Copper-induced oxidative damage to the prophenoloxidase-activating system in the freshwater crayfish Procambarus clarkii

Fish & Shellfish Immunology 52 (2016) 221e229

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Copper-induced oxidative damage to the prophenoloxidase-activating system in the freshwater crayfish Procambarus clarkii Keqiang Wei a, *, Junxian Yang b a b

School of Life Science, Shanxi University, Taiyuan 030006, People's Republic of China School of Economics and Management, Shanxi University, Taiyuan 030006, People's Republic of China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 January 2016 Received in revised form 19 March 2016 Accepted 22 March 2016 Available online 23 March 2016

Previous studies have demonstrated copper-induced proteins damage in gill and hepatopancreas of the freshwater crayfish Procambarus clarkii, but little information is available about its effects on key component of the innate defense in haemolymph. In the present study, we evaluated the relationship between oxidative carbonylation and prophenoloxidase-activating system (proPO-AS) activity, by exposing P. clarkii to sub-lethal concentrations (1/50, 1/12, 1/6 and 1/3 of the 96 h LC50) Cu2þ up to 96 h. Six biomarkers of oxidative stress, i.e. reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), protein carbonyl (PC), malondialdehyde (MDA) and DNA-protein crosslinks (DPCs), and six indicators of immune status, i.e. total hemocyte counts (THCs), differential hemocyte counts (DHCs), hemocyanin (HC), prophenoloxidase (proPO), serine protease (SP) and phenoloxidase (PO), were determined in haemolymph. The results indicated that there was a significant increase (P < 0.05) in the levels of ROS, PC, MDA and DPCs accompanied by markedly decreased (P < 0.05) activities of proPO, SP, PO and HC in a dose and time dependent manner. The significant and positive correlations (P < 0.01) between ROS production and the formation of PC, MDA and DPCs were observed in crayfish at 96 h. There was a significant negative correlation (P < 0.01) between the levels of protein carbonyls and the activities of proPO and SP in hemocyte lysate supernatant and PO and HC in haemolymph. Carbonylated proteins may be recognized not merely as a specific signal in oxidative stress pathways but also as a “non-self” molecule in proPO-AS. In crayfish species, copper-catalyzed protein carbonylation may be one of the main mechanisms for immunity dysfunction in proPO-AS. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Copper Haemolymph Procambarus clarkii Prophenoloxidase Protein carbonylation

1. Introduction Copper (Cu) is one of the most ubiquitous pollutants in aquatic environment. The 2013 Report on the State of the Fishery EcoEnvironment in China indicated that Cu contamination has been more and more serious in the important fishery areas [1]. As an essential trace element, Cu is also required for aquatic animals, serving as a catalytic cofactor in enzymes and as a structural component of proteins such as superoxide dismutase (SOD), phenoloxidase (PO) and hemocyanin (HC). It has been well noted that copper plays vitally important role in the immune system of crustaceans [2]. However, the optimal range between essential and toxic concentrations is found to be rather narrow. Even at low

* Corresponding author. School of Life Science, Shanxi University, 92 Wucheng Road, Taiyuan 030006, People's Republic of China E-mail addresses: [email protected], [email protected] (K. Wei). http://dx.doi.org/10.1016/j.fsi.2016.03.151 1050-4648/© 2016 Elsevier Ltd. All rights reserved.

concentration, Cu could suppress the host's immune response, thus increasing its susceptibility to pathogens [3e5]. In crustaceans, Cu levels are tightly regulated by complex homeostatic mechanisms. Waterborne Cu is absorbed mainly by haemolymph in gill, whereas the hepatopancreas functions as storage and detoxification organ. Once in excess of cellular needs, Cu could catalyze the generation of reactive oxygen species (ROS) through the Haber-Weiss reaction. Small increase of ROS is considered to be beneficial with respect to increased immunity, but overproduction of ROS could induce oxidative damage to cellular macromolecules, including proteins, lipids and nucleic acids [2,5,6]. Normally, the deleterious effect of ROS can be counteracted by endogenous antioxidants like SOD and catalase (CAT). The two enzymes are not only used as biomarkers of oxidative stress but also implicated in the mediation of innate immunity [7]. The immune system of crayfish is a non-adaptive response, based on both cellular and humoral components. Circulating hemocytes are generally classified into three types, i.e. hyaline, semi-

