Marine Environmental Research 101 (2014) 81e90

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The biological importance of glutathione peroxidase and peroxiredoxin backup systems in bivalves during peroxide exposure Rafael Trevisan*, Danielle Ferraz Mello, Marcela Uliano-Silva 1, Gabriel Delapedra, Miriam Arl, Alcir Luiz Dafre
polis 88040-900, Brazil Biochemistry Department, Federal University of Santa Catarina, Floriano

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 July 2014 Received in revised form 15 September 2014 Accepted 19 September 2014 Available online 22 September 2014

Organic peroxide elimination in eukaryotes essentially depends on glutathione peroxidase (GPx) and peroxiredoxin (Prx) enzymes, which are supported by their respective electron donors, glutathione (GSH) and thioredoxin (Trx). This system depends on the ancillary enzymes glutathione reductase (GR) and thioredoxin reductase (TrxR) to maintain GSH and Trx in their reduced state. This study discusses the biological importance of GR and TrxR in supporting GPx and Prx during cumene hydroperoxide (CHP) exposure in brown mussel Perna perna. ZnCl2 or 1-chloro-2,4-dinitrobenze (CDNB) was used to decrease GR and TrxR activities in gills, as already reported with mammals and bivalves. ZnCl2 exposure lowered GR activity (28%), impaired the in vivo CHP decomposition and decreased the survival rates under CHP exposure. CDNB decreased GR (54%) and TrxR (73%) activities and induced glutathione depletion (99%), promoting diminished peroxide elimination and survival rates at a greater extent than ZnCl2. CDNB also increased the susceptibility of hemocytes to CHP toxicity. Despite being toxic and causing mortality at longer exposures, short (2 h) exposure to CHP promoted an up regulation of GSH (50 and 100 mM CHP) and protein-thiol (100 mM CHP) levels, which was blocked by ZnCl2 or CDNB pre-exposure. Results highlight the biological importance of GSH, GR and TrxR in supporting GPx and Prx activities, contributing to organic peroxides elimination and mussel survival under oxidative challenges. To our knowledge, this is the first work that demonstrates, albeit indirectly, the biological importance of GPx/GR/GSH and Prx/TrxR/Trx systems on in vivo organic peroxide elimination in bivalves. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Mussels Perna perna Antioxidants Glutathione reductase Thioredoxin reductase Oxidative stress Cumene hydroperoxide Gills

1. Introduction The result of cellular energy production mediated by aerobic metabolism occurs simultaneously with the generation of potential toxic oxygen intermediates, known as reactive oxygen species (ROS), which in excess may pose a considerable threat to cellular homeostasis (Cadenas and Davies, 2000). Among ROS, peroxides (e.g. H2O2) can be generated by diverse metabolic processes, including dismutation of superoxide anion, or as byproduct of oxidases such as monoamine oxidases (Dringen et al., 2005). Organic peroxides are also generated by cells, for example trough the action of cyclooxygenases and lipoxygenases or by oxidation of polyunsaturated fatty acids (Dringen et al., 2005). At physiological levels, or close to

* Corresponding author. E-mail address: [email protected] (R. Trevisan). 1 Present address: Institute of Biofísica Carlos Chaga Filho, Federal University of Rio de Janeiro, 21044-020 Rio de Janeiro, RJ, Brazil. http://dx.doi.org/10.1016/j.marenvres.2014.09.004 0141-1136/© 2014 Elsevier Ltd. All rights reserved.

that, ROS play an important role activating/deactivating cellular signaling pathways, allowing adaptive responses during stressful situations (Schieber and Chandel, 2014). Excessive ROS production is harmful to the cell, and toxicity is potentiated by its high reactivity. Reaction with biomolecules such as proteins, nucleic acids and lipids, can affect cellular metabolism (Imlay, 2003). The cellular defense against ROS is mediated by enzymatic and non-enzymatic antioxidants, responsible for maintaining the cytosol reductive environment and cellular function (Valko et al., 2007). Intracellular peroxide metabolism is mainly mediated by three different antioxidant enzymes: catalase (Cat), glutathione peroxidase (GPx) and peroxiredoxin (Prx). While the catalytic cycle of Cat is independent of reducing agents and catalyzes only H2O2, GPx and Prx use intracellular electron donors for their peroxidase activity against H2O2 and organic peroxides. GPx uses glutathione (GSH) as reducing agent, generating oxidized glutathione (GSSG). A high GSH/GSSG ratio is maintained by glutathione reductase (GR), using NADPH as a final electron donor (Bindoli et al., 2008). On the other hand, 2-Cys Prxs

