2015) 44–51

reduced glutathione (GSH)) and a cascade of enzymes (such as superoxide dismutase (SOD)) (Blokhina et al., 2003; Nordberg and Arnér, 2001; Valko et al., 2007). SOD is crucial in preventing the formation of lipid peroxidation by catalyzing the disproportionation of the lipid peroxidation initiator and the transformation of superoxide radical (O2  ) into H2O2 and O2 (Tao et al., 2013). The enzyme plays an important role in protecting organisms from oxidative stress. GSH is reported to act as an effective antioxidant in marine animals and as a reactant in conjugation with electrophilic substances. Thus, changes in GSH levels are useful indicators of the detoxification ability of marine animals (Hannam et al., 2009). Overwhelming of the antioxidant capacity can result in mass oxidation of GSH leading to excretion of the oxidized molecule (GSSG) from the cell resulting in a reduced intracellular concentration of total glutathione (Hannam et al., 2010). Exposure to some organic contaminants may result in cellular oxidative stress due to redox cycling and disruption of mitochondrial membranes. Organisms can adapt to increasing ROS production by up-regulating antioxidant defenses, such as the activities of antioxidant enzymes (Livingstone, 2001). But failure in antioxidant defense to detoxify excess ROS production can lead to significant oxidative damage including protein degradation, DNA damage and lipid peroxidation (LPO). Moreover, biotransformation processes not only affect residual contaminant body burden but can also alter the toxicity of certain chemicals (e.g. certain PAH compounds such as BaP), for which metabolites can be more toxic than the parent compound (Buhler and Williams, 1988; Lech and Bend, 1980), and these reactions might bring about the synthesis of more reactive molecules that can interact with the genetic material and cause DNA damage and protein carbonyls. Litopenaeus vannamei is a tropical species that has been widely cultured in extensive, intensive and semi-intensive systems, and it is the most popular shrimp for aquaculture in America, Thailand and China (Hou et al., 2014; Yang et al., 2010). For the past few years, environmental pollution has seriously affected the culture of L. vannamei (Bachère, 2000). Although a great number of studies concerning the effects of environmental pollutants on L. vannamei have been carried out, many aspects of environmental pollutant effects on crustaceans, particularly gills and hepatopancreas remain unclear. In the present study, we used the 4-ringed PAH chrysene (CHR) as representatives of PAHs. The purpose of this study was to determine potential toxic effects of CHR in gills and hepatopancreas on L. vannamei including (i) the accumulation profile of CHR; (ii) phase I detoxification enzyme activity of AHH, EROD, EH and phase II detoxification enzyme activity of GST, SULT, UGT; (iii) antioxidant defense system (T-AOC, SOD) and levels of non-enzymatic glutathione (both reduced (GSH) and oxidized (GSSG) forms); (iv) levels of biomolecule damage parameters (DNA damage, MDA contents and protein carbonyls).

2. Materials and methods 2.1. Chemicals CHR (98% purity) form Supelco (Bellefonte, PA, USA). Acetone (Sigma, USA) was used in this study as a vehicle for CHR. All chemicals for sample preparation and HPLC detection were obtained from E. Merck (Darmstadt, Germany), and ultrapure reduced glutathione (GSH) was purchased from Amresco (American).1-Chloro-2,4-dinitrobenzene(CDNB),3′-phosphoadenosine5′-phosphosulfate (PAPS), disodium salt of reduced form -nicotinamideadenine dinucleotide phosphate (NADPH), pyrogallicacid, 7-ethoxy-resorufin (ERF) were purchased from Fluka (USA). All other chemicals were analytical grade.