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granular (SGC) and large granular (GC) cell, and they are responsible for various protective mechanisms ranging from phagocytosis and encapsulation to melanization [8]. The prophenoloxidaseactivating system (proPO-AS) is an efficient non-self recognition system which consists of several important proteins including prophenoloxidase (proPO), pattern-recognition proteins (PRPs), serine protease (SP), and serine protease inhibitors (serpins). The proPO, an inactive proenzyme of phenoloxidase (PO), is synthesized in hemocytes, localized in the granules of SGC and GC and released into the haemolymph upon activation. An endogenous SP, the so-called prophenoloxidase activating enzyme (ppA), is at the center of this complex and restricted proteolysis cascade [9e11]. Finally PO catalyzes the production of the melanin and toxic reactive intermediates against invading pathogens. When proPO was knocked down via RNAi, invertebrate animals were easily infected by virulent bacteria and viruses [9]. Some research also showed that PO activity may originate from other sources such as HC, although in contrast to proPO, it is synthesized in the hepatopancreas [4,10]. At present, it is completely clarified that the proPO cascade is set off in a stepwise process with the recognition of microbial cell wall components, such as b-1, 3-glucan, lipopolysaccharide and peptidoglycan, by PRPs [12,13]. However, the mechanisms by which ROS regulates the proPO-AS in the case of pollutants exposure are still not fully understood. As a redox active metal, Cu can directly catalyze highly reactive free radicals formation. Oxidative stress occurs when there is an imbalance between ROS and antioxidant defense molecules [5,7]. Proteins are the most abundant cell components (70%) and responsible for most functional processes in cells. So they are possibly the primary target for ROS assault, resulting in protein aggregation, inactivation or degradation [14e16]. Protein carbonylation is widely accepted as a biomarker of oxidative damage due to its relatively early formation and stability compared with other oxidative stress-induced protein modifications. Protein carbonyls (PC) groups can be generated directly by amino acids oxidation and a-amidation pathway or indirectly by forming adducts with lipid peroxidation products [17]. Malonaldehyde (MDA) is the main breakdown product of oxidized lipids, which reacts preferentially with Lys residues of proteins. The lipid peroxidation can influence membrane fluidity as well as the integrity of biomolecules associated with the membrane (membrane bound proteins or cholesterol) [18]. Recent reports indicated that there is a fairly strong correlation between the protein carbonylation and DNA-protein crosslinks (DPCs) induction [19,20]. DPCs are bulky, helixdistorting DNA lesions, which are created when cellular proteins become covalently captured on DNA strands upon exposure to various endogenous, environmental and chemotherapeutic agents. DPCs can interfere with DNA replication, transcription, and repair, potentially contributing to mutagenesis, genotoxicity and cytotoxicity [19]. While higher ROS levels could enhance the risk of protein carbonylation, lipid peroxidation and DNA damage, there is little information on oxidative lesions of hemocytes and their constituents in crustaceans. The red swamp crayfish (Procambarus clarkii) is native to northeastern Mexico and south-central USA. Now it has become a commercially important freshwater aquaculture species in China. The farmed production reached 370 000 tons in 2010. Crayfish has been used as a sensitive bioindicator of heavy metals contaminants in aquatic environments [5,18]. Numerous studies conducted so far have shown that copper exposure could induce immunomodulation in crustaceans, possibly leading to a remarkable change in hemocyte numbers, PO activity and other immune-related proteins levels [2,5,11,21e23]. ROS are known to serve as second messengers in cellular signaling cascades that mediate responses to environmental stress. There was also evidence that carbonylated proteins

are recognized not merely as a specific signal in stress-response but also as a “non-self” molecule in immunology system [6,15]. So we speculated that protein carbonylation may play an important regulatory role in proPO-AS activation. In the present study, we assessed the relationship between oxidative stress and proPO-AS activity after acute exposure to sublethal concentrations of Cu2þ, by determining biomarkers of oxidative stress (i.e. ROS, SOD, CAT, PC, MDA and DPCs) and indicators of immune status (i.e. hemocyte counts, proPO, SP, PO and HC) in haemolymph of the crayfish. The results will provide the cellular and molecular evidence for the possible immunotoxic mechanisms of environmental pollutants. 2. Materials and methods 2.1. Crayfish Adult crayfish P. clarkii (8.5 ± 0.6 cm in length and 24 ± 1.5 g in wet weight) were purchased from a local aquatic product market. They were kept in glass aquaria (45 cm  30 cm  30 cm) filled with continuous aerated and dechlorinated tap water (pH 7.2 ± 0.4 and hardness 43.2 ± 1.3 mg CaCO3/L). The water temperature was maintained at 23 ± 1  C and the photoperiod was set to 12:12 (L:D). The animals were fed with commercial feed and acclimatized for 7 days prior to the experiment. Only apparently healthy males in the intermoult stage were used. 2.2. Chemicals All chemicals were of analytical grade and purchased from Sigma (St. Louis, MO, USA) unless stated otherwise. Cu was added as CuSO4$5H2O (99% purity) in deionised water for stock solution. Test solutions were prepared by dilution of this stock solution with dechlorinated tap water to the desired concentration. 2.3. Exposure Two hundred and twenty-five crayfish were randomly divided into five groups. There were triplicates for each test group with a total number of 45 crayfish (15 per replicate). Four sublethal concentrations of Cu2þ (nominal: 0.5, 2.0, 4.0 and 8.0 mg/L) corresponded to approximately 1/50, 1/12, 1/6 and 1/3 of the 96 h LC50 were chosen for the exposure. The control group was prepared with no additional copper. The 96 h LC50 value (25 mg/L Cu2þ) was based on our previous study of acute toxicity in this species. The Cu content in the dechlorinated tap water was 0.003 ± 0.001 mg/L and the actual Cu2þ level in each treatment group varied between 93 and 97% of the nominal concentrations, as determined with atomic absorption spectrometer (Varian Spectra AA 220, Palo Alto, CA, USA). The bioassays were carried out under static conditions without aeration and solution replacement for a period of 96 h. Crayfish were not fed during the experiment. All other conditions were kept the same as those used for acclimation. 2.4. Sampling No crayfish died in any group during the trials. Three crayfish randomly selected in each aquaria were sampled at the interval of each 24 h. They were anaesthetized on ice for about 15 min. Haemolymph (0.9 mL) was withdrawn from the cardiac cavity of each crayfish into a 1.0 mL sterile syringe (25 gauge) containing 0.1 mL ice-cold anticoagulant solution. A drop of haemolymph was immediately placed on a hemocytometer for total hemocyte counts (THCs) and differential hemocyte counts (DHCs) assay. Hemocyte lysate supernatant (HLS) was prepared following the method described by Pan et al. [24]. The diluted haemolymph