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reduce peroxides with electrons provided by thioredoxin (Trx) (Rhee and Woo, 2011). Oxidized thioredoxin is converted back to its reduced state by thioredoxin reductase (TrxR), which also uses NADPH as final electron donor (Rhee and Woo, 2011). Therefore, peroxide elimination relies on peroxidase enzymes themselves and also on their backup systems composed by the reductase enzymes, such as GR and TrxR, and their ability to reduce their respective substrates, GSSG and
et al., 2011). The reduced forms of GSH and Trx are oxidized Trx (Flohe considered important cellular thiol pools closely associated to ROS detoxification processes. Decreases in the GSH/GSSG or reduced/ oxidized Trx ratios are usually associated with the adverse effects of oxidative compounds, triggering death/survival responses (Circu and Aw, 2010). Therefore, thiol/disulphide ratios can be used as markers of oxidative stress, since the maintenance of GSH and Trx in their reduced forms is essential for a number of cellular functions, including those dependent on GPx and Prx activities (Toledano et al., 2013). The H2O2 steady state levels are considerably low in marine invertebrates when compared to mammals, with mitochondria being taken as a major site of ROS production (see review of Abele and Puntarulo, 2004). During non-stressful situations, these lower ROS generation rates found in invertebrates (including bivalves) are related to their lower metabolic rate (Buttemer et al., 2010). In bivalves, the rate of ROS production, as well as the antioxidant defences, can be modulated by diverse biotic and abiotic factors, including age, size, temperature, salinity and metabolic rate (Frenzilli et al., 2004; Lau and Wong, 2003; Regoli et al., 2000; Sheehan and Power, 1999; Sukhotin et al., 2002). For instance, the spawning season of Mytilus galloprovincialis is marked by an increase in ROS production, such as H2O2 and organic hydroperoxides, generating oxidative stress in gills and digestive gland (Soldatov et al., 2008). Thermal stress in summer season and during reproductive activity is also responsible for an increase in oxygen consumption and ROS production in mussels (Arthur, 2001; Verlecar et al., 2008; Wilhelm Filho et al., 2001). Therefore, elimination of peroxides by redundant peroxidase systems can have a major contribution in the protection of marine invertebrates during both physiological and stressful conditions. It is long known that zinc is able to inhibit GR activity in vitro at the micromolar range (Mize and Langdon, 1962). This effect was also detected in vivo in mussels Perna perna acutely exposed to ZnCl2 (Franco et al., 2006), without alterations on GR protein content (Trevisan et al., 2014). Similarly, it has been shown that 1chloro-2,4-dinitrobenze (CDNB) inhibits TrxR activity in vitro
r et al., 1995). Our group demonstrated that CDNB causes a (Arne marked decrease in both GR and TrxR activities in vivo in oysters Crassostrea gigas (Trevisan et al., 2012). Given that GPx and Prx activities rely on a constant GSH and Trx recycling, the aim of the present study was to evaluate the effects of peroxidase systems impairment of brown mussels during peroxide exposure by previously inhibiting GR and TrxR. Animals were acutely exposed to ZnCl2 or CDNB as an approach to decrease GR and TrxR activities. The role of the peroxidase systems on animal survival, detoxification and cellular viability was investigated in animals subsequently exposed to cumene hydroperoxide (CHP), a model GPx and Prx substrate. Our study discusses the importance of the peroxidase systems and thiol homeostasis in brown mussels under an exogenous pro-oxidant challenge.

GSSG, GR, N-ethylmaleimide (NEM), neutral red, NADPH, perchloric acid (PCA), tris(hydroxymethyl)aminomethane (TRIS), sodium dodecyl sulfate (SDS), xylenol orange and zinc chloride were obtained from SigmaeAldrich (Brazil). All other reagents used were of analytical grade. 2.2. Animals and acclimation conditions Brown mussels P. perna (adults, 8e10 cm shell length) were ~o da Ilha beach, obtained from a commercial mussel farm at Ribeira
polis (Brazil). This area is an important Brazilian bivalve Floriano farming site, with constant water quality analysis and metal levels
enz et al., 2010). Mussels were collected within legislation limits (Sa during different seasons along the experiments, acclimated over 7 days under laboratory conditions (22e25  C, 12 h light/dark) in plastic aquaria (1 L clean seawater/animal) and fed every two days with a commercial food for marine filter-feeding invertebrates (Sera Marin Coraliquid). Experimental procedures were followed in accordance to the Federal University of Santa Catarina ethical policy on the use of animals. 2.3. Acute exposure to ZnCl2 and CDNB Mussels were exposed to different concentrations of ZnCl2 (0; 30; 40; 50 and 100 mM, equivalent to 0, 2.0, 2.6, 3.3 and 6.5 mg Zn2þ/ L) and CDNB (0; 3; 6; 10 and 25 mM) for 18 h in glass aquaria with 1 L of seawater per animal. These concentrations were based on previous studies of our group with mussels and oysters (Franco et al., 2006; Trevisan et al., 2012, 2014). After exposure, animals were sacrificed and gills were collected for biochemical analyses. Tissues were homogenized (1:4 w:v) in 20 mM HEPES buffer pH 7.0 and centrifuged at 20,000 g for 30 min at 4  C. Supernatant was collected and activities of enzymes were analyzed spectrophotometrically. GR activity was measured at 340 nm through the NADPH consumption rate in the presence of GSSG (Carlberg and Mannervik, 1985). TrxR activity was measured at 412 nm through
r et al., the DTNB reduction rate, in the presence of NADPH (Arne 1999). Total GPx activity was measured at 340 nm through the NADPH consumption rate in the presence of GR, GSH and CHP (Wendel, 1981). All assays were performed using a Cary 50 UVeVIS instrument (Agilent Technologies®, USA). 2.4. Survival of brown mussels to CHP exposure Mussels were exposed to CHP (0; 100; 300; 1000 and 3000 mM, prepared in seawater, 1 L/animal) over 96 h and mortality was checked daily. After establishment of the LC50 (96 h), an additional set of experiments was performed using 100 or 300 mM CHP. Based on the results obtained in experiments outlined in section 2.3, a concentration of ZnCl2 (40 mM) and CDNB (10 mM) was chosen for further studies using 18 h as a pre-exposure period. After the preexposure to ZnCl2 or CDNB, mussels were further exposed to CHP 100 or 300 mM for 96 h and the mortality rate was checked daily. An additional group of mussels previously exposed to ZnCl2 or CDNB were also maintained in clean seawater for 96 h, and the mortality rate related only to ZnCl2 or CDNB pre-exposure was assessed. During experiments, water was changed each 24 h and CHP was replaced. Animals were not fed during exposure periods.

