Neurotoxicology and Teratology 33 (2011) 727–734

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Neurotoxicology and Teratology

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Possible role of serotoninergic system in the neurobehavioral impairment induced byacute methylmercury exposure in zebrafish (Danio rerio)

Caio Maximino a,b,⁎, Juliana Araujo a,b, Luana Ketlen Reis Leão a, Alan Barroso Araújo Grisolia a,Karen Renata Matos Oliveira a, Monica Gomes Lima a, Evander de Jesus Oliveira Batista a,c,Maria Elena Crespo-López d, Amauri Gouveia Jr. e, Anderson Manoel Herculano a,b,⁎a Laboratório de Neuroendocrinologia, Instituto de Ciências Biológicas, Universidade Federal do Pará, Brazilb Zebrafish Neuroscience Research Consortium, Brazilc Laboratório de Protozoologia, Núcleo de Medicina Tropical, Universidade Federal do Pará, Brazild Laboratório de Farmacologia Molecular, Instituto de Ciências Biológicas, Universidade Federal do Pará, Brazile Laboratório de Neurociências e Comportamento, Núcleo de Teoria e Pesquisa do Comportamento, Universidade Federal do Pará, Brazil

⁎ Corresponding authors at: Laboratório de NeurCiências Biológicas, Universidade Federal do Pará. R. ABelém/PA, Brazil. Tel.: +5591 3201 7742.

E-mail addresses: caio@ufpa.br (C. Maximino), hercul

0892-0362/$ – see front matter © 2011 Elsevier Inc. Aldoi:10.1016/j.ntt.2011.08.006

a b s t r a c t

a r t i c l e i n f o

Article history:Received 31 March 2011Received in revised form 8 August 2011Accepted 9 August 2011Available online 25 August 2011

Keywords:ZebrafishOxidative stressAnxietyMethylmercurySerotonin

Adult zebrafish were treated acutely with methylmercury (1.0 or 5.0 μg g−1, i.p.) and, 24 h after treatment,were tested in two behavioral models of anxiety, the novel tank and the light/dark preference tests. At thesmaller dose, methylmercury produced a marked anxiogenic profile in both tests, while the greater doseproduced hyperlocomotion in the novel tank test. These effects were accompanied by a decrease inextracellular levels of serotonin, and an increase in extracellular levels of tryptamine-4,5-dione, a partiallyoxidized metabolite of serotonin. A marked increase in the formation of malondialdehyde, a marker ofoxidative stress, accompanied these parameters. It is suggested that methylmercury-induced oxidative stressproduced mitochondrial dysfunction and originated tryptamine-4,5-dione, which could have furtherinhibited tryptophan hydroxylase. These results underscore the importance of assessing acute, low-levelneurobehavioral effects of methylmercury.

oendocrinologia, Instituto deugusto Corrêa, 01, 66075-110,

ano@ufpa.br (A.M. Herculano).

l rights reserved.

© 2011 Elsevier Inc. All rights reserved.

1. Introduction

Methylmercury (MeHg) is a pervasive environmental contaminantthat causes marked neurobehavioral effects, including memorydeficits and anxiety-like effects (Bakir et al., 1980; Carratù et al.,2006; Liang et al., 2009; Maia et al., 2010). This organic mercuryspecies bioaccumulates in the food chain, and its effects arecumulative and dose-dependent (Elhassani, 1982; Mahaffey, 2000).While chronic, high-level poisoning events such as those described inJapan (Harada, 1995) and Iraq (Bakir et al., 1973) certainly requireattention, descriptions of early effects of MeHg poisoning are few. Thecharacterization of these effects can contribute to the development ofmarkers for early deficits caused by methylmercury poisoning(Mahaffey, 2000).