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(0.6 mL) was centrifuged (ZONKIA KDC-140HR, China) at 3200 rpm and 4  C for10 min, and the supernatant fluid was stored at 80  C as plasma sample. The resultant pellet was rinsed, resuspended gently in 0.6 mL ice cold cacodylate-citrate buffer, and then centrifuged again. The pellet was resuspended with 0.6 mL ice cold cacodylate buffer, and the suspension was homogenized (20 kHz/ 100 W, 4  30 s, 4  C) in an ultrasonic homogenizer (Cole Parmer, Chicago, USA). Cell debris was removed by centrifugation at 15 000 rpm and 4  C for 20 min, and the clear supernatant was stored at 80  C as HLS. The plasma and HLS were used for subsequent assays. 2.5. Assay of THCs and DHCs One hundred microliters of diluted haemolymph was fixed with an equal volume of 10% formaldehyde for 30 min at 4  C. A drop of the haemolymph suspension was placed on a haemocytometer, and THCs and DHCs were observed using an inverted phase contrast microscope (Olympus BX51, Tokyo, Japan) [24]. 2.6. Assay of reactive oxygen species (ROS) The levels of ROS in haemolymph were determined by DCFH oxidation method by Keston and Brandt [25] with small modifications. The plasma samples were incubated with 5 mM DCFH-DA (20 , 70 -dichlorofluorescein diacetate) in a final volume of 2 mL Trise HCl for 30 min at room temperature. The DCFH-DA is enzymatically hydrolyzed by intracellular esterases to form nonfluorescent DCFH, which is then rapidly oxidized to form highly fluorescent 20 , 70 dichlorofluorescein (DCF) in the presence of ROS. DCF fluorescence intensity is proportional to the amount of ROS that is formed. Fluorescence was measured (SpectraMax 190 Microplate Reader, Molecular Devices, Sunnyvale, CA, USA) using excitation and emission wavelengths of 485 and 538 nm, respectively. A calibration curve was established with standard DCF (0.1 nm to 1 mm), and results were expressed as nmol of DCF/mg protein. 2.7. Assay of antioxidant enzymes activities Activities of two antioxidant enzymes (SOD and CAT) in plasma were determined with commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's protocol. Briefly, one unit of SOD activity was defined as the amount of enzyme required to inhibit the oxidation reaction by 50% and was expressed as U/mg protein. One unit of CAT activity was defined as the amount of enzyme required to consume 1 mmol H2O2 in 1 s and was expressed as U/ mg protein [26]. 2.8. Assay of oxidative damage Lipid peroxidation products (measured as MDA) in plasma were measured spectrophotometrically at 532 nm by the thiobarbituric acid (TBARS) method according to the instructions of the commercial reagent kit (Nanjing, China), and MDA levels were expressed as nmol/mg protein [26]. Protein carbonyls in plasma were quantified by reaction with 2, 4-dinitrophenylhydrazine (2, 4-DNPH) as described by Levine et al. [27] with slight modifications. In brief, plasma samples were incubated with 10 mM 2, 4-DNPH for 60 min at 37  C. Trichloroacetic acid (TCA, 10% w/v) was added and samples were centrifuged at 12 000 rpm for 10 min at 4  C. The precipitates were washed 3 times with ethanol:ethylacetate (1:1 v/v) and then dissolved in 6 M guanidine chloride (pH 2.3). The absorbance was measured at 360 nm using a SpectraMax 190 Microplate Reader, and the