2. Material and methods 2.5. Analysis of in vivo CHP decomposition rate 2.1. Chemicals CDNB, MTT, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 5,50 -dithiobis-(2-nitrobenzoic acid) (DTNB), bovine serum albumin, CHP, ethylenediamine tetraacetic acid (EDTA), GSH,

CHP decomposition rate was analyzed in seawater in the presence or absence of brown mussels, according to our previously published method (Trevisan et al., 2012). To verify whether preexposure to ZnCl2 (40 mM) or CDNB (10 mM) for 18 h could

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interfere with peroxide elimination, the CHP decomposition rate was evaluated in vivo in control, ZnCl2 or CDNB pretreated animals. Mussels were acclimated for 1 h in individual beakers containing 300 ml of clean seawater with constant aeration. Later, 200 ml of CHP (previously dissolved in seawater) were carefully added to a final concentration of 50 and 100 mM, and animals were maintained in this condition for 2 h. Aliquots of seawater (35e50 mL) were collected over the 2 h exposure for measurements of the remaining CHP level. Two additional groups were analyzed: animals exposed to clean seawater (blank) and beakers containing only CHP but no mussels (CHP auto-decomposition rate). Samples were immediately added to a 96 well microplate and volume was adjusted to 200 mL with ultrapure water. Next, 50 mL of the reaction mixture (2.5 mM xylenol orange, 2.5 mM ammonium iron (II) sulfate and 1.1 mM PCA) were added and the plate was incubated in the dark at room temperature for 30 min (Gay and Gebicki, 2002). Absorbance was estimated at 560 nm using a TP-Reader (Thermo Plate®, Brazil) and remaining CHP level in seawater was calculated based on a CHP standard curve. CHP values obtained over time were plotted in a line chart against their corresponding time period and analyzed using exponential one phase decay curves. The parameters were adjusted as following: plateau constrained to a constant value of zero and initial values constrained to 50 for 50 mM CHP and 100 for 100 mM CHP. The CHP half-life (t1/2) was calculated for each individual data as an indicative of the in vivo CHP decomposition rate. Values from mussels pretreated with ZnCl2 or CDNB and further exposed to CHP (50 or 100 mM) were compared to the t1/2 values from ZnCl2 and CDNB non-treated mussels exposed only to CHP (control).

glutathione) and reduced protein thiol (PSH) measurements, approximately 50 mg of fresh tissue (wet weight) was homogenized in 500 mL of 0.5 M PCA and centrifuged at 15,000 g for 2 min at 4  C. Supernatant was assayed for GSH-t while pellet was assayed for PSH levels. For GSSG measurements, approximately 100 mg of fresh tissue (wet weight) was homogenized in 400 mL of 6.25 mM NEM. Samples were acidified with 0.5 M PCA and centrifuged at 15,000 g for 2 min at 4  C. Excess of NEM was removed following an alkaline hydrolysis protocol (Sacchetta et al., 1986). GSH-t and GSSG analyses were spectrophotometrically assayed following a standard method (Akerboom and Sies, 1981) using a Cary 50 UVeVIS. For PSH assay, the pellet was washed twice with 0.5 M PCA and solubilized in 0.5 M TRIS buffer pH 8.0 containing 1% SDS. Samples were assayed according to a colorimetric assay (Jocelyn, 1987) using a TP-Reader.