In the central nervous system, the main neurochemical effect ofmethylmercury poisoning seems to be excitotoxicity-mediated oxida-tive stress (Aschner et al., 2007; Nascimento et al., 2008; Yee and Choi,

1996). In vivo exposure to MeHg causes its accumulation insidemitochondria followed by a series of biochemical changes, includingreduced cellular respiration and decreases in the activity of mitochon-drial enzymes such as cytochrome C oxidase, superoxide dismutase,monoamine oxidase (MAO) and succinate dehydrogenase (Bernstssenet al., 2003; Beyrouty et al., 2006; Cambier et al., 2009; Chakrabarti et al.,1998; Dreiem and Seegal, 2007; Kirubagaran and Joy, 1990; Levesqueand Atchison, 1992;Mori et al., 2007; Ram and Sathyanesan, 1985). Thedisruption of the electron transport chain in the mitochondria and thedysfunction of energetic metabolism lead to the formation of reactiveoxygen species (ROS) (Ali et al., 1992; Aschner et al., 2007; Carvan et al.,2001;Kusik et al., 2008;Nascimento et al., 2008; Sarafian, 1999; Sarafianet al., 1994; Shanker et al., 2002). One of the main cellular defensesagainst these ROS, glutathione, is also affected by MeHg. This toxicantinhibits cysteine uptake in astrocytes and the activity of glutathioneperoxidase, thereby depleting intracellular glutathione levels (Aschneret al., 2007; Franco et al., 2009; Shanker et al., 2001, 2005). MeHg alsoinhibits glutamate uptake in glial cells, producing a glutamatergicaccumulation in the synaptic cleft and, ultimately, resulting in furtherincreases in ROS formation and excitotoxicity (Aschner et al., 2007;Nascimento et al., 2008).

In addition to the effects of MeHg on glutamate-mediatedexcitotoxicity and ROS formation, alterations were also observed in

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monoaminergic systems. Thus, MeHg is able to inhibit monoamineoxidase activity in vivo and in vitro in both rats and fishes (Berntssenet al., 2003; Beyrouty et al., 2006; Chakrabarti et al., 1998). Likewise,MeHg inhibits serotonin uptake in astrocytes and synaptosomes (Daveet al., 1994; Komulainen and Tuomisto, 1981), and increases serotoninrelease in platelets and synaptosomes (Ally et al., 1993; Hornberger andPatscheke, 1989; Komulainen and Tuomisto, 1981). Another potentialeffect is the production of abnormal, oxidized serotonin metaboliteswhich are formed by reaction with superoxide radicals (Chen et al.,1989; Jiang and Dryhurst, 2002; Jiang et al., 1999; Uemra and Shimazu,1980; Wrona and Dryhurst, 1998, 2001). All of these alterations of theserotonergic systemalterationsmay be partially responsible also for thewell demonstrated anxiety- and depression-like effects of mercurypoisoning (Haut et al., 1999; Liang et al., 2009; Maia et al., 2009, 2010;O'Carroll et al., 1995; Onishchenko et al., 2007, 2008; Scopoli, 1771;Siblerud et al., 1994; Wang et al., 2011).