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carbonyl content was calculated using a molar extinction coefficient of 22 000 M1 cm1. The final result was expressed as nmol carbonyl/mg protein. The DNAeprotein crosslinks (DPCs) of hemocyte in haemolymph were determined by KCl-SDS method of Kuykendall et al. and Liu et al. [28,29] with some modifications. Briefly, the hemocyte suspension was lysed in 2% SDS at 65  C for 10 min. DPCs were precipitated by the addition of 1 M KCl (dissolved in 20 mM TriseHCl, pH ¼ 7.5). The supernatants containing free DNA were collected and the precipitates were resuspended by 1 mL washing buffer (0.1 M KCl, 0.1 mmol L1 EDTA, 20 mmol L1 TriseHCl, pH 7.5). This process was repeated 3 times to collect free DNA and DPC completely. DNA presented in DPC was dissociated with 0.4 mg mL1 proteinase K at 50  C for 3 h, and the supernatant was collected after centrifuging at 12 000 rpm for 10 min and incubating on ice for 5 min. The samples were dyed using Hoechst33258 in the dark for 30 min. The amount of DNA in the samples was determined using a SpectraMax 190 Microplate Reader with excitation and emission wavelengths of 350 and 450 nm, respectively. The DPC coefficient was expressed as the percentage of protein-bound DNA to total DNA (dissociative DNA plus protein-bound DNA). 2.9. Assay of proPO-AS activities PO activity in plasma was measured spectrophotometrically by recording the formation of dopachrome produced from L-3, 4dihydroxyphenylalanine (L-DOPA) following the procedures of
ndez-Lo
pez et al. [30]. Briefly, the supernatant fluid (100 ml) Herna with 100 ml distilled water was added in 3 mL phosphate buffer solution (0.1 mol L1, pH ¼ 6.4) before the addition of 100 ml of LDOPA (0.01 mol L1). Then the mixture was incubated at 30  C for 5 min. The reaction was allowed to proceed and the optical density was measured at 490 nm on a microplate reader per 2 min. One unit of enzyme activity was defined as an increase in absorbance of 0.001/min/mg protein. For measurement of proPO activity, the HLS (100 ml) obtained as described above was incubated with 50 ml trypsin (1 mg mL1), which served as an elicitor, for 10 min at 25  C. Fifty microliters of LDOPA was then added, followed by 800 ml of cacodylate buffer 5 min later. The optical density (OD) at 490 nm was measured using a microplate reader. The control solution, which consisted of 100 ml HLS, 50 ml cacodylate buffer (to replace the trypsin) and 50 ml LDOPA, was used to measure the background PO activity in all test conditions. The background PO activity OD values were subtracted from the PO activity OD values of crayfish. Enzyme activity was expressed as change in absorbance at 490 nm per min per mg of protein [11]. SP activity in HLS was investigated using a synthetic chromogenic substrate BAPNA [31]. A sample of 100 ml of HLS was incubated with 100 ml lipopolysaccharide (1 mg mL1) for 15 min at room temperature. Then, 500 ml of TBS buffer and 50 ml of BAPNA (20 mg mL1) were added and the mixture incubated at 30  C for 30 min. The enzyme reaction was arrested by the addition of 200 mL of 50% (v/v) acetic acid. In the control, the HLS was replaced by TBS buffer. The release of para-nitroanilide (pNA) from the chromogenic peptide was determined spectrophotometrically at 405 nm. 2.10. Assay of hemocyanin level To quantify hemocyanin concentration, plasma was diluted 1:99 with a Tris-Ca buffer (50 mM Tris-HCl þ 10 mM CaCl2, pH ¼ 8.0). Hemocyanin was measured by absorbance of duplicate samples against a blank of pure Tris-Ca buffer at wavelengths of 334 nm.

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Hemocyanin concentration (mg mL1) was calculated using an extinction coefficient of 13.5 for absorbance measurements at 334 nm calculated on the basis of the 74 000-Da functional subunit [32].

All values were expressed as means ± SD and analyzed using SPSS 13.0 for windows (SPSS Inc., Chicago, IL, USA). One-way ANOVA was used to identify the significant difference between the experimental groups and control group. Regression analysis was performed to analyze possible correlations between variables. A confidence limit of 5% was always considered.

(67 h) (Fig. 1E). As shown in Fig. 1 F and G, DPCs coefficient and MDA content displayed similar rising trends according to the exposure time and concentration. The background value of DPCs coefficient in hemocytes was 2.12%e2.18%. Compared to the control, Cu obviously provoked the formation of DPCs in hemocyte, with over 117e186% elevations above background levels following 96 h exposure, respectively (P < 0.05). Meanwhile, MDA content prominently increased by 28.2%, 42.0%, 72.7% and 99.6%, respectively (P < 0.05). Further analysis indicated a strong correlation (P < 0.01) between copper-induced contents of ROS and the levels of protein carbonyl (r ¼ 0.933), DPC (r ¼ 0.969) and MDA (r ¼ 0.971) (Fig. 1H), thus protein carbonyl and DPCs may be viewed as sensitive biomarkers of oxidative stress in haemolymph. In addition, a positive relationship (P < 0.01) between MDA and protein carbonyl (r ¼ 0.945) or DPC (r ¼ 0.948) was found after 96 h exposure (Fig. 1I). This suggested that an oxidative stress leading to membrane peroxidation could rapidly affect protein and DNA in haemolymph, and concomitant oxidative damage on biomolecules may seriously compromise the normal function of hemocytes.