2.6. Hemocyte analysis

3. Results

Animals (previously exposed to ZnCl2, CDNB or untreated control animals) were exposed to CHP as described in Section 2.5. After 2 h, hemolymph was collected via the adductor muscle using a 21G needle coupled with a 1 ml syringe and used for total hemocyte counting (THC) and cellular viability assays. For THC, an aliquot of the hemolymph from each animal was fixed with 4% formaldehyde in a PBS-NaCl (phosphate buffer saline containing 3.3% NaCl) solution and hemocyte cellular density was readily determined using an improved Neubauer chamber. Hemocyte viability was measured as previously described in oysters (Mello et al., 2012), adapted to brown mussels. Briefly, for the MTT assay, hemolymph containing 1  106 cells was centrifuged at 300 g for 10 min at 4  C, resuspended in 300 mL of 0.5 mg/ml MTT (prepared in PBS-NaCl solution) and incubated for 1 h in the dark. After centrifugation at 800 g for 10 min at 4  C, supernatant was removed and insoluble formazan formed from MTT reduction was solubilized with 200 mL of pure dimethyl sulfoxide (DMSO). The absorbance was assessed at 550 nm through a TP-Reader. For the neutral red retention assay, hemolymph containing 4  105 cells was centrifuged at 300 g for 10 min at 4  C, resuspended in 150 mL of 0.004% neutral red (prepared in PBS-NaCl solution) and incubated for 3 h in the dark. After centrifugation at 800 g for 10 min at 4  C, samples were washed with PBS-NaCl and supernatant was removed. Intracellular neutral red was extracted with 200 mL of an ethanol (50%)/acetic acid (1%) solution and absorbance was assessed at 560 nm through a TP-Reader.

3.1. The effect of ZnCl2 and CDNB on GR and TrxR activities

2.8. Statistical analysis Due to the high variability of the biochemical and cellular responses of marine invertebrates, all experiments were repeated three times, with 3e6 animals per group per experiment. For the survival experiments, curves were compared using PROBIT analysis by ManteleCox Test. For the in vivo peroxide detoxification rates analyses (CHP t1/2), data was analyzed by KruskaleWallis followed by the Dunn post hoc. For the other experiments, data was checked for normality and differences in variances and multiple comparisons were made by one or two-way analysis of variance followed by the Duncan post hoc test, when appropriate, except for Data are presented as mean ± SD.

In this study, brown mussels P. perna were acutely exposed to ZnCl2 or CDNB in order to decrease the activity of the ancillary enzymes GR and TrxR in gills, thus probably affecting GSH and Trx recycling as well as GPx and Prx function during peroxide exposures. ZnCl2 caused a decrease in both GR and TrxR activity (Fig. 1): mussels exposed to 40 mM ZnCl2 presented approximately 30% GR activity loss, whereas exposure to 100 mM ZnCl2 decreased GR activity in 92%. TrxR activity was also sensitive to ZnCl2, presenting 48% and 78% activity loss after exposure to 50 and 100 mM, respectively. On the other hand, CDNB caused a decrease in both GR (54%) and TrxR activity (73%) with only 10 mM (Fig. 1). Similar decrease was also detected in animals exposed to 25 mM CDNB (Fig. 1). GPx activity was measured in gills, under the same conditions, but no significant difference was observed between control and ZnCl2 or CNDB exposed animals (data not shown). Based on these results, the concentrations of 40 mM (for ZnCl2) and 10 mM (for CDNB) were chosen for subsequent experiments. At these concentrations, ZnCl2 affected only GR activity (28%), possibly interfering with GSSG reduction and the transference of reducing potential required for completion of the GPx catalytic cycle. Once CDNB decreased both reductase activities (54% GR and 73% TrxR) these lowered activities would impair the transfer of reducing potential to support both GPx and Prx activities. 3.2. Survival of brown mussels after CHP exposure

2.7. Analysis of thiol/disulfide status Mussels (previously exposed to ZnCl2 and CDNB or untreated control animals) were exposed to CHP as described in Section 2.5. Animals were sacrificed and gills were dissected for thiol measurement. For total glutathione (GSH-t, the sum of reduced and oxidized

An oxidative challenge was performed by exposing the mussel P. perna to CHP (0e3000 mM) (Fig. 2A). The CHP toxicity was confirmed, with an LC50 (96 h) of approximately 550 mM (Fig. 2B). In order to investigate the protective effects of GR and TrxR, a second set of experiments was performed. Mussels were previously pretreated

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Fig. 1. Activities of glutathione reductase (GR) and thioredoxin reductase (TrxR) in gills of brown mussels Perna perna exposed to zinc chloride (ZnCl2) or 1-chloro-2,4dinitrobenzene (CDNB) for 18 h. (A) GR and (B) TrxR activities of mussels exposed to ZnCl2; (C) GR and (D) TrxR activities of mussels exposed to CDNB. Data are presented as mean ± SD. Groups presenting distinct letters are statistically different (p < 0.05), according to one way-ANOVA followed by Duncan's post hoc test. Absolute enzymatic activities (mean ± SD) of control (Ctl) animals are GR 75.4 ± 9.0 and TrxR 23.6 ± 4.3 mU/mg protein (n ¼ 6e38).