The zebrafish (Danio rerio) has emerged as a very useful model instudies about genetics, neuropharmacology and behavior (Levin et al.,2007; Serra et al., 1999). The use of this model is increasing due to awide range of advantages that zebrafish provide, such as its low cost,ease of management and accommodation, short life cycle, compre-hensive reproductive performance in laboratory, and extensivehomology at the genetic, neural and endocrine levels with mammals(Champagne et al., 2010; Levin et al., 2007; Tropepe and Sive, 2003).This vertebrate is increasingly being used in toxicological research,including neurobehavioral toxicology (Carvan et al., 2005; Spitsbergenand Kent, 2003; Zhang et al., 2003). A zebrafish cell line (ZEM2S)transfected with luciferase-linked electrophile response element(EPRE) has been used to detect oxidative stress induced by differentcompounds, includingmercuric chloride (HgCl2) (Carvan et al., 2001),and transgenic zebrafish expressing an EPRE-luciferase-green fluores-cent protein (GFP) construct have been used successfully to assessmercury-generated oxidative stress in vivo (Kusik et al., 2008). Withthis strategy, waterborne HgCl2 above 0.3 μM have been shown toinduce oxidative stress in neurons (Kusik et al., 2008). Similarconcentrations increase response latency and reduce maximumvelocity of fast-starts in 5 days post hatch (dph) larvae which wereexposed to mercuric chloride between 0 and 24 h postfertilization(hpf) (Weber, 2006). Waterborne exposure to MeHg dose-depen-dently decreased spontaneous swimming activity in 5 dph larvae(Samson et al., 2001). Interestingly, in 120 hpf zebrafish MeHgexposure downregulates expression of glutathione peroxidase andupregulates expression of peroxiredoxin I, thioredoxin, and glutathi-one S-transferase (Yang et al., 2007). In adult female zebrafish,intraperitoneal injection of 0.5 μg g−1 downregulated thioltransferaseand protein disulfide isomerase, and upregulated selenoproteins andglutathione S-transferase (Richter et al., 2011).

Despite the increased use of adult and larval zebrafish in behavioralstudies (Best and Alderton, 2008; Norton and Bally-Cuif, 2010), fewstudies so far have described behavioral effects of toxic compounds inthis model organism. Given the availability of behavioral assays toevaluate stress- and anxiety-like phenotypes (Maximino et al., 2010a)and the relative conservation of monoaminergic systems in thisspecies (Maximino and Herculano, 2010), the use of adult zebrafish asa model to study MeHg effects on anxiety-like behavior and theirneurochemical underpinnings becomes essential. In the present paper,we show the effects of acute poisoning on geotaxis (novel tank test)and scototaxis (light/dark preference), as well as on extracellularindoleamine levels, and oxidative stress in adult zebrafish.

2. Methods

2.1. Ethical statement

Animals were housed and manipulated minimizing the potentialsuffering, as per the recommendations of the Canadian Council on

Animal Care (Canadian Council on Animal Care, 2005) and of the RoyalSociety for the Prevention of Cruelty to Animals (Reed and Jennings,2010). All procedures complied with the Brazilian Society for Neuro-science and Behavior's (SBNeC) guidelines for the care and use ofanimals in research.

2.2. Subjects and housing

Thirtyfive adult zebrafish ofwild type phenotypewere obtained in alocal ornamental fish shop (AquaNorte, Belém/PA) and brought to thelaboratory facilities, where the animals were left to acclimate for at leasttwo weeks before experiments. Animals of both genders were group-housed in 40 l tanks, with amaximum density of 25 fish per tank. Tankswerefilledwithdeionized and reconstitutedwater at roomtemperature(28 °C) and a pH of 7.0–8.0. Lightingwas provided by fluorescent lampsin a cycle of 14–10 h (light:dark), according to standards of zebrafishhusbandry (Lawrence, 2007). Animals were randomly distributed inthree groups (control: n=15; MeHg groups: n=15 each). The samesubjects were tested in the novel tank and scototaxis tests in acounterbalanced design, and were later euthanized and their brainsprocessed for neurochemical analyses.

2.3. Methylmercury administration

Methylmercury chloride (Sigma, Brazil) stocks were diluted inCortland's salt solution to a concentration of 3.9 mM. Intraperitonealinjections were made using 10 μl syringes with 33 G needles(Hamilton USA), according to Kinkel et al. (2010). Briefly, animalswere individually weighted and cold-anesthetized. Quickly afteranesthesia, animals were transferred to the trough of a surgical tablecomposed of a 20 mm soft sponge embedded in a 60 mm Petri dish,and Cortland's salt solution or MeHg (1.0 or 5.0 μg g−1) was injectedin the midline between the pelvic fins. Injections were always madein the period between 08:00 AM and 12:00 PM in order to allowbehavioral experiments to be made during the same period in thenext day. Animals were then transferred to a collective recovery tank(one tank per treatment) for 24 h before beginning of experiments.Animals were exposed to both behavioral tests as a counterbalanceddesign.