3. Results

3.3. Copper exposure suppressed immune competent

3.1. Copper exposure induced oxidative stress

Fig. 2A showed that Cu resulted in significant time- and concentration-dependent decrease in THC. Compared to the control, THC decreased significantly by 12.2%, 22.7%, 42.0% and 46.3% at 96 h, respectively (P < 0.05). The significant decline in SGC þ GC counts was found in each group following a 24 h exposure (P < 0.05). No obvious differences in SGC þ GC counts were observed among crayfish exposed to Cu2þ in the range of 0.5e4.0 mg/L after 48 h. Only a large increase was detected in crayfish following 72 h exposure to 0.5 mg/L Cu2þ (P < 0.05), but the SGC þ GC counts decreased significantly by 11.3%, 17.0%, 53.0% and 66.3%, respectively, in Cu treatments following 96 h exposure (P < 0.05) (Fig. 2B). Compare to the control, there was no significant difference in HC concentration for crayfish after 24 h exposure to Cu (Fig. 2C). After 48 h, HC level was augmented by lower concentrations of Cu2þ (0.5 mg/L and 2.0 mg/L) but inhibited at higher concentrations (4.0 mg/L and 8.0 mg/L). A significant decrease was found in all experimental groups as the extension of exposure time (P < 0.05). The HC levels decreased significantly by 8.4%, 31.3%, 43.6% and 55.3%, respectively, following 96 h exposure. The changes of proPO activity in HLS were illustrated in Fig. 2D. Only in 0.5 mg/L Cu2þ group, the proPO level returned to normal levels after 96 h. In the Cu2þ range of 2.0e8.0 mg/L, the decline of proPO activity depended significantly on concentration and time of exposure (P < 0.05). Compared to the control, the proPO levels in these crayfish decreased by 30.3%, 48.4% and 51.3%, respectively, after 96 h. When crayfish were exposed to 0.5, 2.0, and 4.0 mg/L Cu2þ, no significant differences in SP activity were observed after 24 h. But it decreased significantly by 42.9% in 8.0 mg/L Cu2þ group compared to the control (Fig. 2E). Then the level of SP significantly decreased with prolongation of exposure time. After 96 h, SP activities decreased by 19.4%, 41.8%, 68.7% and 71.6%, respectively (P < 0.05). Compared to the control, no significant differences in PO activity were observed among crayfish exposed to Cu2þ in the range of 0.5e4.0 mg/L after 24 h. A low concentration of Cu2þ (0.5 mg/L) has no obvious effect on PO activity during a 72 h period. However at higher concentrations, PO activity significantly decreased when crayfish were exposed for 48 h (2.0 and 4.0 mg/L Cu2þ) and 24 h (8.0 mg/L Cu2þ), respectively (P < 0.05). At 96 h, PO activity in treatment groups decreased by 10.7%, 35.0%, 39.3% and 53.6%, respectively (Fig. 2F). A positive correlation (P < 0.01) between SP

2.11. Assay of protein levels Protein concentration in samples was assayed using commercially available kit (Nanjing, China) based on the method described by Bradford [33]. Bovine serum albumin was used as protein standard. 2.12. Statistical analysis

As shown in Fig. 1A, the background level of ROS in haemolymph was 1.26e1.41 nmol DCF/mg protein. When the crayfish were exposed to Cu, an overproduction of ROS was observed, which obviously depended on the concentration and the time of exposure. Compared to the control, ROS levels significantly increased by 0.8efold, 2.0efold, 3.1efold and 4.6efold, respectively, following 96 h exposure to different concentrations Cu2þ (P < 0.05). During a 96-h period, there was a gradual increase in SOD activity in 0.5 and 2.0 mg/L Cu2þ group. At the higher Cu2þ concentration, SOD activity was significantly increased, reaching 115.4% (4.0 mg/L Cu2þ) and 125.9% (8.0 mg/L Cu2þ) following 72 h and 48 h exposure, respectively (P < 0.05). After 96 h, no significant difference was found between those exposed to 4.0 mg/L Cu2þ and the control, but obvious decline occurred only in 8.0 mg/L Cu2þ group (P < 0.05) (Fig. 1B). The activity of CAT in haemolymph was found to be increased within the first 48 h. Obviously, the scavenging activity of CAT on H2O2 was in a concentration-dependent manner (Fig. 1C). As the extension of exposure time, the significant decrease was detected in all experimental groups compared to the control (P < 0.05). After 96 h, CAT activity decreased by 11.9%, 31.1%, 35.2% and 45.9%, respectively. These results indicated that the antioxidant capacity in haemolymph was generally impaired due to a large amount of ROS production, resulting in oxidative stress. 3.2. Copper exposure augmented oxidative damage The background level of protein carbonyl was 1.32e1.38 nmol/ mg protein in plasma. After 72 h exposure to Cu, the highest protein carbonyl content was observed in each group, and there was a strong concentration-dependent induction for the formation of protein carbonyl (P < 0.05) (Fig. 1D). Subsequently, the level of protein carbonyl was found to decrease with increased exposure time. Still, plasma carbonylated protein levels significantly increased by 11.9%, 15.0%, 47.4% and 65.4% at 96 h, respectively, compare to the control. In 8.0 mg/L Cu2þ group, protein carbonyl levels from a total sample size of 36 crayfish were regressed against exposure time. Results revealed that there were highly significant (P < 0.01) correlations between them and the so-called “decarbonylation” may have occurred after carbonylation reached a peak

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Fig. 1. Copper-induced oxidative stress in crayfish haemolymph. A: Reactive oxygen species (ROS) content; B: Superoxide dismutase (SOD) activity; C: Catalase (CAT) activity; D: Protein carbonyls (PC) level; E: Correlation between exposure time and protein carbonyls level at one-third 96 h-LC50 of Cu (n ¼ 36). F: DNAeprotein crosslinks (DPCs) coefficient; G: Malonaldehyde (MDA) level; H: Correlation between ROS levels and biomarkers of oxidative macromolecular damage after a 96-h exposure to Cu2þ (0e8.0 mg/L) (n ¼ 45). I: Correlation between malonaldehyde and the formation of protein carbonyls or the induction of DNAeprotein crosslinks after a 96-h exposure to Cu2þ (0e8.0 mg/L) (n ¼ 45). Asterisks indicate significant difference (P < 0.05) with respect to control. Each point represents an individual crayfish. The t value of regression coefficient is marked in parens.