45

2.2. Animals and treatments Healthy L. vannamei (physical integrity without injury, good viability), averaging 9.5 7 0.5 cm in body length, were obtained from a commercial farm in Nanshan, Qingdao, China. The shrimp were acclimated in tanks (72 cm  56 cm  40 cm) with a water volume of 125 L each, containing aerated seawater (salinity 31%, pH 8.2) at 25 70.5 °C for one week prior to the experiment. During the acclimation period, one third to half of the water in each tank was replaced twice daily and the shrimp were fed with a formulated shrimp diet daily. Only apparently healthy animals at the inter-molt stage were used for the study. The molt stage was decided by the examination of uropoda in which partial retraction of the epidermis could be distinguished (Bonilla-Gómez et al., 2012). In treatment aquarium, the shrimp were exposed to different CHR concentrations (0.3, 2.1 and 14.7 μg/L). There were three replicates for each level and control group, and 60 shrimp in each aquarium (equivalent to 480 shrimp m  3). One third of the water was renewed twice daily, and seawater containing the same concentrations of CHR was added to maintain the corresponding concentrations of CHR during the experiment. CHR was first dissolved in acetone. The final acetone concentration was 0.001% in all tanks including the control ones (the acetone test has been done in a preliminary experiment with the result that there was no influence on shrimp). The exposure concentrations of CHR were based on the concentration of CHR in the coastal seawater, surface sediments in China, as well as CHR solubility (22–25 °C). Shrimp were sampled 0, 1, 3, 6, 10, 15 and 21 days after the end of the acclimatization period, eight shrimp were sampled for each sampling time and concentration, including controls. Gills, muscle and hepatopancreas tissues were collected; 0.1 g hepatopancreas tissue for every sample was grinded in liquid nitrogen, and then was placed in 1.5 mL sterile centrifugal tube. The remaining tissue was dispensed into 5 mL collection tubes, and preserved at  80 °C for CHR concentration and enzyme activities in less than a week prior to use. 2.3. Chemical analysis The analysis of CHR was conducted according to standard method procedures (USEPA, 1996). Freeze-dried shrimp tissue samples were Soxhlet extracted in 150 mL mixture of hexane and dichloromethane (1:1) at 70 °C for 18 h, and extracts for the shrimp tissues were prepared. All extracts were dried using anhydrous sodium sulfate and concentrated by a rotary evaporator. The concentrated extracts were concentrated to 5 mL. A chromatography column of length 50 cm, internal diameter of 1 cm was sequentially packed with glass wool, 12 cm of activated silica gel, 6 cm of alumina, which had been baked at 450 °C for 5 h, and 1 cm of anhydrous sodium sulfate (Wei et al., 2006). The extraction aliquot was added into the column, after that the column was preliminarily eluted with 15 mL dichloromethane and then 30 mL hexane, which were both discarded. 15 mL mixture of dichloromethane and hexane (2:1) were eluted thrice to obtain the combined eluate. The eluate was dried with anhydrous sodium sulfate, evaporated, resuspended in 5 mL acetonitrile and filtered (4 μm pore size). HPLC analysis was performed with a Shimadzu LC20A system. CHR eluate was resolved on an Agilent ZORBAX Eclipse PAH column (4.6 mm  250 mm  5 μm) using a protocol gradient with solvent A: water and B: methanol. The gradient was: 95% B/5% A for 60 min. The column temperature was maintained at 30 °C and the flow rate was set at 1.0 mL/min, with fluorescence (excitation wavelength 295 nm, emission wavelength 430 nm) detection. 5 μL of concentrated extract were injected for each run.

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X. Ren et al. / Ecotoxicology and Environmental Safety 114 (2015) 44–51