2.4. Novel tank test

The novel tank diving test was run between 08:00 AM and12:00 PM, to minimize circadian rhythm effects. The protocol forthe novel tank diving test used was modified from Cachat et al.(2010), and animals were tested individually. Briefly, 24 h after MeHginjection the animals were transferred to the test apparatus, whichconsisted of a 15×25×20 cm (width×length×height) tank lightedfrom above by two 25 W fluorescent lamps, producing an average of120 lm above the tank. As soon as the animal was transferred to theapparatus, a webcam was activated and behavioral recording begun.The webcam filmed the apparatus from the front, thus recording theanimal's lateral and vertical distribution. Animals were allowed tofreely explore the novel tank for 6 min, after which they wereremoved from it and exposed to the scototaxis tank. Video files werelater analyzed by experimenters blind to the treatment using X-Plo-Rat 2005 (http://scotty.ffclrp.usp.br), and the images were divided ina 3×3 grid composed of 10 cm² squares. The following variables wererecorded:

time on top: the time spent in the top third of the tank; squarescrossed: the number of 10 cm² squares crossed by the animal duringthe entire session; erratic swimming: the number of “erraticswimming” events, defined as a per Egan et al. (2009) (that is, sharpchanges in direction or velocity and repeated rapid darting behaviorswith durationb1 s); and freezing: the total duration of freezing

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events, defined as complete cessation of movements with theexception of eye and operculae movements.

2.5. Light/dark preference

Determination of MeHg effects on scototaxis was carried asdescribed elsewhere (Maximino et al., 2010b). Briefly, animals weretransferred to the central compartment of a black and white tank(15 cm×10 cm×45 cm h×d×l) for a 3-min acclimation period, afterwhich thedoorswhichdelimit this compartmentwere removedand theanimal was allowed to freely explore the apparatus for 15 min. Thefollowing spatiotemporal variables were recorded, always in the whitecompartment:

time on the white compartment: the time spent in the top third ofthe tank (percentage of the trial); squares crossed: the number of10 cm² squares crossed by the animal in the white compartment;latency to white: the amount of time the animal spends in theblack compartment before its first entry in the white compart-ment (s); entries in white compartment: the number of entries theanimal makes in the white compartment in the whole session.

In addition to those spatiotemporal measures, the followingswimming ethogram was recorded:

erratic swimming: the number of “erratic swimming” events, definedas per Egan et al. (2009) (that is, sharp changes in direction or

Fig. 1. Effects of methylmercury on (A) time spent on the top third of the tank, (B) number otank test. ***, pb0.0001; **, pb0.01; *, pb0.05. Animals were injected with the indicated do

velocity and repeated rapid darting behaviors with durationb1 s);freezing in white compartment: the proportional duration of freezingevents (in% of time in thewhite compartment), defined as completecessation of movements with the exception of eye and operculaemovements; thigmotaxis in white compartment: the proportionalduration of thigmotaxis events (in% of time in the white compart-ment), defined as swimming in a distance of 2 cm or less from thewhite compartment's walls; andrisk assessment: the number of “riskassessment” events, defined as a fast (b1 s) entry in the whitecompartment followed by re-entry in the black compartment, or as apartial entry in thewhite compartment (i.e., the pectoralfindoes notcross the midline).

These ethogramunitswere scored in thewhite compartment, as theanimal cannot be seen in the black portion of the tank. Therefore,freezing and thigmotaxis were scored as proportional times (i.e.,% oftime in the white compartment). Habituation of white avoidance wasassessed as a cumulative habituation ratio (CHR), i.e. the ratio of timespent in thewhite compartment during thefirst 3-min vs. the last 3-minof the session (Wong et al., 2010).

