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Neuroscience Letters 558 (2014) 103– 108

Contents lists available at ScienceDirect

Neuroscience Letters

jou rn al hom epage: www.elsev ier .com/ locate /neule t

cute restraint stress induces an imbalance in the oxidative status ofhe zebrafish brain

laucia Dal Santoa, Greicy M.M. Conteratoa, Leonardo J.G. Barcellosb,enis B. Rosembergc,d,e, Angelo L. Piatoa,d,e,∗

Laboratório de Psicofarmacologia e Comportamento (LAPCOM), Universidade Comunitária da Região de Chapecó, Avenida Senador Attílio Fontana, 591E,9809-000 Chapecó, SC, BrazilPrograma de Pós-Graduac ão em Bioexperimentac ão, Universidade de Passo Fundo, Campus Universitário, Caixa Postal 611, 99001-970 Passo Fundo, RS,razilLaboratório de Genética e Ecotoxicologia molecular (LAGEM), Universidade Comunitária da Região de Chapecó, Avenida Senador Attílio Fontana, 591E,9809-000 Chapecó, SC, BrazilPrograma de Pós Graduac ão em Ciências Ambientais, Universidade Comunitária da Região de Chapecó, Avenida Senador Attílio Fontana, 591E, 89809-000hapecó, SC, BrazilZebrafish Neuroscience Research Consortium (ZNRC), USA

i g h l i g h t s

Acute restraint stress (ARS) induces oxidative stress in zebrafish brain.ARS increased SOD/CAT activity ratio and lipid peroxides.ARS increased non-protein thiol levels without altering total reduced thiols.Zebrafish is a suitable organism for studying acute stress protocols.

r t i c l e i n f o

rticle history:eceived 16 August 2013eceived in revised form 4 November 2013ccepted 11 November 2013

eywords:cute restraint stressntioxidant defensesxidative stress

a b s t r a c t

The zebrafish (Danio rerio) has become an emergent model organism for translational approaches focusedon the neurobiology of stress due to its genetic, neuroanatomical, and histological similarities with mam-malian systems. However, despite the increasing number of studies using zebrafish, reports examiningthe impact of stress on relevant neurochemical parameters are still elementary when compared to stud-ies using rodents. Additionally, it is important to further validate this model organism by comparing itsstress response with those described in other species. Here, we evaluated the effects of an acute restraintstress (ARS) protocol on oxidative stress-related parameters in the zebrafish brain. Our data revealedthat ARS significantly decreased catalase activity without altering the activity of superoxide dismutase.

ebrafish Oxidative stress was also indicated by increased levels of lipid peroxides. ARS significantly increased thelevels of non-protein thiols, although significant changes in total reduced sulfhydryl content were notdetected. These results suggest that ARS is an interesting strategy for evaluating the mechanisms under-lying the neurochemical basis of the oxidative profile triggered by acute stressors in the zebrafish brain.Furthermore, this protocol may be suitable for screening new compounds with protective properties

hich

against oxidative stress, w

. Introduction

In many vertebrate organisms, stressful stimuli have beenhown to evoke a coordinated pattern of physiological and

∗ Corresponding author at: Universidade Comunitária da Região de Chapecó, LAP-OM – Programa de Pós-Graduac ão em Ciências Ambientais, Avenida Senador Attílioontana, 591E, 89809-000 Chapecó, SC, Brazil. Tel.: +55 49 33218215;ax: +55 49 33218215.

E-mail address: angelopiato@gmail.com (A.L. Piato).

304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.neulet.2013.11.011

plays an increasingly important role in many psychiatric disorders.© 2013 Elsevier Ireland Ltd. All rights reserved.

behavioral changes [1]. When a threatening stimulus is presented,the acute response is “fight or flight”, which consists of a set of phys-iological reactions that culminate in a fast adaptation of cardiac,muscular, and visual functions [2]. The mechanisms underlyingthese adaptive responses are the activation of the hypothalamus-pituitary-adrenal (HPA) axis and the sympathoadrenal system.Both autonomic and neuroendocrine responses are part of the

physiological component of behavioral responses required for themaintenance of homeostasis during stressful events [3].

Zebrafish (Danio rerio) is a suitable model organism for study-ing the deleterious effects promoted by stressors and examining

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he behavioral and neurochemical outcomes [4–8]. This speciesas a low cost, small size, and high fecundity rate, which are valu-ble characteristics for high-throughput analyses [9,10]. The highegree of homology between zebrafish and human genomes [11],he conserved function of brain areas [12] and neurotransmit-er systems [13], and the presence of an HPA axis homolog (theypothalamic-pituitary-interrenal axis) [14,15] make zebrafish anttractive model organism for studying the neurobiology of stressnd complementing the current rodent approaches. Moreover, sim-lar to humans, the main glucocorticoid involved in the stressesponse in zebrafish is cortisol, the level of which increases sig-ificantly after exposure to a wide range of stressors [16–18].