2.4. Enzyme analysis 2.4.1. Sample preparation The supernatant measured for the activities of aryl hydrocarbon hydroxylase (AHH), ethoxyresorufin O-deethylase (EROD), glutathione S-transferase (GST), epoxide hydrolase (EH) and glutathione levels (both reduced (GSH) and oxidized (GSSG) forms), sulfotransferase (SULT) activity, UDP-glucuronosyl-transferase (UGT) activity, malondialdehyde (MDA) contents and protein carbonyl (PC) contents were separated according to the method described by Liu et al. (2014). 2.4.2. Antioxidant defense parameters assays Total antioxidant capacity (T-AOC) was determined using commercial assay kits (Nanjing Jiancheng Institute, China) in a spectrophotometer (DU7400, Beckman Co., USA) according to the manufacturer's instructions. SOD activity was measured by a modification of the method of Marklund and Marklund (1974). This assay was based on the ability of SOD to inhibit the auto-oxidation of pyrogallol (50 mM) in 50 mM Tris–HCl buffer (pH 8.3). The reaction mixture contained 4.5 mL Tris–HCl buffer, 100 μL supernatant and 10 μL pyrogallol. Oxidation of pyrogallol was monitored by measuring absorbance at 325 nm. 1 U of SOD activity was defined as 50% inhibition of the oxidation process (U/mg protein/min). Total amounts of GSH and GSSG were determined spectrophotometrically using 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) according to the method of Anderson (1985) using commercial GSH (Sigma) as a standard. GSH/GSSG ratio, a valuable biomarker of antioxidant status, was systematically calculated. 2.4.3. Detoxification parameters assay AHH activity was assayed by the method developed by Willett et al. (2000). The 1.0 mL incubation mixture consisted of 400 μL supernatant, 510 μL Tris–HCl (50 mM, pH 7.6), 50 μL 0.1 mM NADPH, and 10 μL 3 M MgCl2. Samples were preincubated at 30 °C and the reaction was initiated by the addition of 60 μM CHR. Then samples were incubated for 30 min and stopped with the addition of 1 mL cold acetone followed by 3.25 mL hexane. Samples were vortexed, 2 mL of the organic layer was drawn and extracted with 5 mL aqueous NaOH, and fluorescence was determined with a spectrofluorometer (Molecular Spectroscopy LS 55 from Instruments, P.E., MA, USA) at 396/522 nm (excitation/emission). EROD activity was measured according to the modified method of Pohl and Fouts (1980). The reaction mixture contained 100 μL supernatant, 10 μL 0.2 mM O7-Ethylresorufin, 10 μ L 6 mM NADPH and 1.88 mL phosphate buffer (0.125 M, pH 7.7, containing Na2EDTA 0.05 M, 2–4 °C), allowed to proceed for 10 min at RT, and stopped by the addition of 0.5 mL carbinol. Incubation vials were centrifuged at 11,000g for 1 min to remove precipitated microsomal protein, and supernatants were transferred to vials for measurement of resorufin concentrations in a luminescence spectrometer (Model L S55, Perkin-Elmer of U.K.) at an excitation wavelength of 560 nm and an emission wavelength of 580 nm. Resorufin was identified and concentrations were calculated by comparison to retention times and responses of resorufin standards. Blanks corresponded to t¼ 0 min and quantification was achieved with standard additions of resorufin. Values were expressed as nmol per minutes per mg of microsomal protein (nmol/ min/mg of microsomal protein). EH activity was measured via the protocol of Doderer and Schmid (2004) with modifications. Samples were initially homogenized in ice-cold homogenization buffer (100 mmol/L sodium phosphate, pH 8.0). EH activity was measured with black 96-well microplates designed for fluorimetric applications. In each sample well, 20 μL of tissue fractionate was added to 80 μL of a