to 40 mM ZnCl2 or 10 mM CDNB for 18 h and further exposed to CHP (100 or 300 mM) for 96 h. At these concentrations CHP caused 22% and 42% of mortality, respectively. ZnCl2 pre-treatment increased this rate to 42 and 83% (Fig. 2C and D), while CDNB pre-treatment increased to 82 and 88% (Fig. 2E and F), respectively. ZnCl2 and CDNB did not cause mortality when used alone (data not shown). 3.3. Analysis of in vivo CHP decomposition The significance of lower GR and TrxR activities in gills were also assessed by analyzing the CHP decomposition rate in vivo. CHP levels were monitored over time in seawater during animal exposure to 50 or 100 mM CHP (2 h), and CHP t1/2 was determined by non-linear regression (exponential one-phase decay). Our analysis estimated the CHP t1/2 as approximately 30 min (50 mM, Fig. 3A) and 79 min (100 mM, Fig. 3D). No decrease in CHP levels was detected in seawater in the absence of animals (Fig. 3A and D). Although it was detected a pronounced variation on the CHP t1/2 in ZnCl2 or CDNB pre-treated animals, a general pattern of slower CHP detoxification was observed (Fig. 3B and E). For 50 mM CHP, CDNB increased t1/2 in approximately 15 times (464 min) while no significant effect was detected after ZnCl2 pre-treatment (Fig. 3C). For 100 mM CHP the effects were also evident (Fig. 3F): ZnCl2 increased CHP t1/2 from 79 to 204 min, while CDNB pre-treatment increased it 4 times to 321 min. Therefore, both ZnCl2 and CDNB significantly decreased CHP decomposition capacity in mussels, especially during a more pronounced CHP exposure. 3.4. Effects of CHP exposure on total hemocyte count and viability The results revealed that animals treated with ZnCl2 and subsequently exposed to CHP 100 mM presented 92% increase in the number of circulating hemocytes in comparison to animals with no pre-treatment (Fig. 4A). Hemocyte viability, measured through the MTT assay, decreased (53%) in hemocytes of animals pretreated with CDNB and further exposed to CHP 100 mM for 2 h (Fig. 4B).

Viability was not affected by ZnCl2. Hemocyte viability was also monitored through the neutral red retention assay, however no significant alteration was observed in any treatment (data not shown). It is important to note that although both MTT and neutral red retention assays specificities are debated, they are used to roughly estimate cellular viability. Each assay is related to different cellular compartments and therefore may not present the same responses: while MTT is dependent on mitochondrial dehydrogenases, the neutral red retention ability is associated to lysosome and cytoplasmic membranes stability. 3.5. Thiol/disulfide status of brown mussels exposed to CHP GSH-t, GSSG and PSH levels were also evaluated in this study. Remarkably, CHP exposure alone (50 or 100 mM for 2 h) caused an increase in GSH-t levels (20e25%, Fig. 5A) in non-pretreated animals (Ctl), and PSH levels increased 32% in animals exposed only to 100 mM CHP (Fig. 5C). This thiol increase (GSH-t and PSH) was not detected in animals pretreated with ZnCl2 or CDNB and further exposed to CHP (Fig. 5A and C). Additionally, CDNB pre-treatment (10 mM over 18 h) led to the depletion of almost all GSH-t and GSSG in gills (Fig. 5A and B) but without affecting PSH levels (Fig. 5C) in animals exposed or not to CHP. For GSSG, ZnCl2 pre-treatment or CHP did not cause alteration of the basal levels (Fig. 5B). 4. Discussion Increase in ROS production causes lipid peroxidation, protein oxidation and DNA damage in invertebrates (Ma et al., 2013; Paital and Chainy, 2014; Itziou et al., 2011), which may induce different cellular events, including activation of signaling pathways and gene transcription, altered metabolism, cell survival or death (ParrillaTaylor and Zenteno-Savín, 2011; Qian et al., 2014; Koutsogiannaki et al., 2014). Proteomics studies have identified a wide range of proteins as targets of ROS toxicity in invertebrates, related to metabolism, stress response, protein translation, cell structure and

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Fig. 2. Effects of ZnCl2 and CDNB on the survival rate of brown mussels Perna perna exposed to cumene hydroperoxide (CHP). (A) Survival rate of animals exposed to CHP for 96 h. (B) Representation of the 96 h data from (A) to obtain the distinct LC50(96 h), estimated as ~550 mM. (C, D) Mussels previously exposed to 40 mM ZnCl2 or (E, F) 10 mM CDNB for 18 h and further exposed to CHP for 96 h. No mortality was detected for 96 h in animals only pre-treated with ZnCl2 or CDNB (data not shown). After PROBIT analyses, differences in survival rates were calculated by the ManteleCox test. Groups presenting distinct letters are statistically different at p < 0.001 (n ¼ 12).