Reactive oxygen species (ROS) and free radicals are producedy the mitochondria, which is the major site of their intracellularroduction, during normal aerobic metabolism [19]. The super-xide anion (O2

−) and hydrogen peroxide (H2O2) produced byhe respiratory chain may generate the reactive hydroxyl radi-al (•OH) via the Fenton reaction [20]. Overproduction of •OH iselated to lipid, protein, and DNA oxidation that may trigger sev-ral events, culminating in cell death [21]. However, endogenousntioxidant systems act as scavengers by counteracting these dele-erious effects [22]. Enzymatic antioxidant protection is providedy superoxide dismutase (SOD), catalase (CAT), and glutathioneeroxidase (GPx), while non-enzymatic defense is provided byeduced glutathione (GSH) but also by vitamins from the diet.xidative stress occurs when the antioxidant systems are overcomey ROS production [22]. Nerve cells are particularly susceptible toxidative stress due to elevated polyunsaturated fatty acid content,igh oxygen utilization, and the presence of redox-active metals23]. Indeed, ROS overproduction and oxidative stress have beenmplicated in the pathophysiology of several neurodegenerativend psychiatric diseases [24–26]. It is conceivable that stressfulonditions stimulate intricate cell signal transduction pathwayshat lead to an increase in ROS formation. In this regard, restrainttress can affect central nervous system (CNS) homeostasis byisrupting neurochemical and endocrine parameters, inducing an

mbalance of antioxidant status [27–29]. Because zebrafish is con-idered a promising model organism for translational neuroscienceesearch, the evaluation of oxidative status in the CNS after appli-ation of an acute restraint stress protocol (ARS) is a fundamentaltep for the validation of the model at the construct level. Thus, theoal of the current report was to investigate the effects of ARS onxidative stress parameters in the zebrafish brain.

. Materials and methods

.1. Animals

A total of 100 male and female wild-type short-fin strain adultebrafish (4–6 months old, 50:50 male:female ratio) were obtainedrom the heterogeneous breeding stock of Universidade de Passoundo. The fish were kept in 50-L aquariums (80–100 fish perank) for 2 weeks prior to the experiments to acclimate them toaboratory conditions. All experiments were performed in tankslled with non-chlorinated water that was mechanically and chem-

cally filtered and maintained at 26 ± 2 ◦C with a light/dark cyclef 14/10 h (lights on at 7:00 AM). The fish were fed twice a dayith a commercial flake fish food (Alcon BASIC®, Alcon, Brazil). Allrotocols were approved by the Ethics Committee of Unochapecó#003/2012).

.2. Reagents

Epinephrine, 4,6-dihydroxypyrimidine-2-thiol (TBA), 1,1,3,3-etramethoxypropane (TMP), and 5,5′-dithiobis(2-nitrobenzoic

etters 558 (2014) 103– 108

acid) (DTNB) were purchased from Sigma (St. Louis, MO, USA). Allother reagents were of analytical grade.

2.3. Acute restraint stress protocol (ARS)

In this protocol, which has been previously reported by ourgroup, 50 animals can be tested concomitantly, and only a smallspace is required [6,17]. The protocol involved enclosing each ani-mal in 2-mL plastic microcentrifuge tubes containing openings atboth ends (one at the cap and other at the bottom end of the tube)that were placed in a 20-L tank for 90 min. The openings weremanually shaped and were large enough (approximately 5 mm indiameter) to allow adequate water circulation inside the tube for90 min. Importantly, the time period chosen was previously utilizedfor zebrafish; within this time period, the animals showed signif-icant alterations in CRF levels and increased whole-body cortisol[18]. The control group (non-stressed) remained in the same room.Aeration (8 ppm) and temperature (26 ± 2 ◦C) were controlled dur-ing the test, and pilot experiments showed that the oxygen levelsdid not significantly differ in either the tank water or the watercollected inside the plastic tube. After ARS, the fish were cryoanaes-thetized and euthanized by decapitation to remove the brain.