12.5 mmol/L styrene oxide solution (50 mmol/L phosphate buffer, pH 8.0) and incubated at room temperature for 10 min to allow for substrate–enzyme interaction. After this incubation period, 60 μL of sodium periodate solution (90 mmol/L sodium periodate, 100 mmol/L sodium acetate, pH 4.5) was added to each well and allowed to incubate at room temperature for an additional 10 min. Next, 100 μL of 6-carboxyfluorescein (6-CF) solution (0.425 mmol/ L 6-CF, 100 mmol/L sodium phosphate buffer, pH 8) was added to each well. Plates were then sealed with aluminum foil and incubated for 2 h at 70 °C to initiate the chemical reaction between periodate and 6-CF. To measure residual fluorescence of 6-CF following periodate incubation, plates were briefly shaken and fluorescence was measured (480 nm excitation, 515 nm emission). To allow for background correction, each plate contained three negative control (non-enzymatic) wells without shrimp tissues homogenate. GST activity was determined according to Habig et al. (1974). The reaction mixture contained 200 mL supernatant, 2 mL phosphate buffer (0.125 mol/L, pH 7.7, containing Na2EDTA 0.05 mol/L 4 °C), 400 mL H2O, 200 mL 15 mmol/L of 1-Chloro-2,4-dinitrobenzene (CDNB) dissolved in 95% ethanol and 200 mL 15 mmol/ L ultrapure reduced glutathione (GSH). GST activity was determined following the conjugation of reduced glutathione (GSH) with CDNB at 340 nm (ε ¼9.6 mmol  1). A unit of GST activity was defined as the amount of glutathione conjugate formed using 1 nmol GSH and CDNB/min per mg protein (pmol 2, 4-dinitrophenyl glutathione/mg protein/min). SULT activity was determined according to Beckmann (1991). The reaction mixture contained 0.7 mL of 25 mmol/L Na-succinate, 25 mmol/L Na-phosphate, 100 μmol/L PAPS and 10 μmol/L resorufin. The fluorimeter excitation and emission wavelengths were set at 530 and 585 nm, respectively. The instrument was blanked with control samples lacking resorufin. Alternatively, the blanks contained resorufin concentrations up to 10 nmol/L less than those of the samples to be assayed. This approach effectively expanded the scale of the fluorimeter, increased sensitivity with respect to enzyme, and enabled linear initial rates to be measured. Reactions were initiated by the addition of 2–10 μmol/L enzymes. UGT activity was determined according to Sörgel et al. (1980). The reaction mixture was composed of 0.27 mmol/L 4-methylumbelliferone, 30 mmol/L magnesium chloride, 1.667 mmol/L uridinediphosphate glucuronic acid (as the sodium salt), tris (hydroxymethyl) aminomethane hydrochloride buffer (166.7 mmol/L, pH 8.0), and microsomal suspension equivalent to 50 mg sample fresh weight, in a final volume of 3.0 mL. After starting the enzyme reaction by addition of uridinediphosphate glucuronic acid, the samples were incubated at 37 °C for 30 min, and then rapidly mixed with 6.0 mL ice cold glycine buffer (0.2 mol/L, pH 10.8). After centrifugated at 11,000g for 1 min, fluorescence of the supernatant was measured (385 nm excitation, 445 nm emission). The reference in which uridinediphosphate glucuronic acid had been omitted was used as a standard. It was not necessary to include zero time values. 2.4.4. Damage parameters assay The rate of transition of double-stranded DNA (dsDNA) to single-stranded DNA (ssDNA) under pre-defined alkaline denaturing conditions was proportional to the number of breaks in the phosphodiester backbone, and thus was used as a measure of DNA integrity (Daniel et al., 1985). Methods of DNA extraction and alkaline unwinding assay used in the study were adapted from Ching et al. (2001). In the assay, the DNA sample was diluted and divided into three equal portions for fluorescence determination of dsDNA, ssDNA and alkaline unwound DNA (auDNA). The ratio between double-stranded DNA and total DNA (F value) was determined as follows: F value ¼(auDNA  ssDNA)/(dsDNA  ssDNA).

X. Ren et al. / Ecotoxicology and Environmental Safety 114 (2015) 44–51

Lipid peroxidation (LPO) level was expressed by MDA content. MDA content was evaluated by an improved thiobarbituric method (Wills, 1987). Briefly, 50 μL tissue homogenate was diluted to 1 mL with distilled water and mixed with 500 μL 20% trichloroacetic acid containing 1 mM FeSO4, 1 mL 0.67% thiobarbituric acid was added and the mixture was kept at 90 °C for 10 min. After centrifugation at 3000g for 5 min, 1 mL aliquot was withdrawn and mixed with 2 mL distilled water. Absorbance was measured at 530 nm. MDA contents were expressed as nmol TBARS/mg protein/min. Protein carbonyls (PC) content was evaluated as previously described by measuring the formation of protein hydrazones resulting from the reaction of dinitrophenyl hydrazine (DNPH) with protein carbonyls (Lund et al., 2007). The carbonyl content was measured at 375 nm. The total protein content of gills and hepatopancreas samples was measured in diluted homogenates by the Bradford (1976) method using bovine serum albumin (Sigma) as a standard. The results of antioxidant parameters were expressed as nmol mg  1 protein. 2.5. Statistical analysis All data presented are mean values of three independent sets of the experiment. Each value was presented as means 7standard deviation (S. D.). The significant difference between the control and the exposed shrimp were determined using a one-way ANOVA followed by a multiple comparison of Dunett's tests. The significance level was set at P o0.05. Statistical computations were performed with SPSS 13.0 for Windows (SPSS Inc.).