redox homeostasis (Kumsta et al., 2011; Cole et al., 2014). Therefore, detoxification systems are essential for the survival of organisms and particularly important when animals are exposed to different stressful conditions (Vasseur and Leguille, 2004). The importance of different antioxidants has been documented in bivalves exposed to chemicals such as metals and pesticides: lower GPx activity was associated to higher ROS levels and cellular damage during mercury exposure, while higher activity protects against the oxidative stress induced by copper (Chatziargyriou and Dailianis, 2010; Trevisan et al., 2011); the phospholipid hydroperoxide glutathione peroxidase can protect membrane phospholipid under metal exposure (De Almeida et al., 2004); depletion of GSH was related to pesticide ~ atoxicity, increasing the susceptibility to oxidative stress (Pen Llopis et al., 2002). These data highlight the importance of investigating the biological role of antioxidant enzymes on animal homeostasis and survival during exposure to compounds associated to induction of oxidative stress. 4.1. ZnCl2 and CDNB pre-treatment The biological consequences of GR and TrxR activity loss, in our experimental set up, were achieved by exposing animals to ZnCl2 or CDNB. ZnCl2 caused a decrease in GR activity without affecting thiol

levels or TrxR activity, while CDNB decreased GR and TrxR activities as well as GSH-t levels. Zinc seems to bind to exposed sulfhydryl groups of the protein and inactivates the enzyme as previously demonstrated by in vitro experiments (Mize and Langdon, 1962). €user, CDNB is a known TrxR inhibitor in mammals (Heiss and Gerha 2005; Seyfried and Wüllner, 2007), probably alkylating the reactive protein thiol groups of TrxR and consequently affecting the enzyme
r et al., 1995). activity (Arne Zinc toxicity has been related to GR inhibition and thiol/disulfide impairment in lung and neuronal cells (Bishop et al., 2007; Mitozo et al., 2011; Walther et al., 2000) as well as brown mussels (Franco et al., 2006; Trevisan et al., 2014) and common carp (Franco et al., 2008b). Furthermore, similar to the results obtained in our study with high concentrations of ZnCl2, zinc exposure also caused TrxR inhibition in different mammalian cell models and mitochondria (Bragadin et al., 2004; Gazaryan et al., 2007). In addition, CDNB has already been used to impair the oysters C. gigas antioxidant system, causing GR and TrxR inhibition with parallel GSH depletion, which increased animal susceptibility to oxidative stress (Trevisan et al., 2012). It is predicted that GR inhibition limits GSH regeneration from GSSG, which is necessary to maintain the catalytic cycle of GSHdependent enzymes, such as GPx and other processes that

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Fig. 3. Effects of ZnCl2 and CDNB on the in vivo peroxide detoxification rate in brown mussels Perna perna exposed to cumene hydroperoxide (CHP). Animals were pre-exposed to 40 mM ZnCl2 or 10 mM CDNB for 18 h and further exposed to 50 or 100 mM CHP for 2 h and CHP levels in seawater were analyzed as indicated at Section 3.3. (A) CHP (50 mM) decomposition rate by control (Ctl) animals or in the absence of animals. (B) Graph comparing the 50 mM CHP decomposition rate by Ctl, ZnCl2 or CDNB pre-exposed animals. (C) The 50 mM CHP t1/2 analyzed by exponential one phase decay of Ctl, ZnCl2 or CDNB pre-exposed animals. Results with 100 mM CHP are also presented (D, E and F). Curves are shown only as mean, while t1/2 results are presented as mean ± SD (n ¼ 10e12). Groups presenting distinct letters are statistically different (p < 0.05) according to Kruskal Wallis test followed Dunn's post hoc test.

Fig. 4. Effects of ZnCl2 and CDNB on hemocytes of brown mussels Perna perna exposed to cumene hydroperoxide (CHP). Animals were pre-exposed to 40 mM ZnCl2 or 10 mM CDNB for 18 h and further exposed to 50 or 100 mM CHP for 2 h and hemocytes were analyzed. (A) Total hemocyte count (THC) and (B) MTT reduction assay. Data are presented as mean ± SD (n ¼ 6e12). For each pre-treatment group (control e Ctl, ZnCl2 or CDNB), columns presenting distinct letters are statistically different (p < 0.05). # represents statistical differences when compared to control animals unexposed to CHP (p < 0.05). Analysis were performed by two way-ANOVA followed by Duncan's post hoc test. The absolute value of THC in Ctl animals (mean ± SD) was 3.26 ± 1.00  106 cells/ml.

depend on the reduced form of glutathione (Dickinson and Forman, 2002). Similarly, TrxR inhibition could limit Trx regeneration from its oxidized form, affecting the activity of the fast peroxidases Prx, which present activity comparable to GPx and act as a major peroxide detoxifying enzymes in mitochondria and cytosol (Peskin et al., 2007; Rhee and Woo, 2011; Sies, 2014). Therefore, it is expected that animals pretreated with ZnCl2 presented a slower catalytic cycle of GPx due to the lower GSH regeneration rates in consequence of the GR activity loss. Moreover, both GPx and Prx catalytic cycles could be affected by the CDNB pre-exposure due to the lower GR and TrxR activities as well as on depleted GSH-t levels. Both scenarios (ZnCl2 and CDNB pre-exposure) could lead to impairment in animal's ability to cope with peroxides.