2.6. Subcellular fractionation

After the end of behavioral testing, animals were euthanized andtheir brains were dissected. Zebrafish brains were fractionated bydifferential centrifugation as described by Pradel et al. (Pradel et al.,

f erratic swimming events, (C) freezing duration, and (D) total locomotion in the novelses 24 h before behavioral experiments.

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1999). Briefly, for each sample one brains was quickly removedfrom the skull and incubated in 2 ml of 50 mM TBS, pH 7.4,containing 90 mM NaCl, 2.5 mM CaCl2, 1 mM glutathione for

Fig. 2. Effects of methylmercury on (A) time spent in the white compartment, (B) totalcompartment, (D) habituation of locomotion in white compartment, (E) latency to enterpreference test. The dotted line in (A) signals 50%, the threshold for preference determinationinjected with the indicated doses 24 h before behavioral experiments.

30 min at 4 °C to extract the extracellular fluid (ECF). The brainswere then homogenized in 2 ml TBS, and half of the producedhomogenates (Ho) were centrifuged at 1000×gav for 10 min at 4 °C.

locomotion in the white compartment, (C), habituation of time spent in the whitethe white compartment, and (F) entries in the white compartment of the light/dark. CHR: Cumulative habituation ratio. ***, pb0.0001; **, pb0.01; *, pb0.05. Animals were

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Protein in all fractions was determined as described elsewhere(Bradford, 1976).

2.7. HPLC analysis of indoleamines

Serotonin and 5-HIAA (5 mg) was dissolved in 100 ml of elutingsolution (50 ml MilliQ water, 0.43 ml HClO4 70% [0.2 N], 10 mg EDTA,9.5 mg sodiummetabissulfite) and frozen at−20 °C, to later be used asa standard. Tryptamine-4,5-dione was chemically synthesized accord-ing to Chen et al. (1989) by adding an eight-fold amount of Fremy's saltto a 5-HT·HCl solution (3.5 mg in 2 ml of 0.01 N HCl). This last standardwas used immediately, due to its instability (Wrona and Dryhurst,1998).

TheHPLC systemconsistedof a delivery pump(LC20-AT, Shimadzu),a 20 μl sample injector (Rheodyne), a degasser (DGA-20A5), and ananalytical column(ShimadzuShim-PackVP-ODS, 250×4.6 mminternaldiameter). The integrating recorder was a Shimadzu CBM-20A(Shimadzu, Kyoto, Japan). An electrochemical detector (Model L-ECD-6A) with glassy carbon was be used at a voltage setting of +0.83 V (5-HTand5-HIAA)or+7.5 V (tryptamine-4,5-dione),with a sensitivity setat 8 nA full deflection. The mobile phase consisted of a solution of70 mMphosphate buffer (pH 2.9), 0.2 mM EDTA, 5%methanol and 20%sodium metabissulfite as a conservative. The column temperature wasset at 17 °C, and the isocratic flow rate was 1.8 ml/min 0.5 ml ofextracellular fluid (ECF), extracted as described above, was mixed with0.5 ml of the eluting solution described above.

Fig. 3. Effects of methylmercury on swimming ethogram measures in the light/dark preferencassessment events. ***, pb0.0001; **, pb0.01; *, pb0.05. Animals were injected with the indic

2.8. Lipid peroxidation assay

The degree of lipid peroxidation was assessed by following theformation of thiobarbituric acid-reactive substances (TBARS)(Schmedes and Hölmer, 1989). 100 μl of brain homogenate (Ho)was added to the same volume of TBA reagent (30% TCA, 0.0375%thiobarbituric acid, and 25% 1 N HCl). Sample absorbancies werecompared with a malondialdehyde (MDA) standard curve at 535 nm.

2.9. Statistical analyses

Behavioral variables were first tested for normality using theomnibus K² D'Agostino–Pearson's normality test, and found to benormally distribute (K²b5.99, pN0.05). Therefore, one-way analyses ofvariance (ANOVAs) were used to analyze these data, followed byTukey's tests whenever pb0.05. Results from HPLC, and TBARS assayswere corrected for protein content, normalized as percentage of controlvalues, and analyzed by one-way ANOVA, followed by Tukey's test.