2.4. Oxidative stress analyses

Each of the samples used for biochemical assays consisted ofa pool of ten whole brains, which were gently homogenized in1 mL of phosphate-buffered saline solution (PBS), pH 7.4, contain-ing 137 mM NaCl, 10.1 mM Na2HPO4, and 1.76 mM KH2PO4. Thesamples were further centrifuged at 700 × g for 5 min at 4 ◦C. Theresultant pellets were discarded, and the supernatants were col-lected for the experiments described herein, which were performedsimilarly to experiments reported previously for zebrafish [7].

2.5. Superoxide dismutase (SOD) and catalase (CAT) activities

Superoxide dismutase (EC 1.15.1.1, SOD) activity was quantifiedaccording to Misra and Fridovich [30] by spectrophotometricallydetermining the inhibition of auto-oxidation of epinephrine toadrenochrome at an alkaline pH at 480 nm. Catalase (EC 1.11.1.6;CAT) activity was assessed by measuring the rate of decrease inH2O2 absorbance at 240 nm [31]. For the SOD assay, 20–90 �gof protein was used, whereas CAT activity was determined using50–80 �g protein. All results were calculated and expressed as Uper mg protein.

2.6. Lipid peroxidation

The determination of the lipid redox state was measured basedon the formation of thiobarbituric acid reactive substances (TBARS)[32]. Briefly, 300 �L of sample was mixed with 700 �L of 15%trichloroacetic acid (TCA) and centrifuged at 10,000 × g for 10 min.The supernatants were mixed with 0.67% TBA (at a 1:1 proportion)and heated at 100 ◦C for 30 min. The TBARS levels were determinedin triplicate based on the absorbance at 532 nm using TMP as astandard. The results were expressed as nmol TBARS per mg pro-tein.

2.7. Total content of reduced thiol and non-protein thiol groups

To quantify the total reduced thiol content, 200 �L ofhomogenate was incubated in the presence of 0.2 mM EDTA and

100 mM boric acid buffer (pH 8.5). Subsequently, DTNB (0.01 Mdissolved in ethanol) was added, and after 1 h, the product wasmeasured at 412 nm [33]. The non-protein sulfhydryl groups weresimilarly assessed, except that the samples were centrifuged at

ence Letters 558 (2014) 103– 108 105

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0,000 × g for 10 min in the presence of 15% TCA, and the super-atants were used for the biochemical assay. The results wereormalized and expressed as �mol SH per mg protein.

.8. Protein quantification

Protein was quantified according to the method previouslyescribed by Peterson [34] using bovine serum albumin as atandard.

ig. 1. Effects of ARS on SOD (A) and CAT (B) activities. The effects of ARS on theOD/CAT ratio are also shown (C). The data are presented as the mean ± SEM of fivendependent experiments (n = 5) performed in triplicate (Student’s t test. *P≤0.05s. control).

Fig. 2. Effect of ARS on lipid peroxidation (measured as TBARS formation) in thezebrafish brain. The data are presented as the mean ± SEM of five independentexperiments (n = 5) performed in triplicate (Student’s t test. *P≤0.05 vs. control).

2.9. Statistics

The data were expressed as the means ± standard error ofmean (SEM) of five independent experiments (n = 5) performedin triplicate. The normal distribution of the data was confirmedby Kolmogorov–Smirnov and Levene tests, and the results

Fig. 3. Effect of ARS on total reduced sulfhydryl levels (A) and on non-protein thiolcontent (B) in the zebrafish brain. The data are presented as the mean ± SEM of fiveindependent experiments (n = 5) performed in triplicate (Student’s t test. *P≤0.05vs. control).

106 G. Dal Santo et al. / Neuroscience Letters 558 (2014) 103– 108

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. Results and discussion

The effects of ARS on the enzymatic antioxidant defense systemn the zebrafish brain are demonstrated in Fig. 1. Although the SODctivity in stressed animals did not significantly differ from thatn the control group (Fig. 1A), ARS decreased CAT activity (P < 0.05,pproximately 40%) (Fig. 1B). Thus, the significant increase in theOD/CAT ratio (Fig. 1C, P < 0.05) suggests an imbalance in the enzy-atic antioxidant defense system.Previous studies have shown increased lipid peroxidation

35,36] and modulation of antioxidant enzyme activities in differ-nt brain regions of rodents following restraint stress, dependingn the severity and duration of the protocol [36–38]. SOD is impor-ant for catalyzing the dismutation of the superoxide anion radicalo oxygen and H2O2, which is subsequently decomposed by CAT toater and oxygen [22]. Although the cause of decreased brain CAT

ctivity in zebrafish remains unknown, it is unlikely to occur due to

nactivation by H2O2 overload as previously demonstrated [39,40]ecause SOD activity in both control and ARS groups was similar.he increased ratio of SOD/CAT activities in stressed animals sug-ests a reduction of the brain’s defense mechanisms against H2O2,

of ARS-induced oxidative stress in the zebrafish brain.

which could exert deleterious effects. In fact, H2O2 may be metab-olized to the •OH radical via the Fenton reaction, which is catalyzedby transition metals [20,41]. In addition to perpetuating the chainreaction of polyunsaturated fatty acid oxidation, •OH is involved inthe initiation of lipid peroxidation [42], which could also result inoxidative stress.