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Fig. 2. Copper exposure suppressed crayfish proPO-AS activity. A: Total hemocyte counts (THC); B: Semi-granular (SGC) and large granular cells (GC) counts; C: Hemocyanin (HC) level; D: Prophenoloxidase (proPO) activity; E: Serine protease (SP) activity; F: Phenoloxidase (PO) activity. Asterisks indicate significant difference (P < 0.05) with respect to control. G: Correlation between serine protease activity in hemocyte lysate supernatant and phenoloxidase activity in haemolymph after a 96-h exposure to Cu2þ (0e8.0 mg/L) (n ¼ 45). H: Correlation between phenoloxidase activity and levels of prophenoloxidase and hemocyanin after a 96-h exposure to Cu2þ (0e8.0 mg/L) (n ¼ 45). Each point represents an individual crayfish. The t value of regression coefficient is marked in parens.

and PO activities (r ¼ 0.91) was observed in the haemolymph after 96 h of exposure (Fig. 2G). This supported the causal linkage between SP and PO in proPO-AS. There was a strong correlation (P < 0.01) between PO activity and the levels of proPO (r ¼ 0.879) and HC (r ¼ 0.765) (Fig. 2 H). This indicated that an active PO was affected by proPO and HC, while it is not completely understood how HC was induced to exhibit PO activity in the case of Cu exposure. Pearson's correlation analysis also showed that the activities of proPO, PO and SP were significantly and negatively correlated (P < 0.01) with the levels of protein carbonyl, and the correlation

coefficients (r) were 0.829, 0.820 and 0.857, respectively. This suggested that copper-induced oxidative stress may be seriously deleterious to proPO-AS and may finally inhibit the synthesis or activity of PO. However, HC showed a similar downward trend only when protein carbonyl level was reduced to 2.09 nmol/mg protein (Fig. 3A). In the present study, higher level of Cu2þ would make more severe suppression on immune vigor. So the correlation between PC levels and immune-related proteins activities of crayfish exposed to 8.0 mg/L Cu2þ was analyzed (Fig. 3B). The changes of SP in HLS and HC in plasma exhibited a similar variation trend as those shown in Fig. 3A, however, the activities of proPO in HLS and PO in

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Fig. 3. Copper-induced protein carbonylation regulated crayfish proPO-AS activity. A: Correlation between protein carbonyls contents and levels of immune-related proteins, such as prophenoloxidase (proPO), serine protease (SP), phenoloxidase (PO) and hemocyanin (HC), after a 96-h exposure to Cu2þ (0e8.0 mg/L) (n ¼ 45). B: Correlation between protein carbonyls contents and levels of immune-related proteins, such as proPO, SP, PO and HC, at one-third 96 h-LC50 of Cu (n ¼ 36). The t value of regression coefficient is marked in parens.

plasma presented different change. The proPO activity was stimulated by lower levels of protein carbonyl (below 2.40 nmol/mg protein) and inhibited at higher levels (above 2.40 nmol/mg protein). In contrast, PC levels at 2.38 nmol/mg protein or greater resulted in increased PO activity, whereas PC levels below 2.38 nmol/mg protein caused a significant decrease in PO activity. Even so, an excessive Cu2þ exposure could cause greater damage to PO activity in haemolymph as described in Fig. 2F. 4. Discussion The aquatic ecosystem is the ultimate recipient of various pollution originating from natural and anthropogenic sources. In aquaculture, Cu is extensively used in the form of copper sulphate (CuSO4) as an algaecide, antifungal and antiparasite agent. However, excessive Cu can catalyze the production of highly toxic hydroxyl radical ($OH). It reacts instantly and indiscriminately with all biological macromolecules, resulting in enzyme inactivation, lipid peroxidation and DNA damage. Recently, ROS formation and resultant oxidative stress has been regarded as the major mechanism of copper toxicity in aquatic organisms [5,18]. And the toxic effects could be cell specific, organ specific and immune function specific [34]. The nonspecific immune system appears to be