3. Results

3.1. Novel tank test

Methylmercury decreased the time spent on the 1/3rd top of thetank (F[2, 32]=5.698, pb0.05, Fig. 1A), with post-hoc test indicating thateach MeHg group differed from the control group (pb0.05). Erratic

e test. (A) Thigmotaxis; (B) freezing duration; (C) erratic swimming events; and (D) riskated doses 24 h before behavioral experiments.

Fig. 4. Effects of methylmercury on extracellular contents of (A) serotonin (5-HT), (B) 5-hydroxyindolacetic acid (5-HIAA), and (C) tryptamine-4,5-dione (T-4,5-D). ***, pb0.0001;**, pb0.01; *, pb0.05. Animals were injected with the indicated doses 24 h before braindissection and extracellular fluid extraction.

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swimming events differed among treatment groups (F[2, 32]=3.782,pb0.05; Fig. 1B), with the lowest dose significantly increasing erraticswimming in relation to the control group (pb0.05); no furtherdifferences were found between groups in terms of erratic swimming(pN0.05). Differences were also observed among groups in freezingduration (F[2, 32]=4.446, pb0.05; Fig. 1C), with post-hoc test indicatingthat the highest dose group displayed significantly less freezing(pb0.05), with no other differences detected among groups. Totallocomotion also differed between groups (F[2, 32]=20.78, pb0.05;Fig. 1D), with post-hoc analyses indicating that the animals 5.0 μg g−1

showed hyperactivity in relation to both the control and 1.0 μg g−1

groups (pb0.05).

3.2. Light/dark preference

Differences were observed between treatment groups in the timespent in the white compartment (F[2, 32]=3.742, pb0.05; Fig. 2A); thepost-hoc test showed that the lowest dose group spent significantly lesstime in the white compartment than the control group (pb0.05),without differences between treatments or between the highest dosegroup and controls (pN0.05). No effects were observed on the totallocomotion in the white compartment (F[2, 32]=0.9638, p=0.3922;Fig. 1B). Animals poisoned with both doses showed impairedhabituation of the time spent in the white compartment (F[2, 32]=108.3, pb0.05; Fig. 2C), with all groups differing between each other(pb0.05). Habituation of total locomotion in the white compartmentwas also altered (F2, 32]=9.146, pb0.05; Fig. 2D), with post-hoc testsshowing a difference amongbothdoses and the control group (pb0.05),but not between doses (pN0.05). Latency to enter the whitecompartment was also different among groups (F[2, 32]=3.892,pb0.05; Fig. 2E), with the highest dose showing decreased latency inrelation to the control group (pb0.05), but not the lowest dose group(pN0.05). No effects were observed in the number of entries in thewhite compartment (F[2, 32]=2.457, p=0.1017; Fig. 2F). Regardingethogram measures, thigmotaxis was shown to be altered (F[2, 32]=9.547, pb0.05; Fig. 3A), with the lowest dose producing greaterthigmotaxis than both the control and 5.0 μg g−1 group (pb0.05). Noeffects were observed in freezing duration in the white compartment(F[2, 32]=1.714, p=0.1963; Fig. 3B). Differences were observed in thenumber of erratic swimming events in the white compartment(F[2, 32]=16.49, pb0.05; Fig. 3C), with the lowest dose producingmore erratic swimming than both the control and 5.0 μg g−1 group(pb0.05). Risk assessment eventswere altered (F[2, 32]=9.609, pb0.05;Fig. 3D), with post-hoc tests detecting a difference between the1.0 μg g−1 and the other groups (pb0.05).