3. Result 3.1. CHR bioaccumulation in different tissues The results of the bioaccumulation of juvenile shrimp exposed to CHR were presented in Table 1. The results indicated that a clear time effect with exposure concentration was established for CHR. CHR concentrations in both tissues of all treatment groups increased rapidly during the exposure experiment and became stable at day 6 or day 10, after that the CHR concentrations in both tissues became stable and significantly higher compared to control group. At day 21, CHR concentrations in hepatopancreas were higher than those in gills. There were no apparent changes in control group during the experiment. 3.2. Effects of CHR exposure on detoxification system 3.2.1. Effects of CHR exposure on phase I and phase II detoxification system The detoxification enzyme activities results are shown in Figs. 1 and 2. During the experiment, AHH, EROD, EH, GST, SULT

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and UGT activities of 2.1 and 14.7 μg/L CHR treatments in gills and hepatopancreas increased significantly (P o0.05) and then became stable. At day 21, these enzyme activities in hepatopancreas were positively correlated with CHR exposure concentrations. No apparent changes were found in control group and 0.3 μg/L CHR treatment of all the enzyme activities. 3.2.2. Effects of CHR exposure on the antioxidant status of L. vannamei Antioxidant defenses of T-AOC, SOD, GSH contents in gills and hepatopancreas are shown in Fig. 3. The results showed that the T-AOC and GSH contents of all CHR exposure groups decreased except the 0.3 μg/L CHR exposure group. The T-AOC and GSH contents reached the lowest value at 6 days, as the exposure time progressed to 15 days, the T-AOC and GSH/GSSG remained stable up to the end of the experiment and had significant difference compared with control (P o0.05). The SOD activity increased at 3 days, and peaked at 6 days, afterwards the SOD activity of 2.1 μg/ L CHR treatment in gills decreased to the same level as in control, while the SOD activity of 14.7 μg/L CHR treatment stayed at high level. The 0.3 μg/L CHR exposure group had no significant difference between exposed and control organisms. 3.3. The results of CHR exposure on damage indexes of L. vannamei The results of biomolecule damage of each treatment group were presented in Fig. 4. F value of 0.3 μg/L CHR treatment in gills had no significant change compared to the corresponding controls. As to 2.1 and 14.7 μg/L CHR treatments, F value of both tissues was found to start decreasing during the 1st day, reached the minimum level at 6 days, and remained significantly lower compared to control (P o0.05). It showed that changes of MDA and PC contents in the gills and hepatopancreas shared a similar pattern: no significant changes in MDA and PC contents were observed over the 21-day exposure period between control and 0.3 μg/L CHR treatment (P 40.05). With exposure to 2.1 and 14.7 μg/L CHR treatments, MDA and PC contents in the gills and hepatopancreas increased the highest level at 6 days, and then exhibited a downward trend, but its contents were significantly higher throughout the experiment compared to control group (Po0.05).

4. Discussion When the PAHs enter the organism's system, it will experience a series of oxidative processes mediated by the cytochrome P450 system (phase I), CYP 1A1 isozyme being the predominant, which can be assessed by measuring EROD (Rocha et al., 2012). In phase II, the metabolites are conjugated with polar endogenous constituents such as glucuronic acid, sulfate, or glutathione by UGT-

Table 1 CHR concentration in gills and hepatopancreas of the white shrimp, L. vannamei. Tissue

Gills

Hepatopancreas

CHR (μg/L)

0 0.3 2.1 14.7 0 0.3 2.1 14.7

CHR concentration (μg/g d.w.) 0 day

1 day

3 days

6 days

10 days

15 days

21 days

0.00477 0.0020a

0.0079 70.0007a 0.0463 70.0233a 0.3058 70.0094b 0.8069 70.0009b 0.0025 70.0245a 0.0047 70.0003a 0.1716 70.0153b 0.7620 70.0266c

0.0053 7 0.0006a 0.082 7 0.0053b 0.543 7 0.0138c 1.1007 0.0023c 0.00737 0.0536a 0.06187 0.0071b 0.43077 0.0348c 1.04727 0.1091d

0.0042 7 0.0003a 0.0453 7 0.0574b 0.394 7 0.0592c 3.1447 0.0394d 0.0058 7 0.0002a 0.09917 0.0177b 0.6592 7 0.0311c 2.7164 7 0.0343d