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particularly sensitive to chemical pollutants. There was substantial evidence indicating that Cu2þ could modulate the levels of THCs, DHCs, PO, and respiratory burst in crustaceans, such as Macrobrachium rosenbergii and Palaemon elegans, and down-regulate the expression of immune-related genes in Puntius gonionotus and Morone saxatilis, which seems mostly to be a consequence of oxidative stress [11,22,35]. In order to combat elevated levels of ROS, crustacean hemocytes could express a large number of antioxidants like SOD and CAT, which constitute the first defense line against excess free radicals. SOD is responsible for detoxifying O2to the less-reactive H2O2, and subsequently it was converted into H2O by CAT [14]. But O2- can inhibit CAT activity and the consequent excess of H2O2 resulting from CAT inhibition could finally inhibit SOD activity, thus feeding a pernicious cycle [36]. Some studies also indicated that their decreased activities could be attributed to enzyme and/or protein synthesis inhibition [37]. As a result, severe or persistent stress caused the imbalance between ROS production and scavenging, and irreversible damage and death of hemocytes occurred [38]. Reactive oxygen species $OH and H2O2 are not merely damagecausing but also important signaling molecules involved in the immune defenses. Protein carbonylation is an irreversible oxidative damage, which is often formed via peroxide-dependent Fenton reaction [6,15]. Wong et al. (2008) considered that it may represent a regulatory mechanism to finely tune oxidative stress response [15]. Therefore, protein carbonylation and subsequent degradation (termed decarbonylation) may be important events in stressresponse pathways. Generally, proteins contain carbonyl groups. During oxidative stress, direct damage to proteins or chemical modification of amino acids in proteins will only give rise to new ones [39]. Since elevated PCs were first found in blue mussels Mytilus edulis in 2005 by McDonagh et al., Cu-mediated carbonylation has been confirmed in aquatic organisms such as corkwing wrasse Symphodus melops, zebrafish Danio rerion and crayfish Procambarus clarkii [5,18]. It has been reported that PCs may be generated by the oxidation of several amino acid side chains (e.g. in Lys, Arg, Pro, and Thr); by the formation of Michael adducts between Lys, His, and Cys residues and a, b-unsaturated aldehydes, forming advanced lipoxidation end products; or by glycation/glycoxidation of Lys amino groups, forming advanced glycation end products [40,41]. Subsequent to the peak of carbonylation, carbonylated proteins were subjected to proteasome-dependent degradation, as shown in Fig. 1E. This process was termed “decarbonylation” [15,40]. Even though the nature of decarbonylation remains unclear, it has been observed in some bacteria and several human diseases [42e44]. Actually, only a subset of proteins is prone to carbonylation [45], resulting in an inhibition of their enzymatic activity or an increased susceptibility to proteolysis [15,17]. An increase in PCs levels may indicate that normal protein metabolism had been altered by the accumulation of damaged molecules [46]. In particular, the carbonylated modification of protein may be recognized as non-self by the immune system. The elevated levels of PCs in blood or tissues is closely related to dysfunction and defective immune response [47,48]. It could be found in Fig. 3 that there was a close relationship between proPO-AS activities and PC levels. Further, proteomic analysis will provide deeper insights into the mechanisms of their carbonylation. In addition to protein carbonylation, higher ROS could enhance the risk of lipid peroxidation and DNA damage. MDA is the most abundant by-product of oxidized lipid breakdown, which may then, in turn, attack nearby proteins, causing the formation of excessive PC [40]. DNA is a particularly good target for metal ions due to its electron-rich structure allowing several ligands and complexation sites for positively-charged metal ions. Oxidative damage, in the form of DNA strand breaks and base modifications

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such as 8-OHdG, has been observed following exposure to Cu2þ. In normal cells, DNAeprotein crosslinks (DPCs) are usually induced at basic level. Oxidative stress is able to affect the formation of 50% of DPCs, which is mainly caused by $OH generated from a Fentontype reaction mediated by copper ion [46,49,50]. Several evidences suggested that PC and MDA may also be involved in DPCs formation [20,50]. The transcriptional arrest by persisting DPCs may attenuate the expression of proteins that are essential for cell function [51]. As shown in Fig. 1, Cu2þ-induced oxidative damage to cellular macromolecules, such as proteins, lipids and nucleic acids, may be concomitant and all seriously deleterious [20]. MDA has been proposed to appraise the health status of exposed species. PC and DPCs might be served as potential biomarkers for oxidative perturbations in crayfish. Most immunoreactive proteins like proPO and PO are stored within circulating hemocytes and released in the haemolymph following stimulation with non-self molecules [8,12,13]. The hemocyte number and PO activity have functioned as reliable indicators of immune status in crustaceans [2,7]. Different hemocyte sub-populations undertake different functions with respect to protective immunity of crustaceans. Some authors thought that SGCs and GCs have higher phagocytic ability and ROS production than hyalinocytes upon exogenous stimuli [8]. The proportion change of hemocyte subgroups has even influenced the overall immunocompetence of the organism [52]. Previous studies showed that Cu could improve the percentage of granulocytes in oysters and mussels and reduce THC in the white shrimp Litopenaeus vannamei [21,23]. But there was no significant change in THC and DHC for the giant freshwater prawn Macrobrachium rosenbergii following a 96 h exposure to CuSO4 in the range of 0e0.4 mg L-l [11]. These different responses in hemocyte count could be due to different species, exposure concentration and exposure time. When the shrimps were exposed to high dose of Cu2þ, it was found that the elevated ROS correlated well with increased apoptotic and necrotic hemocytes and decreased THC [2], which may be attributed to lipid peroxidation and DNA damage [53]. But some studies suggested necrosis in gills and hepathopancreas could lead to the recruitment of circulating hemocytes to the site of injury, reducing their number in the haemolymph [5,11,18]. In crayfish haemolymph, ROS may be generated through multiple sources such as copper exposure, activated haemocytes (known as respiratory burst) [7] and increased PO [9,10]. The exact mechanism by which copper influences hemocyte population is still unknown, however, because superfluous ROS is cytotoxic, it exerts a direct effect on the hemocytes, perhaps causing cell death or degranulation [2,7,23]. The decline of THC, especially SGC and GC counts, could cause the lower proPO and PO levels [21,54]. Crustaceans contain two types of protein generating PO activity: one being its precursor proPO and the other being HC [55]. The proPO is activated by restricted proteolysis cascade called the proPO-AS involving SP. PO is a bifunctional copper containing enzyme that promotes the melanization, during which many toxic molecules-such as cytotoxic quinones and reactive oxygen-are produced [9,10]. Thus, it is quite plausible that increased PO activity could indirectly result in elevated levels of free radicals, which could produce oxidative stress and other free radical-mediated cellular damage. The active components of proPO-AS, in particular PO, has to be controlled and regulated to avoid the deleterious effects. Several proteinase inhibitors for preventing over-activation of prophenoloxidase-activating enzyme (ppA) and a phenoloxidase inhibitor (POI), which can directly inhibit the PO activity, have been reported from several arthropod species [10]. It is well known that PO is synthesized as an inactive zymogen called proPO, and conversion of proPO to PO occurs through ppA, a serine protease. Phylogenetic analysis revealed that proPO of P. clarkii is distinctly