3.3. Extracellular indoleamines levels

ECF serotonin content was different among groups (F[2, 32]=5.971,pb0.05; Fig. 4A), with the smallest dose decreasing serotonin levels byabout 65% in relation to control levels (pb0.05); no further differenceswere observed (pN0.05). No effect in the extracellular levels of 5-HIAA(F[2, 32]=0.5363, p=0.5901; Fig. 4B) was observed. Our results alsohave showed that MeHg treatment induced significant increases in theextracellular levels of tryptamine-4,5-dione (F[2, 32]=12.39, pb0.05;Fig. 4C), with both dose groups being different from control (pb0.05)but from each other (pN0.05).

3.4. Lipid peroxidation

Our results demonstrated that both doses of methylmercuryinduced an intense increase in the amount of MDA, a product oflipid peroxidation, in brain homogenates (F[2, 32]=16.05, pb0.05;Fig. 5). The post-hoc analysis revealed differences between bothMeHg groups and controls (pb0.05), but not between doses (pN0.05).

4. Discussion

In the present article, we demonstrated neurochemical andbehavioral effects of acute methylmercury poisoning in adult zebrafish.Animals injected with MeHg 24 h prior to behavioral testing showedanxiety-like behavior in both the novel tank and light/dark preferencetests at the smaller dose (1.0 μg g−1), with more marked effects on thesecond test. The higher dose (5.0 μg g−1) produced a hyperlocomotor

Fig. 5. Effects ofmethylmercury poisoning on lipid peroxidation. ***, pb0.0001; **, pb0.01; *,pb0.05. Animals were injected with the indicated dose 24 h before brain dissection andhomogenization.

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effect in thenovel tank test, not detect in the light/dark preferenceassay.The anxiogenic-like effect of the smaller dose of methylmercury isdemonstrated by increased bottom-dwelling and white avoidance,increased erratic swimming, and increased thigmotaxis and riskassessment. The effects on white avoidance habituation suggestincreased arousal/anxiety/stress, while the effects on locomotionhabituation suggest either increased arousal or working memoryimpairments. Similar results have also been observed recently in adultzebrafish developmentally exposed to silver nanoparticles, which showa dose-dependent decrease in 5-HT levels and faster acquisition ofinhibitory avoidance (Powers et al., 2011).

These behavioral effects were accompanied by decreased extracel-lular serotonin and increased levels of the partially oxidized serotoninmetabolite, tryptamine-4,5-dione. Methylmercury also produced oxi-dative stress, as assessed by the lipid peroxidation assay. The increase inoxidative stress supports a “depletion” time-course, in which MeHgproduces reactive oxygen species which oxidize intra- and extracellularserotonin into tryptamine-4,5-dione, which will produce serotoninefflux and inhibit tryptophanhydroxylase (Chenet al., 1989;Wrona andDryhurst, 2001); serotonin efflux has been observed inMeHg-poisonedplatelets and synaptosomes (Ally et al., 1993; Komulainen andTuomisto, 1981), but a link with ROS formation and partial oxidationof 5-HT has not been reported. The resulting anxiogenic-like effect issimilar to what is observed with serotonin-depleting drugs such asPCPA, which enhances behavioral suppression mediated by the brainaversive system (Graeff, 2002; Graeff and Rawlings, 1980; Guimarãeset al., 2008; Lowry and Hale, 2010).

Overall, this article presents the first demonstration of an acuteanxiogeniceffect ofmethylmercurypoisoning inzebrafishassociatedwithchanges in the serotonin system; also novel is the demonstration of theproduction, in vivo, of tryptamine-4,5-dione, a putative serotonergicendotoxin (WronaandDryhurst, 1998). These results suggest that anxietyis an early effect of low-level methylmercury poisoning, and that theseeffects are associated with changes in the serotonin system. Anxiety-likesymptoms could be used to detect early effects of MeHg poisoning.

Conflict of interest

The authors declare no conflict of interests.

Acknowledgments

This work was financially supported by a CNPq/Brazil grant(Process 483336/2009-2). CM is the recipient of a CAPES studentship.AGJ and MECL are recipients of a CNPq productivity fellowship.

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