To investigate this hypothesis, CHP was chosen as model organic peroxide. This hydroperoxide has been used as an oxidative agent for biochemical studies in different organisms, such as bacteria (Chen et al., 1998), yeast (Lewinska et al., 2004), rat (Jou et al., 2004), mouse (Sultana et al., 2003) and cultured cells (Mitozo et al., 2011), where it was related to oxidative damage as well as to mitochondrial and cellular dysfunction. Therefore, CHP could be used to investigate the effectiveness of the bivalve antioxidant system on peroxide removal and protection. Indeed, the antioxidant impairment caused by ZnCl2 or CDNB pre-treatment decreased the CHP in vivo decomposition rate, in agreement with our a priori hypothesis: control mussels efficiently detoxified CHP,

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Fig. 5. Effects of ZnCl2 and CDNB on the cellular thiol levels in gills of brown mussels Perna perna exposed to cumene hydroperoxide (CHP). Animals were pre-exposed to 40 mM ZnCl2 or 10 mM CDNB for 18 h and further exposed to 50 or 100 mM CHP for 2 h and gills were analyzed (A) Total glutathione (GSH-t), (B) protein thiols (PSH) and (C) oxidized glutathione (GSSG) levels. Data are presented as mean ± SD (n ¼ 12). For each pre-treatment group (control e Ctl, ZnCl2 or CDNB), columns presenting distinct letters are statistically different (p < 0.05). # represents statistical differences when compared to control animals unexposed to CHP (p < 0.05). Analysis were performed by two way-ANOVA followed by Duncan's post hoc test. The absolute values of control animals (mean ± SD) are GSH-t 0.44 ± 0.09 (mmol/g wet weight), PSH 1.81 ± 0.29 (mmol/g wet weight) and GSSG 51.45 ± 14.92 (nmol/g wet weight), respectively (n ¼ 12).

while ZnCl2, and particularly CDNB, increased the CHP half-life and the CHP body burden. 4.2. Thiols and disulfide status An interesting finding of this study is the adaptive response of mussels under peroxide exposure, with a fast increase in GSH-t and PSH levels after 2 h of exposure to CHP. Several studies have demonstrated that PSH can be used as a responsive marker of oxidative stress in bivalves, with increased and specific PSH oxidation after exposure to several classes of stresses (Cole et al., 2014; Hu et al., 2014; Tedesco et al., 2010). Therefore, the increased PSH values in the present study were unanticipated, although we already detected increased PSH values in P. perna exposed to zinc for 21 days (Trevisan et al., 2014), as well as decreased PSH values in this same species after 2 days of zinc exposure (Franco et al., 2006). Some events would lead to increased PSH levels, such as protein unfolding or deglutathionylation processes, exposing previously inaccessible thiols, as well as an increase in synthesis of thiol-containing proteins. The short exposure period (2 h) may be a limiting barrier for these biochemical changes to occur, requiring additional experiments to address this issue. Regarding the GSH-t increase after CHP exposure in control animals, it is not clear what is the molecular or biochemical mechanism underlying such responses, but it is already known that longer exposures (12e48 h) to oxidative stress-inducing compounds, such as metals, quinones and polycyclic aromatic hydrocarbons (Dafre et al., 2004; Gravato et al., 2004) can cause GSH synthesis in bivalves. In eukaryotes, such a response is usually related to the activation of genes involved in glutathione synthesis, antioxidant and biotransformation defenses, mediated by the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling pathway. Some indirect evidences suggest the occurrence of a similar signaling pathway in bivalves. Increase in glutathione S-transferase activity and transcription, quickly GSH de novo synthesis and coordinated increase in activity of antioxidant enzymes was found after exposure to electrophilic substances, polycyclic aromatic compounds, pesticides or metals (Geret et al., 2013; Lüchmann et al., 2014; Trevisan et al., 2012, 2014). Therefore, the increase in GSH-t levels after CHP exposure detected in this study could be related to an adaptive response to oxidative stress by the Nrf2