0.0058 7 0.0001a 0.03487 0.0068b 0.829 7 0.0088c 4.081 7 0.0139d 0.00647 0.0745a 0.2784 7 0.0345b 1.51907 0.4689c 4.1609 7 1.6459c

0.0053 7 0.0002a 0.08647 0.0016b 1.0167 0.0032c 4.092 7 0.0146d 0.0040 7 0.0005a 0.43387 0.0193b 2.1234 7 0.0857b 6.2546 7 1.1420c

0.00787 0.0004a 0.0639 7 0.0153b 1.1887 0.0052c 4.1167 0.0128d 0.0058 7 0.0006a 0.54357 0.0606b 3.41877 0.1751b 7.21707 0.8774c

0.0052 7 0.0008a

Values are presented as the mean 7 S.D. (n¼ 3). Significant differences from control in the same time of sampling are indicated with different letters mean significant difference (Po 0.05).

X. Ren et al. / Ecotoxicology and Environmental Safety 114 (2015) 44–51

Control

16 12 8

0.3μg/L

2.1μg/L

14.7μg/L

c b a aaa

b a a

aa a

a a

bb

a a

ab

b aaa

b a a a

4

EROD activity (pmol /min/mg Pro)

EROD activity (pmol /min/mg Pro)

48

1

3

6

Control

0.3

0.2 a a aa

bb

a a

b

10

0.3μg/L c c b

a a

15

15

b a aaa

c

c

ab

aa

2.1μg/L

d

c

20

d

14.7μg/L c

a

c

bc

b

b

b

aa

aa

aa

10 5

2.1μg/L b

aa

0

21

14.7μg/L

c

b

b a aa

a a

1

3

a aa

0.1

0

0.6 aa aa

b

aa

a aa

10

0.3μg/L

15

2.1μg/L

c

c b

0.4

6

Control

0.8

AHH activity (U/mg Pro)

0

AHH activity (U/mg Pro)

0.3μg/L

25

0

0

14.7μg/L

b c

b aa

21

b

c

c b

a a

a a

aa

0.2 0

1

3

6

Control

0.3

c

0.2 aaaa

a aa a

a

10

0.3μg/L

d

2.1μg/L

21

0

1

3 Control

0.5

14.7μg/L

6

10

0.3μg/L

0.4

b

b

b

15

b

c aa

a a

a a

aa

a aaa

0.1

EH activity (U/mg Pro)

0

EH activity (U/mg Pro)

Control

30

0.3

a a a a

a

a a

b

b

a a

2.1μg/L

c

c a

b

a

15

ab b

a a

21

14.7μg/L

b a a a

a a a

15

21

b

0.2 0.1 0

0 0

1

3 6 Experimental time (d)

10

15

0

21

1

3

6

10

Experimental time (d)

Fig. 1. The phase I enzyme activities of EROD, AHH, EH in gills and hepatopancreas of L. vannamei exposed to CHR (0.3, 2.1 and 14.7 μg/L) for 1, 3, 6, 10, 15 and 21 days. Each bar represents the mean value from four replicate tanks with the standard error. Means ( 7S.D.) with different letters are significantly different (Po 0.05).

Control

32 24 aaa a 16

ab a a

aa

0.3μg/L

b c

2.1μg/L c

b

aa

a aa

D

14.7μg/L b

b a aa

b a

a a

8

GST activity (nmol /min/mg Pro)

GST activity(nmol /min/mg Pro)

A

0 3

6

Control

10

0.3μg/L

15

24

a aa a

bc

aa

a a

bc

2.1μg/L b

14.7μg/L c

c

a a

c

b

b

aa

a a

16 8

2.1μg/L

21

0

E

14.7μg/L

c

20 c

c

16 a aaa

aa

b

b

b

b

aa

a a

aa

aa

c

c b

b aaa

8

SULT activity (U/mg Pro)

SULT activity (U/mg Pro)