far away from that of insecta, and is also distinct from that of penaeid shrimps, lobster, and freshwater prawn [13]. Among arthropods, serine proteases act as potent activators of diverse immune cascades [8]. A serine protease cascade for insect proPO activation has been proposed in 1986. Subsequently, many proteins, such as ppA and proPO-activating proteinase (PAP), have been found to regulate proPO activation using B. mori, Manducasexta, Drosophila melanogaster, Holotrichiadiomphalia, and mosquitoes as models [9]. Now an endogenous SP that can activate proPO has been isolated from crayfish hemocytes [56]. In haemolymph, adequate numbers of circulating protein is a prerequisite for maintenance of hemocytes stability, oxygen transport, cell integrity etc [54]. Crustacean HC accounts for 90e95% of the haemolymph protein [3,55]. It is the largest copper pool in shrimp and estimated to be up to 40% of the whole-body Cu load [4]. Recently, arthropod HC has been demonstrated to originate from ancient proPO-like proteins, and they can be induced to exhibit PO activity by some reagents or endogenous molecules [12,55]. All arthropod proPOs possess sequences similar to those of HC, which contains two copper-binding motifs, and six histidine residues within two copper-binding motifs. These features are also highly conserved in arthropod proPOs [12]. Therefore, the activation of proPO-AS seems to be more complex than we realize. From Figs. 1E and 3B, we speculated that intracellular protein carbonylation and subsequent “decarbonylation” may regulate proPO activation. As shown in Fig. 2G and H, the levels of PO in haemolymph was directly related to at least three factors: SP and zymogenic proPO existed in hemocyte and HC, which was synthesized in the hepatopancreas and secreted in the haemolymph. But it can be noticed that the activities of these proteins were obviously affected by PC level (Fig. 3). In our previous study, tissue-specific formation of protein carbonyls in crayfish digestive gland has been reported [5], however, it seemed that carbonylation in hepatopancreas and haemolymph showed different effects on hemocyanin. Apparently, this series of changes was the biochemical perturbation in hemocytes induced by copper exposure. Copper-catalyzed protein carbonylation may be one of the main mechanisms behind immunity dysfunction of crayfish proPO-AS. In recent years, the potential relationship between environmental pollution and disease incidence in aquatic organisms has received increasing attention. A number of nutrients, such as vitamin E, ascorbic acid, beta-carotene etc., that possess antioxidant capabilities may provide protection against free radical-mediated molecular damage [57]. It is very important to alleviate stresses and enhance the animal's innate immunity through nutritional methods. Acknowledgements This work was supported by grants from the Research Fund for the Doctoral Program of Higher Education (RFDP) of China (200801081012) and the Special Fund for Talent Introduction and Development of Shanxi Province, China (2006). References [1] Ministry of Agriculture, State Environmental Protection Administration, Report on State of the Fishery Eco-environment in China, Ministry of Agriculture, State Environmental Protection Administration, Beijing, China, 2012. [2] J.A. Xian, A.L. Wang, C.X. Ye, X.D. Chen, W.N. Wang, Phagocytic activity, respiratory burst, cytoplasmic free-Ca2þ concentration and apoptotic cell ratio of haemocytes from the black tiger shrimp, Penaeus monodon under acute copper stress, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 152 (2010) 182e188. [3] B. Soedarini, L. Klaver, I. Roessink, B. Widianarko, N.M. van Straalen, C.A. van Gestel, Copper kinetics and internal distribution in the marbled crayfish (Procambarus sp.), Chemosphere 87 (2012) 333e338. [4] H.H. Taylor, J.M. Anstiss, Copper and haemocyanin dynamics in aquatic invertebrates, Mar. Freshw. Res. 50 (1999) 907e931.

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