ARS significantly increased (P < 0.05, approximately 135%) thelevel of lipid peroxides in the zebrafish brain (Fig. 2). The effects ofARS have previously been widely studied in different model orga-nisms [43]. For example, in rodents, restraint stress is associatedwith modulation of the hypothalamic-pituitary-adrenal axis andsympathetic nervous system [44]. Despite widespread knowledgeregarding the stress response in mammals, few studies have inves-tigated the acute response to restraint stress in zebrafish. Ghisleniet al. [17] and Piato et al. [6] demonstrated that ARS altered behav-ioral, biochemical, and molecular parameters in zebrafish. The ARSprotocol increased the time that fish spent in the bottom area of anovel tank, which is indicative of anxiety-like behavior [43]. More-over, a recent study performed by Braga and colleagues [45] showedthat hypoxia decreased the locomotor activity of zebrafish but did

not significantly change the time spent in the bottom area of thetank. Therefore, the biochemical changes described here can beattributed to restraint stress and not to hypoxic conditions. Takinginto consideration the fact that ARS alters central nervous system

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nd neuroendocrine functions, the occurrence of lipid peroxidationould also be attributed to a complex modulatory effect on brainomeostasis.

One interesting strategy that could be used to assess the redoxrofile of biological samples is the measurement of thiol levels.RS did not promote significant changes in total reduced thiol lev-ls (Fig. 3A). However, non-protein thiol levels were substantiallyncreased after ARS (Fig. 3B, P < 0.05). These data could indicate thexistence of an adaptive response to ROS production during stress-ul conditions in zebrafish. Non-protein thiols are mostly composedf GSH, an important non-enzymatic antioxidant that maintainsntracellular redox homeostasis [46]. This tripeptide is involved inetoxification of xenobiotics as well as in protection against ROSeneration [22,47]. The intracellular increase of non-protein thiolroup levels is frequently observed in animals exposed to toxicantshat induce oxidative stress [48,49]. The induction of ARS in ratsncreases non-protein thiol levels in the cerebellum after immo-ilization [50]. Additionally, ARS in rodents either abolished or

essened changes in GSH levels in different brain structures [35,51].his finding could be attributed to the use of distinct models androtocols.

The mechanism proposed for ARS-induced oxidative stress inebrafish is presented in Fig. 4. The inhibitory effect on CATctivity could lead to increased levels of H2O2, which, in turn,ould be converted to the •OH radical via the Fenton reaction.his reactive species could exert deleterious effects on cellulartructures via lipid peroxidation, suggesting oxidative stress. Con-ersely, the levels of non-protein thiols detected could be a resultf increased GSH levels. Previous studies have shown that thencrease in GSH levels under oxidative stress may be related toctivation of NF-E2-related factor 2 (Nrf2) pathways in an attempto restore redox homeostasis [10,15,27,46]. Although Nrf2 path-ay activation has been previously demonstrated in zebrafish

mbryos and larvae exposed to a different oxidant agents [52], thenvolvement of GSH and the Nrf2 pathway in ARS is a topic thatequires further investigation. Independent of this mechanism, thencreased levels of non-protein thiols in our study did not preventhe lipid peroxidation observed in the stressed zebrafish, whichndicates that ROS production overcame the cellular antioxidantapacity.

. Conclusion

In summary, our results reveal that ARS induces an imbalancen the antioxidant status in the zebrafish brain, which is evidencedy increases in the SOD/CAT activity ratio and lipid peroxida-ion. The increase in non-protein thiol levels may be interpreteds a defense response against ROS generated in animals underRS. Taken together, these results indicate that zebrafish is an

nteresting model organism in which to study the neurochemicallterations induced by stress. Moreover, the ARS protocol may beuitable for screening new compounds with the ability to protectgainst oxidative stress.

cknowledgments

This work was supported by Conselho Nacional de Desen-olvimento Científico e Tecnológico – Brazil (CNPq, Proc.72715/2012-7). The authors are grateful to Daiane Ferreira (PPGarmacologia, UFSM) for manuscript revision.

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