1

24

12

32

0.3μg/L d c b a

0

0

B

Control

40

1

3 Control

32

10

0.3μg/L

a a aa

a a

ab

b

b a a

b

b

aa

aa

21

14.7μg/L

c

b

c

15

2.1μg/L

c

24 16

6

c

c

aa

a a

b

8

4

UGT activity (U/mg Pro)

C

0

1

60 40

3

Control aa aa

a aa a

aa

bb

6

10

0.3μg/L aa

b

2.1μg/L

15

F

14.7μg/L

a a

0

1

3

Control

80

6

0.3μg/L

10

15

21

2.1μg/L

14.7μg/L

c

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Fig. 2. The phase II detoxification enzyme activities GST, SULT and UGT in gills and hepatopancreas of L. vannamei exposed to CHR (0.3, 2.1 and 14.7 μg/L) for 1, 3, 6, 10, 15 and 21 days. Each bar represents the mean value from four replicate tanks with the standard error. Means ( 7 S.D.) with different letters are significantly different (P o0.05).

X. Ren et al. / Ecotoxicology and Environmental Safety 114 (2015) 44–51

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Fig. 3. Antioxidant activities of T-AOC, SOD and GSH/GSSG in gills and hepatopancreas of L. vannamei exposed to CHR (0.3, 2.1 and 14.7 μg/L) for 1, 3, 6, 10, 15 and 21 days. Each bar represents the mean value from four replicate tanks with the standard error. Means ( 7 S.D.) with different letters are significantly different (P o0.05).

glucuronyl transferase, sulfotransferase, and glutathione-S-transferase, respectively, to produce water-soluble conjugates that are easily excreted. Gonçalves-Soares et al. (2012) reported that after injection with a sub-lethal dose of a toxic Microcystis aeruginosa extract in L. vannamei, MCs caused up-regulation of GST, and increases in the total GST enzyme activity. Our results were in accordance with previous study. In our study, we have observed a significant increase in enzyme activity of EROD and GST activities in both tissues to higher CHR dose in comparison with control. These results suggested that enzymes of EROD and GST may play an important role in detoxification of xenobiotics. In aquatic animals, PAHs detoxication metabolism largely occurs through radical oxidation involving reactive oxygen species (ROS), which can be generated at various stages along the metabolic pathway (Livingstone, 1991). Shrimp possess an integrated antioxidant system, containing some enzymatic and non-enzymatic antioxidants to maintain normal status against natural or induced stressors (Parrilla-Taylor and Zenteno-Savín, 2011). Oxidative stress seems to be a common mechanism of environmental stress. Under stress, the balance between the production of ROS and the antioxidant defense is always disturbed. The antioxidant enzymes are vital tools for detoxifying harmful ROS. And some ROS may function as important signaling molecules that alter gene expression and modulate the activity of specific defense proteins. In this study, the effects of pollutants on non-enzymatic antioxidant molecules in two shrimp tissues were investigated. The results showed that SOD activity was equally induced by CHR exposure. Similar changes in antioxidant enzymes in response to xenobitotics exposure have been reported in cultured Neocaridina heteropoda (Wang et al., 2011). However, in our present study,

GSH/GSSG ratios were inhibited at CHR exposure, which is consistent with previous study on the Macrobrachium borellii (Crustacea: Palaemonidae) reported by Lavarías et al. (2011). GST also plays an important role in detoxification reactions as a key to conjugate electrophilic intermediates which are generate by antioxidant defense system. Phase II of xenobiotic metabolism (conjugation) is characterized by addition reactions in which large and often polar compounds (e.g., GSH) are covalently added to xenobiotic compounds facilitating their excretion. Therefore, the conjugation of CHR intermediates with the glutathione molecule may explain the low levels of GSH/GSSG ratios observed in this present study. Although SOD activity was triggered by CHR exposure, a reduction of T-AOC was detected following CHR exposure. Generally, the antioxidant capability of an organism under certain condition can reflect its health status (Xu and Pan, 2013). We speculate that the reduction of the T-AOC is one of the reasons that lead to the disease susceptibility of L. vannamei. Damage to cellular macromolecules occurs due to reactive oxygen species (ROS) and/or reactive metabolites formation (Pan et al., 2009). As the pollutants are metabolized, reactive molecules are produced and interact with the genetic material and cause DNA damage as a toxic terminal of the induction of a cascade of cellular events. DNA integrity reduction represents the genotoxicity which may have a long-term effect on the sustainability of a particular population. DNA damage is a primary concern for the assessment of pollution-related stress in living organisms (Klobučar et al., 2003). Lipid peroxidation (LPO) is one of the major indicators associated with failure of the antioxidant system and hence quantification of MDA is the way to evaluate the LPO level frequently used as a biomarker of oxidative damage to lipids