pathway. Instead, under a more intense stress, other signaling pathways related to cellular disturbances and death are activated (Kaspar et al., 2009). This could be the case in animals pretreated with ZnCl2, where the CHP toxicity was higher and the increase in GSH-t was not observed. GSSG levels did not increase as expected in organisms challenged with organic peroxide in parallel to impaired GR activity. This lack of GSSG build up has also been found in mammals or cell models treated with zinc, even though presenting significant GR inhibition (Bishop et al., 2007; Franco et al., 2008a). Cells can export GSSG in an ATP-dependent manner by ATP-binding cassette (ABC) export proteins (Ellison and Richie, 2012), a possible explanation for the lack of GSSG increase in our experiments. The overexpression of P-glycoprotein (Pgp), an ABC export protein, has already been reported in brown mussel P. perna exposed to 30 mM of zinc for 48 h (Franco et al., 2006). Although Pgp is not necessarily responsible for GSSG efflux, we speculated that other ABC proteins can be overexpressed by zinc treatment in mussels, probably increasing GSSG efflux, which remains to be confirmed. For animals pretreated to CDNB, the lower GSH-t and GSSG levels are expected, and are probably related to the consumption of GSH by conjugation reactions with CDNB as already suggested in oysters (Trevisan et al., 2012), fish erythrocytes (Stoelting and Tjeerdema, 2000) and humans cells (Rebbeor et al., 1998). Therefore, even with a lower GR activity caused by CDNB pre-exposure, the GSSG formation rate during a CHP exposure can be restricted by the low availability of GSH. 4.3. Consequences of ZnCl2 and CDNB pre-treatments in mussel health status and survival Invertebrates rely on an innate immune system to cope with microorganism invasion, having hemocytes as its main cellular effector. Hemocyte analyzes are widely used in ecotoxicological studies as general indicators of animal physiological and health status (Mydlarz et al., 2006). Therefore, cellular assays were conducted with hemocytes from mussels with an antioxidant system impaired by GSH depletion, GR and TrxR inhibition as consequence of ZnCl2 or CDNB pre-exposure. Bivalve THC may undergo significant variations due to several environmental conditions, including chemical contaminants such as metals (Galloway and Depledge,

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2001). This may be the case for the synergetic effect observed on THC values from animals exposed to ZnCl2 and CHP. Since it was used a short exposure period (18 h ZnCl2 þ 2 h CHP), this higher number of circulating hemocytes may indicate their migration from tissues, a bivalve adaptive mechanism to stressful conditions, as already detected in mussels exposed to copper (Pipe et al., 1999). On the other hand, CDNB pre-treatment increased the susceptibility of hemocytes to CHP, as detected by the viability assays, suggesting that a higher CHP body burden could be occurring, increasing peroxide levels in the hemolymph and thus negatively affecting the immune cells. The animal predisposition to CHP was also investigated in organisms pre-exposed to ZnCl2 or CDNB. Zinc significantly increased the mortality rate caused by 50 and 100 mM CHP, and due to their impaired ability to decompose CHP, it is reasonable to assume that this increase in mortality is related to a lower CHP detoxification rate caused by GR inhibition, a hypothesis remaining to be confirmed. CDNB decreased animal survival to CHP exposure in a more preeminent manner, suggesting that disruption in the GPx and Prx peroxidase systems, caused by GR, TrxR and GSH-t impairment, is associated with increased mussel susceptibility to peroxides. 5. Conclusions Our data contribute to the discussion on the relative importance of GR, TrxR and GSH in protecting brown mussels P. perna against peroxide exposure. By using ZnCl2 and CDNB as antioxidant depleting agents, we integrated biochemical, cellular and toxicity assays to evaluate the effects of these chemical stressors in increasing organic peroxide toxicity. Data suggest that impairment of the peroxidase systems and thiol status lead to lower peroxide detoxification rate, affects immune cells and lower animal chances to survive when facing a pro-oxidant challenge. Furthermore, results reinforce the importance of the peroxidase systems in bivalve adaptability to oxidative stress conditions, an area still underrepresented in the literature. Competing interests All authors declare to have no actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations. The work described has not been published previously (except in the form of an abstract); it is not under consideration for publication elsewhere; and its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out, and if accepted, it will not be published elsewhere in the same form, in English or in any other language, without the written consent of the copyright holder. Author contributions RT: experiment design, execution of assays, data analysis and manuscript writing; DFM: execution of assays with hemocytes, data analysis and manuscript writing; MUS: execution of biochemical assays, data analysis; GD: execution of biochemical assays, data analysis; MA: execution of assays with hemocytes, data analysis; ALD: experiment design, data analysis and manuscript writing. Funding This work was supported by CNPq (Conselho Nacional de
gico, #481488/2012-0), Desenvolvimento Científico e Tecnolo

~o de Amparo a  Pesquisa e Inovaç~ FAPESC (Fundaça ao do Estado de Santa Catarina, #7033/2010-5 and #1348/2010-0) and INC-TA ^ncia e Tecnologia de Toxicologia Aqua
tica, (Instituto Nacional de Cie #573949/2008-5). The provided scholarships are highly appreciated (RT and DFM e CAPES; MUS, GD and MA e CNPq). ALD is a research fellow of CNPq. List of symbols and abbreviations ABC Cat CDNB CHP Ctl DMSO DTNB EDTA GPx GR GSH GSH-t GSSG HEPES MTT

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