X. Ren et al. / Ecotoxicology and Environmental Safety 114 (2015) 44–51

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(Lavarías et al., 2011; Parrilla-Taylor et al., 2013). During LPO, aldehydes such as malonaldehyde are formed as by products, and these can react with DNA bases forming DNA adducts. PC are considered to be the main indicators of oxidative damage to proteins. Moreover, metabolic intermediates can also cause protein damage. Recently, PC is used as a maker of oxidative stress in shrimp (Lushchak, 2011). Pan et al. (2008) studied the effect of BaP on the Scallop Chlamys farreri found serious DNA strand breaks in the gills and digestive gland. Parrilla-Taylor et al. (2013) reported that after injection with white spot syndrome virus in L. vannamei, LPO and PC levels were higher in infected specimens compared to uninfected controls. A significant reduction in antioxidant enzyme activities was found at 48 h post-infection in all tissues analyzed. Our results were in accordance with previous study. In our study, F value in gills and hepatopancreas of 2.1 and 14.7 μg/L CHR treatments decreased until day 6 and then showed a gradual increase trend until the end of the experiment. An increase in LPO and PC was observed in this current study following exposure to CHR concentrations. Considering that the EROD and GST activities showed a plateau at the end of the experiment, it is implied that the reactive intermediates may be maintained at a comparative persistent level, initiating the biomolecule damage repair system. Chang et al. (2009) reported that after 6 h exposure to Cd2 þ , the degree of DNA damage in L. vannamei declined, suggesting that DNA repair pathways could have been activated during this period. Our former results showed a significant decline of DNA damage and induction of protein carbonyls in gills and hepatopancreas of L. vannamei after Bap exposure (Ren et al., 2014). Our results also showed the same trend after CHR exposure, increased F value, decreased MDA and PC contents demonstrated that biomolecule damages were repaired.

The results of CHR concentration in the gills and hepatopancreas show that CHR was rapidly taken up in 3 days. CHR bioaccumulation in two tissues rose parallel with the exposure concentration. Pan et al. (2008) reported that BaP bioaccumulation in gills and digestive gland of scallops (Chalmys farreri) increased first and showed an incoming plateau exposed to 0.5, 3 and 10 μg/L BaP for 20 days. Wang et al. (2011) found that in 0.01 μg/L BaP exposure treatment, the BaP concentration of gills and digestive gland of clam Ruditapes philippinarum increased significantly after exposure for 6 days and there was no significant difference in BaP bioaccumulation in these two tissues. Our results are consistent with the former. The rate of CHR bioaccumulation in gills and hepatopancreas was high at the beginning of the experiment, and then became lower, eventually reaching equilibrium at day 10 or 15, and these are in agreement with the process of CHR metabolism.

5. Conclusion In conclusion, the results presented here confirmed that the CHR was accumulated and metabolized in the L. vannamei, subsequently resulting antioxidant system changes and the magnitude of biomolecule damage. Our results indicated that the detoxification function in gills was weaker than hepatopancreas. We concluded that the detoxification enzyme activities and biomolecule damage indexes can be suitable biomarkers to evaluate the toxicity of CHR. Results of present study were expected to provide additional contribution to the emerging field in invertebrate about understanding the physiological and biochemical basis of adaptation to aquatic environmental hydrocarbon pollution. Further

X. Ren et al. / Ecotoxicology and Environmental Safety 114 (2015) 44–51

studies would be needed for a better understanding of the effects and mechanisms of toxicity in L. vannamei exposed to CHR.

Acknowledgments This research was supported by the National Marine Public Industry Research Project “park aquaculture environment engineering ecological optimization technology integration and demonstration” (201305005). We thank the staff at the Laboratory of Environmental Physiology of Aquatic Animal for help with sampling and taking care of the shrimp.

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